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Volume 9
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Biology, Volume 9, Issue 7 (July 2020) – 39 articles

Cover Story (view full-size image): Mesenchymal stem cells (MSCs) have been considered a promising cell population for cell-based therapy and tissue regeneration. Recently, dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) have been isolated, and have attracted substantial attention in terms of their clinical application because of their MSC-like qualities including robust self-renewal ability, multilineage differentiation potential, and immunomodulatory effects. In addition, various growth factors, extracellular vesicles, and epigenetic regulators which can facilitate the therapeutic potential of these cells have been identified, although their signaling interactions are still under investigation. Further clarification of the biological attributes of DPSCs and SHEDs and the development of safe and effective therapeutic approaches are needed to translate them into a clinical setting. View this [...] Read more.
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45 pages, 3962 KiB  
Review
Circadian Photoentrainment in Mice and Humans
by Russell G. Foster, Steven Hughes and Stuart N. Peirson
Biology 2020, 9(7), 180; https://doi.org/10.3390/biology9070180 - 21 Jul 2020
Cited by 91 | Viewed by 9223
Abstract
Light around twilight provides the primary entrainment signal for circadian rhythms. Here we review the mechanisms and responses of the mouse and human circadian systems to light. Both utilize a network of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). In [...] Read more.
Light around twilight provides the primary entrainment signal for circadian rhythms. Here we review the mechanisms and responses of the mouse and human circadian systems to light. Both utilize a network of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). In both species action spectra and functional expression of OPN4 in vitro show that melanopsin has a λmax close to 480 nm. Anatomical findings demonstrate that there are multiple pRGC sub-types, with some evidence in mice, but little in humans, regarding their roles in regulating physiology and behavior. Studies in mice, non-human primates and humans, show that rods and cones project to and can modulate the light responses of pRGCs. Such an integration of signals enables the rods to detect dim light, the cones to detect higher light intensities and the integration of intermittent light exposure, whilst melanopsin measures bright light over extended periods of time. Although photoreceptor mechanisms are similar, sensitivity thresholds differ markedly between mice and humans. Mice can entrain to light at approximately 1 lux for a few minutes, whilst humans require light at high irradiance (>100’s lux) and of a long duration (>30 min). The basis for this difference remains unclear. As our retinal light exposure is highly dynamic, and because photoreceptor interactions are complex and difficult to model, attempts to develop evidence-based lighting to enhance human circadian entrainment are very challenging. A way forward will be to define human circadian responses to artificial and natural light in the “real world” where light intensity, duration, spectral quality, time of day, light history and age can each be assessed. Full article
(This article belongs to the Special Issue Biological Clocks)
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<p>(<b>A</b>): phase response curve (PRC) for a nocturnal animal such as a mouse. In the upper part of this figure (A–D) the light/dark cycle is shown and the dark line illustrates the duration of activity (also called “alpha”) on subsequent days. For the first four days the animal is kept under a light/dark cycle of 12 h of light and 12 h of dark (L:D 12:12). On day 5, the lights were switched off and the animal was kept under constant darkness (DD), and it freeran with a period slightly shorter than 24 h. To provide reference points under freerunning conditions, activity onset in a nocturnal animal is termed “circadian time 12” or CT 12. The CT 0–12 is considered as the “subjective day,” and CT 12–24 is considered “subjective night.” If the animal is exposed to a single one-hour pulse of light during its subjective circadian day, as shown in (A), there is usually no or little phase shifting effect on the freerunning rhythm. This is called the “dead zone.” At (B) the light pulse is given early in the subjective night, the effect is to start activity slightly later the next day (a delaying phase shift). In (C) the light exposure is later into the night and there is an increased delaying effect the following day. When light is given during the second half of the night (D), the effect is to advance the freerunning rhythm. If the phase shifts (A–D) are plotted against the circadian time the result produces a phase response curve (PRC). (<b>B</b>): One version of the human phase response curve (PRC) derived from human subjects [<a href="#B4-biology-09-00180" class="html-bibr">4</a>]. In this figure, phase advances (positive values) and delays (negative values) have been plotted against the timing of light exposure relative to the measured phase of melatonin, which, in humans, is frequently used as a routine measure of circadian phase. The light “pulse” consisted of 6.7 h bright light exposure alternating between 6 min fixed gaze (approximately 10,000 lux) and free gaze (approximately 5000–9000 lux) exposures. Redrawn from Khalsa et.al. 2003. See text for details.</p> Full article ">Figure 2
<p>(<b>A</b>): Diagram of the Mouse Retina. The rods and cones have differing spectral maxima (λ<sub>max</sub>): rod photoreceptors (R) colored grey, λ<sub>max</sub> ~ 498 nm; green cones (M) colored green, λ<sub>max</sub> ~ 508 nm; ultraviolet sensitive cones (UVS) colored purple, λ<sub>max</sub> ~ 360 nm. These photoreceptors convey visual information to the retinal ganglion cells via the second order neurons of the inner retina (INL), and the bipolar (BC), horizontal (HC) and amacrine (AC) cells. The optic nerve is formed from the axons of all the ganglion cells and this large nerve takes light information into the brain. A subset of photosensitive retinal ganglion cells (pRGC—shown in blue) detects light directly by using the “blue” light sensitive photopigment called melanopsin or OPN4. Thus, photodetection in the retina occurs in three types of cell: the rods, cones and pRGCs. The eye itself has an independent clock, which changes the sensitivity or the rods and cones to light, and to complicate matters still further, the pRGCs also receive signals from the rods and cones, via inner retinal neurons, and can help drive light responses by the pRGCs. Counter-intuitively, light passes to the rods, cones and pRGCs by passing through the inner to the outer retina. (<b>B</b>): A least five, and possibly six, subtypes of melanopsin-expressing pRGCs have been identified to date. Images showing the pRGC subtypes (1–5) identified in the mouse retina are based upon their intensity of labeling with melanopsin antibodies (indicated as dark to light blue) and their anatomy; specifically, their dendritic projections to the “ON” and “OFF” layers of the sublaminae of the inner plexiform layers (IPL). Most recently, a potential M6 cell has been identified which has a small bistratisfied dendritic field with spiny, highly branched dendrites (similar to M5 cells). Like other non-M1 pRGCs (including M4 cells), M6 cells project to the dorsal lateral geniculate nucleus, suggesting they contribute to pattern vision [<a href="#B16-biology-09-00180" class="html-bibr">16</a>]. <span class="html-italic">Abbreviations</span>: inner nuclear layer (INL) which comprises multiple types of horizontal cells (H), bipolar cells (BC) and amacrine cells (AC); ganglion cell layer (GCL); optic nerve (ON); outer nuclear layer (ONL); outer plexiform layer (OPL); outer segments (OS); pigmented epithelium (PE); Off and On denote the ON and OFF sublaminae of the IPL.</p> Full article ">Figure 3
<p>Action spectra for circadian entrainment. Action spectra were derived using the magnitude of the phase shift in a freerunning locomotor activity rhythm by a 15 min light stimulus. Wheel running activity rhythms of singly housed male C3H/He <span class="html-italic">rd/rd cl</span> and wildtype mice aged between 80 and 250 days were monitored. Animals were entrained to a L:D 12:12 cycle for seven days then placed under constant darkness. After 7 to 10 days of constant darkness, a single monochromatic (half band width = 10nm), 15 min light pulse of defined irradiance was applied four hours after activity onset (Circadian Time 16) to generate maximum phase delays. Animals were returned to constant darkness for a further 10 days. The magnitude of the phase shift was calculated by comparing the time of activity onset before and after the light pulse. Pre-pulse phase was calculated from the seven days prior to the light pulse application, and the post-pulse stable freerunning activity was calculated from the seven days after the light pulse, taken from the second day after the light pulse. Monochromatic and neutral density filters were used to regulate the wavelength and intensity of the stimulus allowing irradiance response curves to be compiled at 420, 460, 471, 506, 540, 560 and 580 nm. Irradiance response curves (IRCs) were compiled at seven wavelengths of near-monochromatic light (n = 4 to 7 animals for each data point) between 420 and 580 nm in: (<b>A</b>) the <span class="html-italic">rd/rd cl</span> and (<b>B</b>) wildtype mice. (<b>C</b>) The derived action spectrum for circadian entrainment in <span class="html-italic">rd/rd cl</span> is well approximated an opsin-retinal photopigment with a novel λ<sub>max</sub> at 481 nm (R-squared = 0.976). (<b>D</b>) The wildtype action spectrum is also well approximated by an opsin-vitamin A photopigment (R-squared = 0.896), but with a λmax of ~500 nm. This is consistent with the involvement of a rod (498 nm) and/or cone (508 nm) absorption spectra.</p> Full article ">Figure 4
<p>A comparison of <span class="html-italic">rd/rd cl</span> and wildtype responses at 471 nm (15 min exposure) The data show a similar irradiance range of responses in the two genotypes, from ~1 × 10<sup>9</sup> photons cm<sup>2</sup> s<sup>−1</sup> to a saturating response at ~1 × 10<sup>14</sup> photons cm<sup>2</sup> s<sup>−1</sup>. This dynamic range corresponds to both the rod and cone activation ranges. A significant difference is identified in the slopes of the response relationship of the irradiance response curves (<span class="html-italic">p</span> &lt; 0.002), and photobiology formalisms suggest that this represents responses driven by different photoreceptors in the two genotypes.</p> Full article ">Figure 5
<p>Comparison of flavin-photopigment-based and opsin/vitamin A-based spectral responses. The well-defined action spectra for flavin-photopigment-based responses correspond closely to each other but not to the action spectrum for circadian phase shifts in <span class="html-italic">rd/rd cl</span> mice. See text for details.</p> Full article ">Figure 6
<p>Visualization of melanopsin expressing photosensitive retinal ganglion cells (pRGCs) of the adult mouse retina. (<b>A</b>) Confocal microscopy image of a flat mounted mouse retina showing antibody labeling of melanopsin in an M1 type pRGC with high levels of melanopsin expression and large but sparse dendritic fields. Additionally, note the presence of a weaker stained processes from neighboring non-M1 cells. (<b>B</b>) Cross section image of the mouse retina showing the dendrites of M1 type pRGCs extending to the OFF layers of the inner plexiform layers (IPL). <span class="html-italic">Abbreviations</span>: Outer nuclear layer (ONL); inner nuclear layer (INL); inner plexiform layer (IPL); ON and OFF mark the ON and OFF sublaminae of the IPL; ganglion cell layer (GCL).</p> Full article ">Figure 7
<p>Data showing that the progressive loss of 24 h rhythmicity in a subject with a grade IV glioblastoma that infiltrated the anterior hypothalamus. Case study: Patient identifier (JJB), male, 67 years of age diagnosed with grade IV glioblastoma with progressive infiltration into the anterior hypothalamus. The Figure shows rest/activity recordings measured in the home environment using actigraphy and plotted on a 48 h time base from 25th February 2010 to the 1st July 2010, and the death of JJB. Actigraphy profiles are shown across eleven periods of analysis (times indicated on left of the actigraphy profile). Periodogram analysis of the activity profiles indicated diurnal rest/activity profiles close to a period of 24 h until 26th March 2010 (five periods); beyond this time the subject showed increasingly non-24 h (arrhythmic) behavior, as defined by periodogram analysis. Post-mortem analysis of the brain of JJB showed significant tumor (glioma) infiltration of the suprachiasmatic nuclei (SCN) and compression of this area of the brain due to basal brain swelling. The single peak of activity between ~10.00 and 12.00 seen in periods 10 and 11 corresponds to the daily visits to the home by a nurse. Unpublished data collected by Emma Cussans, Katharina Wulff, Olaf Ansorge and Russell Foster. Sincerest thanks are expressed to the family of JJB for their help and participation in the collection of these data during a very difficult time.</p> Full article ">Figure 8
<p>An action spectrum for pupillary constriction in a woman lacking functional rods and cones. Irradiance-response curves (IRCs) were generated at eight wavelengths for both eyes to define the action spectrum. The resulting action spectrum of pupil responses provided a poor fit to rod and cone photopigments (rod R2 = 0.35; short wave-sensitive (SWS) cone, mid wave-sensitive (MWS) cone, long wave-sensitive (LWS) cone R2 = 0). An optimum fit to the pupil response to light was provided by an opsin/vitamin A-based template with λ<sub>max</sub> 476 nm (R2 = 0.89), corresponding closely to the pRGC system. The data shown were not corrected for pre-retinal lens absorption. When this correction was applied, the λ<sub>max</sub> shifted from 476 nm to 480 nm [<a href="#B93-biology-09-00180" class="html-bibr">93</a>].</p> Full article ">Figure 9
<p>The human retina functions over a very wide range of light intensities. Scotopic vision is light detection by the rod photoreceptors of the eye under low light conditions. In the human eye cone photoreceptors are nonfunctional in low light and rods mediate scotopic vision. Photopic vision is light detection by the cone photoreceptors of the eye under bright light conditions. In humans and many other animals, photopic vision allows color perception, mediated by cone cells, and a significantly higher visual acuity and temporal resolution than available with scotopic vision. Mesopic vision is a combination of photopic vision and scotopic vision in low but not quite dark lighting situations and involves an input from both rod and cone photoreceptors. As light levels increase, and as rods become saturated, melanopsin photoreception is activated. Whilst this diagram gives some sense of the sensory thresholds for the different photoreceptor classes, it is also misleading in that it fails to take into consideration the differences in effective stimulus durations for the rods, cones and melanopsin-pRGCs. Rods and cones detect light over the millisecond range whilst melanopsin-based pRGCs require long duration exposure to light to elicit a biological response. See text for details.</p> Full article ">
22 pages, 53194 KiB  
Article
Biocontrol Potential of Purified Elicitor Protein PeBL1 Extracted from Brevibacillus laterosporus Strain A60 and Its Capacity in the Induction of Defense Process against Cucumber Aphid (Myzus persicae) in Cucumber (Cucumis sativus)
by Khadija Javed, Humayun Javed and Dewen Qiu
Biology 2020, 9(7), 179; https://doi.org/10.3390/biology9070179 - 21 Jul 2020
Cited by 16 | Viewed by 3352
Abstract
The Cucumber aphid (Myzus persicae), a destructive cucumber aphid usually managed by chemical pesticides, is responsible for enormous annual agricultural losses. A protein elicitor, PeBL1, was investigated in the present work for its ability to induce a defense response against M. [...] Read more.
The Cucumber aphid (Myzus persicae), a destructive cucumber aphid usually managed by chemical pesticides, is responsible for enormous annual agricultural losses. A protein elicitor, PeBL1, was investigated in the present work for its ability to induce a defense response against M. persicae in cucumber. The rates of population growth (Intrinsic rate of increase) of M. persicae (second and third generations) decreased with PeBL1-treated cucumber seedlings as compared to positive (water) and negative 70.58 μg mL−1 controls (50 mM Tris-HCl, pH 8.0). In an assay on host selection, M. persicae had a preference for colonizing control plants as compared to the PeBL1-treated cucumber seedlings. The nymphal development time of the aphid was extended with the PeBL1-treated cucumber seedlings. Likewise, fecundity was reduced, with less offspring produced in the PeBL1-treated cucumber seedlings as compared to the positive (water) and negative 70.58 μg mL−1 controls (50 mM Tris-HCl, pH 8.0). The cucumber leaves treated with PeBL1 had a hazardous surface environment for M. persicae, caused by trichomes and wax formation. Jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) levels were significantly higher, exhibiting significant accumulation in the PeBL1-treated cucumber seedlings. The following results showed that PeBL1 considerably altered the height of the cucumber plant and the surface structure of the leaves to minimize M. persicae reproduction, and it prevented colonization. Defensive processes also included the activation of pathways (JA, SA, and ET). This study provides evidence of biocontrol for the use of PeBL1 in cucumber defense against M. persicae. Full article
(This article belongs to the Special Issue Plant–Bacterial Interaction: From Molecule to Ecosystem)
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<p>PeBL1 recombinant protein purification. (<b>A</b>) Evaluation of recombinant protein by using the Äkta explorer (<b>B</b>) His-Trap HP column used for the purification of total <span class="html-italic">E. coli</span> proteins. An elution buffer—25 mM Tris, 200 mM NaCl, 500 mM imidazole, and pH 8.0—was used to elute peak P2, which comprised recombinant PeBL1 with a flow rate of 5 mL/min. A HiTrap desalting column was used to load P2. The purified and desalted PeBL1 protein formed a single band of the molecular mass of 12 kDa on Tricine SDS-PAGE. Lane M: protein molecular mass marker; lane P2: peak P2.</p> Full article ">Figure 2
<p>The number of <span class="html-italic">M. persicae</span> colonized on control and PeBL1-treated cucumber seedlings (<b>A</b>) 24 h after the infestation colonization of <span class="html-italic">M. persicae</span> (mean ± SD). Data were compared statistically by an ANOVA and LSD in Statistix, version 8.1. Significant differences can be seen in lower style alphabet letters among all treatments (<span class="html-italic">p = 0.05</span>) (<b>B</b>) <span class="html-italic">M. persicae</span> colonized and preferred feeding on control cucumber seedlings as compared to the PeBL1-treated seedlings.</p> Full article ">Figure 3
<p>Prolonged nymphal development time of aphid instars (1st, 2nd, 3rd, and 4th). (<b>A</b>) Cucumber seedlings treated with control, buffer, and four PeBL1 protein concentrations prepared by BSA. (<b>B</b>) Nymphal developmental time of <span class="html-italic">M. persicae</span> from the prolonged 1st to 4th instar’s in PeBL1-treated cucumber seedlings. Data are shown as the mean (±SE) of different nymphal instars of (<span class="html-italic">M. persicae</span>) on cucumber plants by the PeBL1 elicitor protein at different concentrations and different temperature regimes (<span class="html-italic">n</span> = 10). Data were compared statistically by a factorial ANOVA and LSD at α = 0.05 in Statistix, version 8.1. Different alphabets above bar tops specify significant differences among treatments.</p> Full article ">Figure 4
<p><span class="html-italic">M. persicae</span> fecundity reduced in PeBL1-treated cucumber seedlings. (<b>A</b>) Seedlings treated with control, buffer, and PeBL1 protein concentrations prepared by BSA. (<b>B</b>) PeBL1 and control seedlings for aphid development (20, 24, and 27 °C). Average fecundity of <span class="html-italic">M. persicae</span> on cucumber plant in relation to various PeBL1 concentrations at different temperature regimes (<span class="html-italic">n</span> = 10). Data are shown as mean (±SE). Data were compared statistically by a factorial ANOVA and LSD at α = 0.05 in Statistix, version 8.1. Letters on each bar’s top show significant differences among treatments.</p> Full article ">Figure 5
<p><span class="html-italic">Myzus persicae</span> that fed on PeBL1 showed a reduction in the number of offspring as compared to the control plants.</p> Full article ">Figure 6
<p>Influence of PeBL1 on the growth of PeBL1-treated and control cucumber seedlings. (<b>A</b>) Plant height increased in PeBL1-treated cucumber seedlings as compared to control (<b>B</b>) Physical characteristics of PeBL1-treated seedlings enhanced as compared to control (<b>C</b>) Data were shown in mean (±SD) of cucumber plants in PeBL1-treated and control seedlings (n = 10). Data were compared statistically by one-way analysis of variance (ANOVA) and Least significant difference (LSD) in Statistix 8.1 version. Different lower style alphabets letters indicate significant differences among treatments (<span class="html-italic">p</span> = 0.05).</p> Full article ">Figure 7
<p>Jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) contents in cucumber seedlings (mean ± SD). Treatment with PeBL1 was collected one day after spraying. In both treatments, the aphids were inoculated one day after seedlings were sprayed, and the samples were collected one day after inoculation. Data were compared statistically by the LSD, ANOVA, and Leven’s test using Statistix, version 8.1. Different lower-case letters show significant differences among various treatments performed in JA, SA, or ET (<span class="html-italic">p</span> = 0.05).</p> Full article ">Figure 8
<p>Relative expression of plant defense from the JA, SA, and ET pathway genes found after treatment with the PeBL1 elicitor, PeBL1 and aphid, and aphid infestation alone. For each gene, an asterisk on the bar shows a significant difference from the buffer control, as found by Student’s <span class="html-italic">t</span>-test (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
18 pages, 801 KiB  
Review
The Relationship between Sperm Oxidative Stress Alterations and IVF/ICSI Outcomes: A Systematic Review from Nonhuman Mammals
by Jordi Ribas-Maynou, Marc Yeste and Albert Salas-Huetos
Biology 2020, 9(7), 178; https://doi.org/10.3390/biology9070178 - 21 Jul 2020
Cited by 24 | Viewed by 5263
Abstract
Achieving high embryo quality following IVF and ICSI procedures is a key factor in increasing fertility outcomes in human infertile couples. While the male factor is known to underlie infertility in about 50% of cases, studies performed in human infertile couples have not [...] Read more.
Achieving high embryo quality following IVF and ICSI procedures is a key factor in increasing fertility outcomes in human infertile couples. While the male factor is known to underlie infertility in about 50% of cases, studies performed in human infertile couples have not been able to define the precise effect of sperm affectations upon embryo development. This lack of consistency is, in most cases, due to the heterogeneity of the results caused by the multiple male and female factors that mask the concrete effect of a given sperm parameter. These biases can be reduced with the use of animal gametes, being a good approach for basic researchers to design more homogeneous studies analyzing the specific consequences of a certain affectation. Herein, we conducted a systematic review (March 2020) that assessed the relationship between sperm oxidative stress alterations and IVF/ICSI outcomes in nonhumans mammals. The review was conducted according to PRISMA guidelines and using the MEDLINE-PubMed and EMBASE databases. Thirty articles were included: 11 performed IVF, 17 conducted ICSI, and two carried out both fertilization methods. Most articles were conducted in mouse (43%), cattle (30%) and pig models (10%). After IVF treatments, 80% of studies observed a negative effect of sperm oxidative stress on fertilization rates, and 100% of studies observed a negative effect on blastocyst rates. After ICSI treatments, a positive relationship of sperm oxidative stress with fertilization rates (75% of studies) and with blastocyst rates (83% of studies) was found. In conclusion, the present systematic review shows that sperm oxidative stress is associated with a significant reduction in fertilization rates and in vitro embryo development. Full article
(This article belongs to the Special Issue Oxidative Stress in Gametes and Embryos)
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<p>Flowchart of the literature search and selection process.</p> Full article ">
46 pages, 4512 KiB  
Review
Phytoremediation of Cadmium: Physiological, Biochemical, and Molecular Mechanisms
by Ali Raza, Madiha Habib, Shiva Najafi Kakavand, Zainab Zahid, Noreen Zahra, Rahat Sharif and Mirza Hasanuzzaman
Biology 2020, 9(7), 177; https://doi.org/10.3390/biology9070177 - 21 Jul 2020
Cited by 174 | Viewed by 15447
Abstract
Cadmium (Cd) is one of the most toxic metals in the environment, and has noxious effects on plant growth and production. Cd-accumulating plants showed reduced growth and productivity. Therefore, remediation of this non-essential and toxic pollutant is a prerequisite. Plant-based phytoremediation methodology is [...] Read more.
Cadmium (Cd) is one of the most toxic metals in the environment, and has noxious effects on plant growth and production. Cd-accumulating plants showed reduced growth and productivity. Therefore, remediation of this non-essential and toxic pollutant is a prerequisite. Plant-based phytoremediation methodology is considered as one a secure, environmentally friendly, and cost-effective approach for toxic metal remediation. Phytoremediating plants transport and accumulate Cd inside their roots, shoots, leaves, and vacuoles. Phytoremediation of Cd-contaminated sites through hyperaccumulator plants proves a ground-breaking and profitable choice to combat the contaminants. Moreover, the efficiency of Cd phytoremediation and Cd bioavailability can be improved by using plant growth-promoting bacteria (PGPB). Emerging modern molecular technologies have augmented our insight into the metabolic processes involved in Cd tolerance in regular cultivated crops and hyperaccumulator plants. Plants’ development via genetic engineering tools, like enhanced metal uptake, metal transport, Cd accumulation, and the overall Cd tolerance, unlocks new directions for phytoremediation. In this review, we outline the physiological, biochemical, and molecular mechanisms involved in Cd phytoremediation. Further, a focus on the potential of omics and genetic engineering strategies has been documented for the efficient remediation of a Cd-contaminated environment. Full article
(This article belongs to the Section Plant Science)
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<p>Depiction of major transporters present on the root, shoot, and leaves for Cd sequestration and storage (these processes are related to phytoremediation). Read text and <a href="#biology-09-00177-t002" class="html-table">Table 2</a> for more information.</p> Full article ">Figure 2
<p>Representation of phloem and xylem transporters. Low-affinity calcium transporter 1 (LCT1) and metal-transporting ATPases (HMA2) that function in the translocation of Cd into the phloem. The major transporters of Cd loading into the xylem are HMA2 and HMA4. However, the role of transporters between the xylem and phloem is unclear.</p> Full article ">Figure 3
<p>A hypothetical model of phytoremediation by adding chelating agents to remediate Cd-contaminated soil.</p> Full article ">Figure 4
<p>Schematic overview of Cd tolerance and its accumulation by PCs in leaves cells of Cd-hyperaccumulating plants. (<b>a</b>) Elevated Cd concentration inside the cytosol. (<b>b</b>) The Cd can be directly bound to the PCS and stimulate the biosynthesis of PCs from GSH by activating this enzyme. (<b>c</b>) Once the Cd-PCs complexes are formed, they can finally be sequestered within vacuoles by transferring ABC transporters through the tonoplast. (<b>d</b>) Due to the low pH of vacuoles, Cd-PCs complexes disassociate, and Cd can be stabilized in vacuoles by binding to ligands, including organic acids and probably amino acids. (<b>e</b>) PCs may be by destroyed hydrolase enzymes inside the vacuole or returned to the cytosol, where they able to keep their role as Cd shuttles.</p> Full article ">Figure 5
<p>Schematic overview of Cd detoxification and its accumulation by MTs in leaves cells of Cd hyperaccumulator plants. (<b>a</b>) By increasing the Cd concentration in the cytosol, (<b>b</b>) Cd can stimulate the expression of the MTs gene. (<b>c</b>) By binding MTs to Cd, they can play a substantial role in detoxifying and homeostasis Cd within the cytosol. (<b>d</b>) Additionally, MTs stimulate GSH biosynthesis as the main constituent of PCs. (<b>e</b>) MTs play an essential role in ROS scavenging and its tolerance.</p> Full article ">Figure 6
<p>Schematic illustration of Cd phytoremediation by microbes. In the phytoextraction process, the production of siderophores and organic acids by the soil microbes influences the phytoextraction capacity of the accumulator in a positive manner. The generation of polymeric substances by soil microbes keeps the Cd metal in a static form, which facilitates the biosorption and bioaccumulation process.</p> Full article ">
14 pages, 2291 KiB  
Article
Testosterone Decreases Placental Mitochondrial Content and Cellular Bioenergetics
by Jay S. Mishra, Chellakkan S. Blesson and Sathish Kumar
Biology 2020, 9(7), 176; https://doi.org/10.3390/biology9070176 - 20 Jul 2020
Cited by 11 | Viewed by 3330
Abstract
Placental mitochondrial dysfunction plays a central role in the pathogenesis of preeclampsia. Since preeclampsia is a hyperandrogenic state, we hypothesized that elevated maternal testosterone levels induce damage to placental mitochondria and decrease bioenergetic profiles. To test this hypothesis, pregnant Sprague–Dawley rats were injected [...] Read more.
Placental mitochondrial dysfunction plays a central role in the pathogenesis of preeclampsia. Since preeclampsia is a hyperandrogenic state, we hypothesized that elevated maternal testosterone levels induce damage to placental mitochondria and decrease bioenergetic profiles. To test this hypothesis, pregnant Sprague–Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg/day) from gestation day (GD) 15 to 19. On GD20, the placentas were isolated to assess mitochondrial structure, copy number, ATP/ADP ratio, and biogenesis (Pgc-1α and Nrf1). In addition, in vitro cultures of human trophoblasts (HTR-8/SVneo) were treated with dihydrotestosterone (0.3, 1.0, and 3.0 nM), and bioenergetic profiles using seahorse analyzer were assessed. Testosterone exposure in pregnant rats led to a 2-fold increase in plasma testosterone levels with an associated decrease in placental and fetal weights compared with controls. Elevated maternal testosterone levels induced structural damage to the placental mitochondria and decreased mitochondrial copy number. The ATP/ADP ratio was reduced with a parallel decrease in the mRNA and protein expression of Pgc-1α and Nrf1 in the placenta of testosterone-treated rats compared with controls. In cultured trophoblasts, dihydrotestosterone decreased the mitochondrial copy number and reduced PGC-1α, NRF1 mRNA, and protein levels without altering the expression of mitochondrial fission/fusion genes. Dihydrotestosterone exposure induced significant mitochondrial energy deficits with a dose-dependent decrease in basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. In summary, our study suggests that the placental mitochondrial dysfunction induced by elevated maternal testosterone might be a potential mechanism linking preeclampsia to feto-placental growth restriction. Full article
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<p>Plasma T levels and placental and fetal weights in control and T-treated pregnant rats. Pregnant rats were treated with vehicle (sesame oil) or T propionate from gestation day 15 to 19 and euthanized on day 20. (<b>A</b>) Plasma T levels were quantified using ELISA. (<b>B</b>) Placental and (<b>C</b>) Fetal weights were measured. Data presented as mean ± SEM of 6 rats in each group. * <span class="html-italic">p &lt;</span> 0.05 vs. control.</p> Full article ">Figure 2
<p>Characterization of mitochondrial structure in the placenta. (<b>A</b>) Representative electron micrographs of placental mitochondria from control (left) and T-treated pregnant rats (right). Images show less abundant mitochondria and abnormal mitochondrial structure with condensed matrix and cristae in the placenta of T-treated rats. (<b>B</b>) Quantification of the percentage of morphologically abnormal mitochondrial showing condensed matrix and cristae in the placenta of control and T-treated rats. <span class="html-italic">n</span> = 4 in each group. * <span class="html-italic">p &lt;</span> 0.05 vs. control.</p> Full article ">Figure 3
<p>Mitochondrial copy number and ATP levels in the placenta of control and T-treated pregnant rats. (<b>A</b>) Mitochondrial DNA copy number was quantified using qRT-PCR based analysis. Placental (<b>B</b>) ATP and (<b>C</b>) ADP content was quantified using ApoSENSOR ADP/ATP kit. (<b>D</b>) Measurement of the ATP/ADP ratio. Data presented as mean ± SEM of 6 rats in each group. * <span class="html-italic">p</span> &lt; 0.05 vs. control.</p> Full article ">Figure 4
<p>Expression of mitochondrial biogenesis indicators in the placenta of control and T-treated pregnant rats. Real-time PCR was used to assess (<b>A</b>) Pgc-1a and (<b>B</b>) Nrf1 mRNA expression in the placenta. Quantitation of placental Pgc-1a and Nrf1 mRNA expression was normalized relative to β-actin. (<b>C</b>) Representative Western blots for Pgc-1α, Nrf1, and β-actin are shown at top; blot density obtained from densitometric scanning of Pgc-1α and Nrf1 normalized to β-actin is shown at the bottom. Data presented as means ± SEM of 6 rats in each group. * <span class="html-italic">p</span> &lt; 0.05 vs. control.</p> Full article ">Figure 5
<p>Mitochondrial copy number and expression of fission/fusion and biogenesis indicators. Trophoblasts cells were treated with vehicle (ethanol) or dihydrotestosterone (DHT) for 24 h. (<b>A</b>) Mitochondrial DNA copy number was quantified using qRT-PCR based analysis. (<b>B</b>) Cell viability after exposure to DHT was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. The LDH levels were measured and expressed as the fold change compared to vehicle control. Real-time PCR was used to assess the relative mRNA expression levels of (<b>C</b>) fission/fusion genes (FIS-1, DRP-1, MFN-1, MFN-2, and OPA-1), and biogenesis indicators (<b>D</b>) PGC-1α and (<b>E</b>) NRF1, normalized to β-actin. (<b>F</b>) Representative Western blots for PGC-1α, NRF-1, and β-actin are shown at the left; blot density obtained from densitometric scanning of PGC-1α and NRF-1 normalized to β-actin is shown at the right data presented as means ± SEM of 4 biologically independent replicates. * <span class="html-italic">p</span> &lt; 0.05 vs. control.</p> Full article ">Figure 6
<p>Bioenergetics profile of trophoblasts. Trophoblasts cells were treated with vehicle (ethanol) or dihydrotestosterone (DHT) for 24 h; mitochondrial respiratory parameters were measured using Seahorse. (<b>A</b>) Representative traces of mitochondrial respiration, (<b>B</b>) basal oxygen consumption rates (OCR), (<b>C</b>) proton leak, (<b>D</b>) ATP production-linked respiration (OCR after oligomycin administration), (<b>E</b>) maximal respiration (OCR after carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) administration) and (<b>F</b>) spare respiratory capacity (Difference between basal and maximal OCR). Data are presented as means ± SEM. The studies were done in duplicate from samples obtained from 4 biologically independent replicates. * <span class="html-italic">p</span> &lt; 0.05 vs. vehicle control.</p> Full article ">
18 pages, 1750 KiB  
Article
Impairments of Synaptic Plasticity Induction Threshold and Network Oscillatory Activity in the Hippocampus Underlie Memory Deficits in a Non-Transgenic Mouse Model of Amyloidosis
by Jennifer Mayordomo-Cava, Guillermo Iborra-Lázaro, Souhail Djebari, Sara Temprano-Carazo, Irene Sánchez-Rodríguez, Danko Jeremic, Agnès Gruart, José María Delgado-García, Lydia Jiménez-Díaz and Juan D. Navarro-López
Biology 2020, 9(7), 175; https://doi.org/10.3390/biology9070175 - 20 Jul 2020
Cited by 15 | Viewed by 4938
Abstract
In early Alzheimer disease (AD) models synaptic failures and upstreaming aberrant patterns of network synchronous activity result in hippocampal-dependent memory deficits. In such initial stage, soluble forms of Amyloid-β (Aβ) peptides have been shown to play a causal role. Among [...] Read more.
In early Alzheimer disease (AD) models synaptic failures and upstreaming aberrant patterns of network synchronous activity result in hippocampal-dependent memory deficits. In such initial stage, soluble forms of Amyloid-β (Aβ) peptides have been shown to play a causal role. Among different Aβ species, Aβ25–35 has been identified as the biologically active fragment, as induces major neuropathological signs related to early AD stages. Consequently, it has been extensively used to acutely explore the pathophysiological events related with neuronal dysfunction induced by soluble Aβ forms. However, the synaptic mechanisms underlying its toxic effects on hippocampal-dependent memory remain unresolved. Here, in an in vivo model of amyloidosis generated by intracerebroventricular injections of Aβ25–35 we studied the synaptic dysfunction mechanisms underlying hippocampal cognitive deficits. At the synaptic level, long-term potentiation (LTP) of synaptic excitation and inhibition was induced in CA1 region by high frequency simulation (HFS) applied to Schaffer collaterals. Aβ25–35 was found to alter metaplastic mechanisms of plasticity, facilitating long-term depression (LTD) of both types of LTP. In addition, aberrant synchronization of hippocampal network activity was found while at the behavioral level, deficits in hippocampal-dependent habituation and recognition memories emerged. Together, our results provide a substrate for synaptic disruption mechanism underlying hippocampal cognitive deficits present in Aβ25–35 amyloidosis model. Full article
(This article belongs to the Special Issue Brain Damage and Repair: From Molecular Effects to CNS Disorders)
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<p>Surgery and temporal sequence of <span class="html-italic">icv.</span> injections, electrophysiological recordings. (<b>A</b>) Implantation of a guide cannula in the left ventricle for <span class="html-italic">icv.</span> vehicle or A<span class="html-italic">β</span><sub>25–35</sub> administration. The photomicrograph serves as histological verification of cannula position (black arrow). (<b>B</b>) Bipolar stimulating electrodes were implanted at the right <span class="html-italic">Schaffer</span> collateral-commissural pathway of the dorsal hippocampus, and a recording electrode was placed at the ipsilateral <span class="html-italic">stratum radiatum</span> underneath the CA1 area. Photomicrographs illustrate the location (black arrows) of the stimulating (right photomicrograph) and recording (left photomicrograph) electrodes. (<b>C</b>) Representation of the fPSPs evoked in the CA1 hippocampal region after paired-pulse stimulation (interval of 40 ms) of the Schaffer collaterals. The recording was obtained from a representative animal and illustrates the averaged (<span class="html-italic">n =</span> 50) profile of the postsynaptic response. Two different components were identified for amplitude analysis: (1) A glutamatergic fEPSP (Glut), with a latency of appearance of 2.25–4 ms after <span class="html-italic">Schaffer</span> collateral stimulation; and (2) a GABAergic fIPSP dependent on GABA<sub>A</sub> receptors (GABA<sub>A</sub>), with a latency of 12–15 ms. For each component or postsynaptic potential, the maximum amplitude (peak-to-peak value) was measured for the analysis. (<b>D</b>) Vehicle or A<span class="html-italic">β</span><sub>25–35</sub> administration was performed on days 1–3 (D1–D3). Local field potential (LFP) recordings were collected before (D0) and after A<span class="html-italic">β</span><sub>25–35</sub> administration (D1-D6). From days 6 to 9 (D6–D9), the effect of A<span class="html-italic">β</span><sub>25–35</sub> on long-term potentiation (LTP) was checked. LTP was induced by high-frequency stimulation (HFS) of the <span class="html-italic">Schaffer</span> collateral pathway. The responses to paired-pulse stimulation were collected before LTP induction (Baseline; BL) and after the HFS session (LTP1-4). Scale bars, 500 μm. RV, right ventricle; LV, lateral ventricle; DG, dentate gyrus; St., stimulus; D, dorsal; M, medial; L, lateral; V, ventral; Glut, glutamate.</p> Full article ">Figure 2
<p>Long-term synaptic plasticity in behaving mice. The amplitude of the field postsynaptic potentials (fPSPs) evoked in vehicle and A<span class="html-italic">β</span>-injected mice by the first (St. 1) and second (St. 2) stimulus of paired-pulse stimulation was examined before (Baseline) and after (Day 1–Day 4) LTP induction through a HFS session. (<b>A</b>) Representative examples (averaged five times) of fEPSPs evoked before the HFS session (baseline; 1), 10 min after HFS (2) and 48 h after HFS (3) are shown above. Below, LTP of fEPSPs (mean ± SEM). * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001 vs. vehicle. (<b>B</b>) Representative examples (averaged five times) of GABA<sub>A</sub>-dependent fIPSPs evoked in vehicle and A<span class="html-italic">β</span>-injected mice are shown above. Below, LTP of fIPSPs (mean ± SEM). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. vehicle.</p> Full article ">Figure 3
<p>Oscillatory activity in CA1 region of behaving mice. Spectral power of 180 s LFP recordings at the CA1 area obtained on 6 consecutive days in the absence of any electrical stimulation for two experimental groups: vehicle and A<span class="html-italic">β</span><sub>25–35</sub>. (<b>A</b>) Representative example of LFP recorded from CA1 hippocampal region in behaving mice. Note the different time scales for the traces illustrated on the left and right. (<b>B</b>) Low-frequency and (<b>C</b>) high-frequency bands were analyzed. Recordings on day 0 were collected previous to <span class="html-italic">icv.</span> administration (purple). Injections were performed on days 1, 2, and 3, recording 1 h after each one. LFP evolution was also followed 24, 48, and 72 h post <span class="html-italic">icv.</span> (days 4–6). Additionally, each day (D0-D6) spectral power of frequency intervals corresponding to hippocampal <span class="html-italic">theta</span> ((<b>D</b>); 4–12 Hz) and <span class="html-italic">gamma</span> ((<b>E</b>); 60–80 Hz) bands is represented (on top) along with the average value for that interval (below; mean ± SEM). After <span class="html-italic">icv.</span> administration, A<span class="html-italic">β</span>-injected mice showed increased spectral power at both low and high frequencies compared to vehicle-injected animals, and the same result was found for theta and gamma rhythms. *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Hippocampal-dependent memory tasks. (<b>A</b>) On top, open field habituation test. Mice underwent a training session by being exposed to an open field. 24 h later, and 1 h after <span class="html-italic">icv.</span> administration, animals were re-exposed to the open field (habituation session). On each session, mice freely explored the environment for 15 min. Movements in the X, Y, and Z axis were detected with an infrared system. Below, total activity (movements in the XYZ axis), horizontal activity (XY axis), and vertical activity (Z axis) during training and habituation sessions (mean ± SEM) are represented. *** <span class="html-italic">p</span> &lt; 0.001. Asterisks indicate differences between training and habituation sessions within each experimental group. ‡‡ <span class="html-italic">p</span> &lt; 0.01, ‡‡‡ <span class="html-italic">p</span> &lt; 0.001. Crosses reflect differences between A<span class="html-italic">β</span> and vehicle (control animals) in habituation. Note that exploratory activity decreases in the vehicle group during the habituation session compared to the training session, but also compared to the A<span class="html-italic">β</span> group during the habituation session. (<b>B</b>) On top, novel object recognition task. Three 5-min habituation trials were performed on Day 1. On Day 2, a 10 min training session with two identical objects occurred. A test session (NOR1) was performed 3 h later for evaluation of short-term memory, substituting one familiar object for a novel one. On days 3 and 4, NOR2 and NOR3 trials were performed to evaluate long-term memory. <span class="html-italic">Icv.</span> injections took place one hour before NOR2 trial. Below, discrimination index (defined as the difference in exploration time between the two objects divided by the total time spent exploring both objects) for each experimental group during trials. After <span class="html-italic">icv.</span> injections, only vehicle-injected mice showed positive discrimination toward the novel object. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. Tr., training; Hab., habituation. (<b>C</b>) On top, Rotarod performance test. 1 h after <span class="html-italic">icv.</span> injections, animals performed a single session consisting of six trials. Mean latency to fall off the rod was calculated for each trial (bottom left graph) and the whole session (bottom bar plot). (<b>D</b>) Locomotion test. 1 h after <span class="html-italic">icv.</span> administration, mice freely explored an empty chamber for 15 min. Mean travelled distance was recorded by a LABORAS<sup>®</sup> system. Right, representative movement tracking of animals from both experimental groups. No differences were found between treatments in C or D.</p> Full article ">
16 pages, 627 KiB  
Review
Sperm Quality Assessment in Honey Bee Drones
by Jesús L. Yániz, Miguel A. Silvestre and Pilar Santolaria
Biology 2020, 9(7), 174; https://doi.org/10.3390/biology9070174 - 18 Jul 2020
Cited by 32 | Viewed by 6603
Abstract
The quality of honey bee drone semen is relevant in different contexts, ranging from colony productivity to pathology, toxicology and biodiversity preservation. Despite its importance, considerably less knowledge is available on this subject for the honey bee when compared to other domestic animal [...] Read more.
The quality of honey bee drone semen is relevant in different contexts, ranging from colony productivity to pathology, toxicology and biodiversity preservation. Despite its importance, considerably less knowledge is available on this subject for the honey bee when compared to other domestic animal species. A proper assessment of sperm quality requires a multiple testing approach which discriminates between the different aspects of sperm integrity and functionality. Most studies on drone semen quality have only assessed a few parameters, such as sperm volume, sperm concentration and/or sperm plasma membrane integrity. Although more recent studies have focused on a broader variety of aspects of semen quality, some techniques currently used in vertebrates, such as computer-assisted sperm analysis (CASA) or multiparametric sperm quality testing, still remain to be developed in the honey bee. This may be attributed to the particular sperm morphology and physiology in this species, requiring the development of technologies specifically adapted to it. This article reviews the present knowledge of sperm quality in honey bee drones, highlighting its peculiarities and proposing future lines of research. Full article
(This article belongs to the Special Issue Factors Affecting In Vitro Assessment of Sperm Quality)
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<p>A combined phase-contrast and fluorescence (Hoechst) image of a honey bee spermatozoon showing the acrosome (a), nucleus (b) and flagellum (c). Scale-bar = 10 µm.</p> Full article ">
12 pages, 8648 KiB  
Article
Effect of Edge-to-Edge Mitral Valve Repair on Chordal Strain: Fluid-Structure Interaction Simulations
by Milan Toma, Daniel R. Einstein, Keshav Kohli, Sheridan L. Caroll, Charles H. Bloodworth IV, Richard P. Cochran, Karyn S. Kunzelman and Ajit P. Yoganathan
Biology 2020, 9(7), 173; https://doi.org/10.3390/biology9070173 - 18 Jul 2020
Cited by 14 | Viewed by 4757
Abstract
Edge-to-edge repair for mitral valve regurgitation is being increasingly performed in high-surgical risk patients using minimally invasive mitral clipping devices. Known procedural complications include chordal rupture and mitral leaflet perforation. Hence, it is important to quantitatively evaluate the effect of edge-to-edge repair on [...] Read more.
Edge-to-edge repair for mitral valve regurgitation is being increasingly performed in high-surgical risk patients using minimally invasive mitral clipping devices. Known procedural complications include chordal rupture and mitral leaflet perforation. Hence, it is important to quantitatively evaluate the effect of edge-to-edge repair on chordal integrity. in this study, we employ a computational mitral valve model to simulate functional mitral regurgitation (FMR) by creating papillary muscle displacement. Edge-to-edge repair is then modeled by simulated coaptation of the mid portion of the mitral leaflets. in the setting of simulated FMR, edge-to-edge repair was shown to sustain low regurgitant orifice area, until a two fold increase in the inter-papillary muscle distance as compared to the normal mitral valve. Strain in the chordae was evaluated near the papillary muscles and the leaflets. Following edge-to-edge repair, strain near the papillary muscles did not significantly change relative to the unrepaired valve, while strain near the leaflets increased significantly relative to the unrepaired valve. These data demonstrate the potential for computational simulations to aid in the pre-procedural evaluation of possible complications such as chordal rupture and leaflet perforation following percutaneous edge-to-edge repair. Full article
(This article belongs to the Special Issue Computational Biology)
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<p>The geometry of unfolded MV; the papillary muscles attach via chordae tendineae to the anterior, posterior and commissural leaflets [<a href="#B2-biology-09-00173" class="html-bibr">2</a>].</p> Full article ">Figure 2
<p>The detailed MV model extracted from DICOM <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>CT images with all chords preserved.</p> Full article ">Figure 3
<p>The fluid particles are confined in a pipe-like rigid structure surrounding the MV model and prescribed velocity boundary conditions are applied to the open ends via the use of moving pistons in z-direction.</p> Full article ">Figure 4
<p>The effect of edge-to-edge (E-to-E) repair on the regurgitant orifice area (ROA) that resulted from dilated papillary muscles. (<b>a</b>) Without E-to-E repair. (<b>b</b>) With E-to-E repair.</p> Full article ">Figure 5
<p>Regurgitant orifice area versus papillary muscles (PM) displacement (where ‘d’ is the distance between papillary muscles of the healthy valve model, i.e., without PM dilation).</p> Full article ">Figure 6
<p>Average strain values in the chordae near the papillary muscles (PM) versus PM displacement (where ‘d’ is the distance between papillary muscles of the healthy valve model, i.e., without PM dilation).</p> Full article ">Figure 7
<p>Average strain values in the chordae near the mitral valve leaflets versus papillary muscles (PM) displacement (where ‘d’ is the distance between papillary muscles of the healthy valve model, i.e., without PM dilation).</p> Full article ">
19 pages, 2956 KiB  
Article
Impact of the Static Magnetic Field on Growth, Pigments, Osmolytes, Nitric Oxide, Hydrogen Sulfide, Phenylalanine Ammonia-Lyase Activity, Antioxidant Defense System, and Yield in Lettuce
by Arafat Abdel Hamed Abdel Latef, Mona F. A. Dawood, Halimeh Hassanpour, Maryam Rezayian and Nabil A. Younes
Biology 2020, 9(7), 172; https://doi.org/10.3390/biology9070172 - 17 Jul 2020
Cited by 41 | Viewed by 5144
Abstract
Magnetic fields are an unavoidable physical factor affecting living organisms. Lettuce seeds (Lactuca sativa var. cabitat L.) were subjected to various intensities of the static magnetic field (SMF) viz., MF0 (control), SMF1 (0.44 Tesla (T), SMF2 (0.77 T), and SMF3 (1 T) [...] Read more.
Magnetic fields are an unavoidable physical factor affecting living organisms. Lettuce seeds (Lactuca sativa var. cabitat L.) were subjected to various intensities of the static magnetic field (SMF) viz., MF0 (control), SMF1 (0.44 Tesla (T), SMF2 (0.77 T), and SMF3 (1 T) for three exposure times (1, 2, and 3 h). SMF-treated seedlings showed induction in growth parameters and metabolism comparing to control. All photosynthetic pigments were induced markedly under SMF, especially chlorophyll a. SMF at different intensities boosted osmolytes, non-enzymatic antioxidants, and the phenylalanine ammonia-lyase activity over non-magnetized seedlings. Oxidative damage criteria viz., hydrogen peroxide, superoxide radical, and lipid peroxidation, as well as polyphenol oxidase activity, were kept at low values under SMF-treated seeds relative to control, especially SMF2. Electron donors to antioxidant enzymes including nitrate reductase, nitric oxide, and hydrogen sulfide induced via SMF exposure and consequently the activities of superoxide dismutase, glutathione-S-transferases, catalase, and peroxidases family enzymes were also stimulated under SMF, whatever the intensity or the exposure period applied. All these regulations reflected on the enhancement of lettuce yield production which reached 50% over the control at SMF3. Our findings offered that SMF-seed priming is an innovative and low-cost strategy that can improve the growth, bioactive constituents, and yield of lettuce. Full article
(This article belongs to the Section Plant Science)
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<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on chlorophyll a and b and carotenoids contents of lettuce. Different letters of the same format indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). FW—fresh weight; T—Tesla; h—hour.</p> Full article ">Figure 2
<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on the (<b>A</b>) H<sub>2</sub>O<sub>2</sub> and O<sub>2</sub><sup>−</sup> and (<b>B</b>) malondialdehyde (MDA) contents of lettuce. Different letters indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). FW—fresh weigh; T—Tesla; h—hour.</p> Full article ">Figure 2 Cont.
<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on the (<b>A</b>) H<sub>2</sub>O<sub>2</sub> and O<sub>2</sub><sup>−</sup> and (<b>B</b>) malondialdehyde (MDA) contents of lettuce. Different letters indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). FW—fresh weigh; T—Tesla; h—hour.</p> Full article ">Figure 3
<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on (<b>A</b>) nitrate reductase (NR) activity, (<b>B</b>) nitric oxide (NO), and (<b>C</b>) hydrogen sulfide (H<sub>2</sub>S) contents of lettuce. Different letters indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). FW—fresh weigh; T—Tesla; h—hour.</p> Full article ">Figure 3 Cont.
<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on (<b>A</b>) nitrate reductase (NR) activity, (<b>B</b>) nitric oxide (NO), and (<b>C</b>) hydrogen sulfide (H<sub>2</sub>S) contents of lettuce. Different letters indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). FW—fresh weigh; T—Tesla; h—hour.</p> Full article ">Figure 4
<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on the activities of (<b>A</b>) superoxide dismutase (SOD), polyphenol oxidase (PPO), and glutathione-S-transferase (GST); (<b>B</b>) ascorbate peroxidase (APX); and (<b>C</b>) catalase (CAT), peroxidase (POD), and glutathione peroxidase (GPX) of lettuce. Different letters of the same format indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). T—Tesla; h—hour.</p> Full article ">Figure 4 Cont.
<p>Effect of different intensities of the static magnetic field (SMF) for three exposure periods on the activities of (<b>A</b>) superoxide dismutase (SOD), polyphenol oxidase (PPO), and glutathione-S-transferase (GST); (<b>B</b>) ascorbate peroxidase (APX); and (<b>C</b>) catalase (CAT), peroxidase (POD), and glutathione peroxidase (GPX) of lettuce. Different letters of the same format indicate statistically significant values following Duncan’s multiple range test at <span class="html-italic">p</span> &lt; 0.05. Bars represent means of three (<span class="html-italic">n</span> = 3) replicates with standard errors (SEs). SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), and SMF3 (1 T). T—Tesla; h—hour.</p> Full article ">Figure 5
<p>Effect of the static magnetic field (SMF) on the principal component analysis (PCA) of different variable relationships in lettuce. The PCA test identified 62.62% of the total variation, and axis 1 and axis 2 accounted for 46.35% and 16.27%, respectively. Growth parameters displayed positive correlations with H<sub>2</sub>S, NO, nitrate reductase, osmotic adjustments, and enzymatic and non-enzymatic antioxidants. DW (dry weight), Chl (chlorophyll), Car (carotenoids), H<sub>2</sub>O<sub>2</sub> (hydrogen peroxide), MDA (malondialdehyde), NR (nitrate reductase), NO (Nitric oxide), hydrogen sulfide (H<sub>2</sub>S), SOD (superoxide dismutase), PPO (polyphenol oxidase), GST (glutathione-S-transferase), APX (ascorbate peroxidase), CAT (catalase), POD (peroxidase), GPX (glutathione peroxidase), ASA (ascorbic acid), GSH (reduced glutathione), PAL (phenylalanine ammonia-lyase), SMF0 (control), SMF1 (0.44 T), SMF2 (0.77 T), SMF3 (1 T), T (Tesla), and h (hour).</p> Full article ">
11 pages, 1323 KiB  
Article
Development of a Low-Cost and High-Efficiency Culture Medium for Bacteriocin Lac-B23 Production by Lactobacillus plantarum J23
by Jianming Zhang, Yushan Bu, Chengcheng Zhang, Huaxi Yi, Daqun Liu and Jingkai Jiao
Biology 2020, 9(7), 171; https://doi.org/10.3390/biology9070171 - 17 Jul 2020
Cited by 14 | Viewed by 4136
Abstract
At present, De Man, Rogosa and Sharpe (MRS) broth is the medium of choice for promoting bacteriocin production. However, this medium is expensive and not applicable for large-scale production. Therefore, a low-cost and high-efficiency culture medium for bacteriocin Lac-B23 production by Lactobacillus plantarum [...] Read more.
At present, De Man, Rogosa and Sharpe (MRS) broth is the medium of choice for promoting bacteriocin production. However, this medium is expensive and not applicable for large-scale production. Therefore, a low-cost and high-efficiency culture medium for bacteriocin Lac-B23 production by Lactobacillus plantarum J23 was developed. First, the effects of the composition of MRS broth on bacteriocin Lac-B23 production and bacterial growth were researched by a one variable at a time approach. Then, a Plackett-Burman design was used to screen significant components for production. Finally, the steepest ascent and central composite designs were used to obtain an optimum medium. The final composition of the modified MRS was much simpler than MRS broth, and the modified MRS contained only glucose, yeast extract, dipotassium phosphate, manganese sulfate monohydrate, Tween 80 and sodium acetate anhydrous. The highest bacteriocin Lac-B23 production reached 2560 activity units (AU)/mL in the modified MRS, which is nine times higher than that in MRS broth (280 AU/mL). Meanwhile, the cost per liter of the modified MRS (8.56 Ren Min Bi (RMB)/L) is 34.70% the cost of MRS broth (13.11 RMB/L), and the cost per arbitrary units of bacteriocin Lac-B23 in the modified MRS is approximately fourteen times more convenient (3.34 RMB/106 AU) than in the MRS broth (46.82 RMB/106 AU). Full article
(This article belongs to the Section Microbiology)
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<p>Effects of the ingredients of the De Man, Rogosa and Sharpe (MRS) broth on the growth of <span class="html-italic">Lactobacillus plantarum</span> J23 and bacteriocin Lac-B23 production. Note: (<b>a</b>) is dipotassium phosphate, (<b>b</b>) is magnesium sulfate, (<b>c</b>) is manganese sulfate, (<b>d</b>) is Tween 80, (<b>e</b>) is sodium acetate and (<b>f</b>) is diammonium hydrogen citrate. Values listed are the averages of three experiments.</p> Full article ">Figure 2
<p>Response surface plot (<b>A</b>) and contour plot (<b>B</b>) of bacteriocin Lac-B23 production. Values listed are the averages of three experiments.</p> Full article ">
17 pages, 5120 KiB  
Article
Peptide Profiling and Biological Activities of 12-Month Ripened Parmigiano Reggiano Cheese
by Lisa Solieri, Andrea Baldaccini, Serena Martini, Aldo Bianchi, Valentina Pizzamiglio and Davide Tagliazucchi
Biology 2020, 9(7), 170; https://doi.org/10.3390/biology9070170 - 16 Jul 2020
Cited by 26 | Viewed by 3511
Abstract
Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than [...] Read more.
Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than samples having low-salt and low-fat content (LL). This positive correlation between salt/fat reduction and proteolysis was also confirmed by the analysis of biological activities, as the LL group showed higher average values of angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. UHPLC/HR-MS allowed the identification of 805 unique peptides: LL and HH groups shared 59.3% of these peptides, while 20.9% and 19.9% were LL and HH specific, respectively. Frequency analysis of peptides identified a core of 183 peptides typical of 12-month ripened PR cheeses (corresponding to the 22.7% of total peptides), but no significant differences were detected in peptide patterns between LL and HH groups. Forty bioactive peptides, including 18 ACE-inhibitors and 12 anti-microbial peptides, were identified, of which 25 firstly found in PR cheese. Globally, this work contributed to unraveling the potentially healthy benefits of peptides fraction in PR cheese and provided prior evidence that PR with reduced fat/salt content showed the highest antihypertensive and antioxidant activities. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Total peptides concentration and biological activities of 12-month ripened Parmigiano-Reggiano (PR) peptide fractions. (<b>A</b>) Total peptides concentration. (<b>B</b>) Angiotensin-converting enzyme inhibitory activity. (<b>C</b>) Dipeptidyl-peptidase IV-inhibitory activity. (<b>D</b>) Antioxidant activity. Light grey bars represent PR samples belonging to the high-salt and high-fat group (HH). Dark grey bars represent PR samples belonging to the low-salt and low-fat group (LL). HH represents the average values, considering the samples belonging to the high-salt and high-fat group. LL represents the average values, considering the samples belonging to the low-salt and low-fat group. Average represents the mean values considering all of the 12 PR samples. The x-axis reports the code number of PR samples. Values are means of three assay replications ± standard deviation (SD). Different letters indicate significantly different values (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 2
<p>Venn diagrams of peptides obtained from Parmigiano-Reggiano (PR) peptide fractions. (<b>A</b>) Venn diagram created with all the identified peptides in the 12 PR peptide fractions (see online <a href="#app1-biology-09-00170" class="html-app">supplementary material Table S1</a> for the peptide sequences). (<b>B</b>) Venn diagram created with bioactive peptides identified in the 12 PR peptide fractions (see <a href="#biology-09-00170-t001" class="html-table">Table 1</a> for the peptide sequences). HH represents the samples belonging to the high-salt and high-fat group. LL represents the samples belonging to the low-salt and low-fat group.</p> Full article ">Figure 3
<p>Frequency of identification of the peptides in the Parmigiano-Reggiano (PR) peptide fractions. (<b>A</b>) Frequency of identification of the peptides considering all of the 12 PR samples. (<b>B</b>) Frequency of identification of the peptides in the 6 PR samples belonging to the high-salt and high-fat group. (<b>C</b>) Frequency of identification of the peptides in the 6 PR samples belonging to the low-salt and low-fat group. (<b>D</b>) Frequency of identification of the bioactive peptides considering all of the 12 PR samples. The numbers from 1 to 12 or from 1 to 6 indicate the number of PR samples in which the peptides were identified. The percentage values are referred to the % of peptides found in the PR samples. For example, in <a href="#biology-09-00170-f003" class="html-fig">Figure 3</a>A, 31.9% of single peptides were found in an individual PR sample, whereas the 10.3% of peptides were common in all of the 12 PR samples.</p> Full article ">Figure 4
<p>Distribution of peptides characterized by angiotensin-converting enzyme (ACE) inhibitory IC<sub>50</sub> values lower than 300 µmol/L along with principal components 1 (PC1) and 2 (PC2). IC<sub>50</sub> represent the concentration of peptide able to inhibit the enzymatic activity by 50%. Yellow diamonds represent LL class samples, red diamonds represent HH class samples, green circle represents the biological activity (ACE-inhibition), and blue triangles represent the bioactive peptides.</p> Full article ">
12 pages, 1004 KiB  
Article
Comparison of the Photoautotrophic Growth Regimens of Chlorella sorokiniana in a Photobioreactor for Enhanced Biomass Productivity
by Elvira E. Ziganshina, Svetlana S. Bulynina and Ayrat M. Ziganshin
Biology 2020, 9(7), 169; https://doi.org/10.3390/biology9070169 - 16 Jul 2020
Cited by 15 | Viewed by 3852
Abstract
Microalgae have a wide industrial potential because of their high metabolic diversity and plasticity. Selection of optimal cultivation methods is important to optimize multi-purpose microalgal biotechnologies. In this research, Chlorella sorokiniana AM-02 that was isolated from a freshwater lake was cultured under various [...] Read more.
Microalgae have a wide industrial potential because of their high metabolic diversity and plasticity. Selection of optimal cultivation methods is important to optimize multi-purpose microalgal biotechnologies. In this research, Chlorella sorokiniana AM-02 that was isolated from a freshwater lake was cultured under various high photosynthetic photon flux density (PPFD) conditions and CO2 gas levels in standard Bold’s basal medium (BBM). Furthermore, a wide range of nitrate levels (180–1440 mg L−1) was tested on the growth of C. sorokiniana. Microalgae growth, pigment concentration, medium pH, exit gas composition, as well as nitrate, phosphate, and sulfate levels were measured during an experimental period. The preferred high PPFD and optimal CO2 levels were found to be 1000–1400 μmol photons m−2 s−1 and 0.5–2.0% (v/v), respectively. The addition of nitrate ions (up to 1440 mg L−1) to the standard growth medium increased final optical density (OD750), cell count, pigment concentration, and total biomass yield but decreased the initial growth rate at high nitrate levels. Our findings can serve as the basis for a robust photoautotrophic cultivation system to maximize the productivity of large-scale microalgal cultures. Full article
(This article belongs to the Special Issue The Path to Sustainable Production and Application of Algae)
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<p>Phylogenetic tree based on <span class="html-italic">rbcL</span> gene sequences. Analysis was conducted in MEGA7 [<a href="#B30-biology-09-00169" class="html-bibr">30</a>] using the neighbor-joining method. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method. <span class="html-italic">Meyerella planktonica</span> and <span class="html-italic">Parachlorella kessleri</span> were used as outgroup.</p> Full article ">Figure 2
<p>Growth of <span class="html-italic">C. sorokiniana</span> AM-02 (OD<sub>750</sub> (<b>a</b>–<b>c</b>) and cell number (<b>d</b>–<b>f</b>)) and total pigment concentration in cells (<b>g</b>–<b>i</b>) cultured under various light and CO<sub>2</sub> regimens (1000, 1200, and 1400 μmol photons m<sup>−2</sup> s<sup>−1</sup> and atmospheric CO<sub>2</sub>, 0.5, 1.0, and 2.0% CO<sub>2</sub>) in standard Bold’s basal medium (BBM).</p> Full article ">Figure 3
<p>Changes in culture medium pH (<b>a</b>–<b>c</b>) and the released carbon dioxide level (<b>d</b>–<b>f</b>) observed during the growth of <span class="html-italic">C. sorokiniana</span> AM-02 cultured under various light and CO<sub>2</sub> regimens (1000, 1200, and 1400 μmol photons m<sup>−2</sup> s<sup>−1</sup> and atmospheric CO<sub>2</sub>, 0.5, 1.0, and 2.0% CO<sub>2</sub>) in standard BBM.</p> Full article ">Figure 4
<p>Changes in nitrate (<b>a</b>–<b>c</b>), phosphate (<b>d</b>–<b>f</b>), and sulfate (<b>g</b>–<b>i</b>) concentrations in culture medium during the growth of <span class="html-italic">C. sorokiniana</span> AM-02 cultured under various light and CO<sub>2</sub> regimens (1000, 1200, and 1400 μmol photons m<sup>−2</sup> s<sup>−1</sup> and atmospheric CO<sub>2</sub>, 0.5, 1.0, and 2.0% CO<sub>2</sub>) in standard BBM.</p> Full article ">Figure 5
<p>Growth of <span class="html-italic">C. sorokiniana</span> AM-02 (<b>a</b>), total pigment concentration in cells (<b>b</b>), culture medium pH (<b>c</b>), as well as nitrate (<b>d</b>), phosphate (<b>e</b>), and sulfate (<b>f</b>) removal from the medium in different treatments with modified BBM (PPFD and CO<sub>2</sub> concentration values: 1200 μmol photons m<sup>−2</sup> s<sup>−1</sup> and 2.0% CO<sub>2</sub>, respectively).</p> Full article ">
3 pages, 211 KiB  
Editorial
The Unknown Process Osseointegration
by Nansi López-Valverde, Javier Flores-Fraile and Antonio López-Valverde
Biology 2020, 9(7), 168; https://doi.org/10.3390/biology9070168 - 16 Jul 2020
Cited by 7 | Viewed by 2153
Abstract
Although it was already described more than fifty years ago, there is yet no in-depth knowledge regarding the process of osseointegration as far as its mechanism of action is concerned. It could be one of the body’s ways of reacting to a foreign [...] Read more.
Although it was already described more than fifty years ago, there is yet no in-depth knowledge regarding the process of osseointegration as far as its mechanism of action is concerned. It could be one of the body’s ways of reacting to a foreign body, where the individual’s immune response capacity is involved. It is known that the nervous system has an impact on bone health and that the role of the autonomic nervous system in bone remodeling is an attractive field for current research. In the future, immuno/neuromodulatory techniques will open new and exciting lines of research. Full article
(This article belongs to the Section Evolutionary Biology)
20 pages, 2810 KiB  
Article
Impact of Cytokines and Phosphoproteins in Response to Chronic Joint Infection
by Nicole Prince, Julia A. Penatzer, Matthew J. Dietz and Jonathan W. Boyd
Biology 2020, 9(7), 167; https://doi.org/10.3390/biology9070167 - 16 Jul 2020
Cited by 2 | Viewed by 2812
Abstract
The early cellular response to infection has been investigated extensively, generating valuable information regarding the mediators of acute infection response. Various cytokines have been highlighted for their critical roles, and the actions of these cytokines are related to intracellular phosphorylation changes to promote [...] Read more.
The early cellular response to infection has been investigated extensively, generating valuable information regarding the mediators of acute infection response. Various cytokines have been highlighted for their critical roles, and the actions of these cytokines are related to intracellular phosphorylation changes to promote infection resolution. However, the development of chronic infections has not been thoroughly investigated. While it is known that wound healing processes are disrupted, the interactions of cytokines and phosphoproteins that contribute to this dysregulation are not well understood. To investigate these relationships, this study used a network centrality approach to assess the impact of individual cytokines and phosphoproteins during chronic inflammation and infection. Tissues were taken from patients undergoing total knee arthroplasty (TKA) and total knee revision (TKR) procedures across two tissue depths to understand which proteins are contributing most to the dysregulation observed at the joint. Notably, p-c-Jun, p-CREB, p-BAD, IL-10, IL-12p70, IL-13, and IFN-γ contributed highly to the network of proteins involved in aseptic inflammation caused by implants. Similarly, p-PTEN, IL-4, IL-10, IL-13, IFN-γ, and TNF-α appear to be central to signaling disruptions observed in septic joints. Ultimately, the network centrality approach provided insight into the altered tissue responses observed in chronic inflammation and infection. Full article
(This article belongs to the Special Issue Using Omics Data and Systems Biology Approaches Based on Network Analysis to Understand Biological Systems)
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<p>Map of approximate tissue collection locations, shown with prosthetic implant. Seven tissue samples were taken for each patient; (1) the solid circle represents the medial femoral condyle (denoted as F); (2) the dashed circle represents the medial tibial plateau (denoted as T); (3) the solid square represents the lateral gutter (denoted as LG); (4) the dashed square represents the posterior capsule (denoted as PC). Locations 1–4 were taken for the ATL layer, and locations 1–3 were taken for the RTL layer; separation between ATL (unhealthy tissue, closer to joint) and RTL (healthy tissue, further from joint) was approximately 1 cm, depending on individual patient.</p> Full article ">Figure 2
<p>Relative cytokine levels measured in tissues from primary TKA, aseptic TKR, and septic TKR at adjacent tissue layer (ATL) and radial tissue layer (RTL) debridement depths. Relative cytokine responses (normalized to highest cytokine signal) were observed for all three patient groups: primary, aseptic, and septic at two debridement depths: ATL is closer to the knee joint, and RTL is approximately 1 cm removed from the knee joint. Statistically significant differences (<span class="html-italic">p</span> &lt; 0.05) were determined by two-way ANOVA with Bonferroni’s post-test to examine group-dependent and spatially-dependent differences in cytokine relative response. Differences for the same group (i.e., septic) between ATL and RTL are marked with an asterisk (*). Differences between groups within a tissue layer are denoted with bars. Responses are shown as the mean ± SEM.</p> Full article ">Figure 3
<p>Relative levels of phosphoproteins associated with the proliferative processes in acute wound healing. Relative phosphoprotein responses (normalized to highest signal) were observed for all three patient groups: primary, aseptic, and septic at two debridement depths: ATL is closer to the knee joint, and RTL is approximately 1 cm removed from the knee joint. Statistically significant differences (<span class="html-italic">p</span> &lt; 0.05) were determined by two-way ANOVA with Bonferroni’s post-test to examine group-dependent and spatially-dependent differences in protein phosphorylation. Differences for the same group (i.e., septic) between ATL and RTL are marked with an asterisk (*). Differences between groups within a tissue layer are denoted with bars. Responses are shown as the mean ± SEM.</p> Full article ">Figure 4
<p>Relative phosphoprotein levels associated with cell migration processes in acute wound healing. Relative phosphoprotein responses (normalized to highest signal) were observed for all three patient groups: primary, aseptic, and septic at two debridement depths: ATL is closer to the knee joint, and RTL is approximately 1 cm removed from the knee joint. Statistically significant differences (<span class="html-italic">p</span> &lt; 0.05) were determined by two-way ANOVA with Bonferroni’s post-test to examine group-dependent and spatially-dependent differences in protein phosphorylation. Differences for the same group (i.e., septic) between ATL and RTL are marked with an asterisk (*). Differences between groups within a tissue layer are denoted with bars. Responses are shown as the mean ± SEM.</p> Full article ">Figure 5
<p>Relative levels of pro-apoptotic and inhibitory wound healing phosphoproteins in acute wound healing. Relative phosphoprotein responses (normalized to highest signal) were observed for all three patient groups: primary, aseptic, and septic at two debridement depths: ATL is closer to the knee joint, and RTL is approximately 1 cm removed from the knee joint. Statistically significant differences (<span class="html-italic">p</span> &lt; 0.05) were determined by two-way ANOVA with Bonferroni’s post-test to examine group-dependent and spatially-dependent differences in protein phosphorylation. Differences between groups within a tissue layer are denoted with bars. Responses are shown as the mean ± SEM.</p> Full article ">Figure 6
<p>Ingenuity Pathway Analysis (IPA)-generated networks for primary TKA, aseptic TKR, and septic TKR groups based on cytokine and phosphoprotein datasets. Proposed networks used relative cytokine and phosphoprotein responses in the ATL depth, illustrating the differences in tissue responses for the three groups. The nodes are illustrated in a “heat map” coloring scheme, with red denoting up-regulation, green denoting down-regulation, and the intensity of color correlates to the intensity of relative response. The networks are supplemented with other nodes likely to be involved, as identified in the Ingenuity Knowledge Base. A solid line represents a direct interaction between two nodes, while a dotted line denotes an indirect relationship.</p> Full article ">Figure 7
<p>Changes in significant nodes between groups for low radiality outcomes. Nodes with low radiality outcomes that differed between primary TKA response and aseptic/septic TKR responses are shown (significance threshold: the average radiality ± standard deviation). Boxes indicate significance at varying depths. IL-10 is shown in red to highlight its presence in all three groups: primary TKA, aseptic TKR, and septic TKR. IL-13 (green) and IFN-γ (blue) are also colored to highlight overlap in both aseptic TKR and septic TKR groups.</p> Full article ">
11 pages, 1702 KiB  
Article
Plasma CAF22 Levels as a Useful Predictor of Muscle Health in Patients with Chronic Obstructive Pulmonary Disease
by Rizwan Qaisar, Asima Karim and Tahir Muhammad
Biology 2020, 9(7), 166; https://doi.org/10.3390/biology9070166 - 15 Jul 2020
Cited by 31 | Viewed by 2969
Abstract
Skeletal muscle dysfunction and reduced physical capacity are characteristic features of chronic obstructive pulmonary disease (COPD). However, the search for a reliable biomarker to assess muscle health in CODP remains elusive. We analyzed the course of hand-grip strength (HGS) and appendicular skeletal mass [...] Read more.
Skeletal muscle dysfunction and reduced physical capacity are characteristic features of chronic obstructive pulmonary disease (COPD). However, the search for a reliable biomarker to assess muscle health in CODP remains elusive. We analyzed the course of hand-grip strength (HGS) and appendicular skeletal mass index (ASMI) in COPD in relation to spirometry decline and plasma extracellular heat shock protein-72 (eHSP72) and c-terminal fragment of agrin-22 (CAF22) levels. We evaluated male, 62–73 years old patients of COPD (N = 265) and healthy controls (N = 252) at baseline and after 12 and 24 months for plasma biomarkers, spirometry and HGS measurements. HGS declined significantly over time and plasma CAF22, but not eHSP72 levels, had a significant negative association with HGS and ASMI in COPD. Plasma CAF22 also had an association with walking speed and daily steps count in advanced COPD. Lower ASMI was associated with reduced HGS at all time-point. Narrow age-span of the study cohort and exclusion of lower-limb muscles from the analysis are limitations of this study. Taken together, we report that the plasma CAF22 may be a useful tool to assess muscle weakness and atrophy in COPD patients. Full article
(This article belongs to the Section Medical Biology)
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<p>Study flow. FEV<sub>1</sub>%: Forced expiratory volume in the first second and GOLD: global initiative for obstructive lung diseases.</p> Full article ">Figure 2
<p>Relationship of serum levels of eHSP72 and CAF22 with hand-grip strength (HGS; <b>A</b>–<b>C</b>) and appendicular skeletal muscle mass (ASMI; <b>D</b>–<b>F</b>) at the baseline (<b>A</b>,<b>D</b>), 12 (<b>B</b>,<b>E</b>) and 24-month (<b>C</b>,<b>F</b>) time points in healthy controls and patients with COPD GOLD stages 1 and 2 and 3 and 4 (<span class="html-italic">N</span> = 64–68 participants/group/time point for pooled data).</p> Full article ">Figure 3
<p>Relationship of hand-grip strength (HGS) with appendicular skeletal muscle mass (ASMI) at the baseline (<b>A</b>), 12 (<b>B</b>) and 24-month (<b>C</b>) time points in healthy controls and patients with COPD GOLD stages 1 and 2 and 3 and 4 (<span class="html-italic">N</span> = 64–68 participants/group/time point for pooled data).</p> Full article ">
21 pages, 3943 KiB  
Article
Potential Involvement of lncRNAs in the Modulation of the Transcriptome Response to Nodavirus Challenge in European Sea Bass (Dicentrarchus labrax L.)
by Patricia Pereiro, Raquel Lama, Rebeca Moreira, Valentina Valenzuela-Muñoz, Cristian Gallardo-Escárate, Beatriz Novoa and Antonio Figueras
Biology 2020, 9(7), 165; https://doi.org/10.3390/biology9070165 - 15 Jul 2020
Cited by 16 | Viewed by 2825
Abstract
Long noncoding RNAs (lncRNAs) are being increasingly recognised as key modulators of various biological mechanisms, including the immune response. Although investigations in teleosts are still lagging behind those conducted in mammals, current research indicates that lncRNAs play a pivotal role in the response [...] Read more.
Long noncoding RNAs (lncRNAs) are being increasingly recognised as key modulators of various biological mechanisms, including the immune response. Although investigations in teleosts are still lagging behind those conducted in mammals, current research indicates that lncRNAs play a pivotal role in the response of fish to a variety of pathogens. During the last several years, interest in lncRNAs has increased considerably, and a small but notable number of publications have reported the modulation of the lncRNA profile in some fish species after pathogen challenge. This study was the first to identify lncRNAs in the commercial species European sea bass. A total of 12,158 potential lncRNAs were detected in the head kidney and brain. We found that some lncRNAs were not common for both tissues, and these lncRNAs were located near coding genes that are primarily involved in tissue-specific processes, reflecting a degree of cellular specialisation in the synthesis of lncRNAs. Moreover, lncRNA modulation was analysed in both tissues at 24 and 72 h after infection with nodavirus. Enrichment analysis of the neighbouring coding genes of the modulated lncRNAs revealed many terms related to the immune response and viral infectivity but also related to the stress response. An integrated analysis of the lncRNAs and coding genes showed a strong correlation between the expression of the lncRNAs and their flanking coding genes. Our study represents the first systematic identification of lncRNAs in European sea bass and provides evidence regarding the involvement of these lncRNAs in the response to nodavirus. Full article
(This article belongs to the Special Issue Transcriptome and Genome Analyses Applied to Aquaculture Research)
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<p>Features of predicted lncRNAs in <span class="html-italic">D. labrax</span>. (<b>a</b>) Guanine-cytosine (GC) content and (<b>b</b>) length distribution of the 12158 predicted lncRNAs. (<b>c</b>) LncRNA abundance and localisation per chromosome. (<b>d</b>) Correlation between chromosome length and lncRNA abundance.</p> Full article ">Figure 2
<p>LncRNAs identified per tissue. (<b>a</b>) Venn diagram reflecting the number of lncRNAs with a transcripts per million (TPM) value in at least one of the samples from each tissue. Most of the predicted lncRNAs were detected in both the head kidney and brain. In contrast, a total of 30 predicted lncRNAs obtained a TPM value of 0 in all the samples. (<b>b</b>,<b>c</b>) The neighbouring coding genes of the lncRNAs expressed in the head kidney but not in the brain (<b>b</b>) and vice versa (<b>c</b>) were analysed by GO enrichment analyses (biological processes). Only the 30 most significant terms were represented.</p> Full article ">Figure 3
<p>Temporal expression of the predicted lncRNAs after nodavirus infection in head kidney and brain. (<b>a</b>) Number of lncRNAs up- and downregulated in each tissue at 24 and 72 hpi with nervous necrosis virus (NNV). (<b>b</b>) Venn diagram representing the shared and unshared lncRNAs modulated after NNV challenge in both tissues at the different sampling points.</p> Full article ">Figure 4
<p>Modulation of lncRNAs in (<b>a</b>) head kidney and (<b>b</b>) brain after nodavirus challenge. Venn diagrams represent the number of shared and unshared lncRNAs significantly modulated at each sampling point (FC &gt; 2, FDR &lt; 0.05). Heat maps of the lncRNAs significantly affected by the infection in each tissue and hierarchical clustering of the different samples constructed with the TPM values.</p> Full article ">Figure 5
<p>GO enrichment analysis (biological processes) of the neighbouring coding genes of the differentially modulated lncRNAs (FC &gt; 2, FDR &lt; 0.05) in head kidney after viral challenge. Only the 30 most significant terms were represented.</p> Full article ">Figure 6
<p>GO enrichment analysis (biological processes) of the neighbouring coding genes of the differentially modulated lncRNAs (FC &gt; 2, FDR &lt; 0.05) in the brain after viral challenge. Only the 30 most significant terms were represented.</p> Full article ">Figure 7
<p>Correlation between differentially modulated coding genes after NNV infection and their flanking lncRNAs. (<b>a</b>) Heat map representing the TPM values of four genes and their neighbouring lncRNAs across the different head kidney and brain samples. Expression levels are represented as row-normalised values on a blue–yellow colour scale. (<b>b</b>) Correlation values (Pearson’s correlation coefficient and Spearman’s rank correlation coefficient) between the lncRNAs and their nearby coding genes. * represents significant correlation and ns non-significant correlation.</p> Full article ">Figure 8
<p>Calcium homeostasis-related genes and their relationship with lncRNAs. (<b>a</b>) Protein–protein interaction network of the lncRNAs neighbouring coding genes involved in calcium homeostasis. Numerous lncRNAs significantly modulated in the brain after nodavirus infection were flanked by genes directly related to calcium homeostasis. (<b>b</b>) Gene representation of two genes mediating cellular calcium concentration after NNV challenge and genomic location of their neighbouring lncRNAs. Heap maps represent the TPM values of the different contigs. Expression levels are represented as row-normalised values on a blue–yellow colour scale. * represents significant correlation and ns non-significant correlation.</p> Full article ">
22 pages, 3021 KiB  
Article
Vegetation- and Environmental Changes on Non-Reclaimed Spoil Heaps in Southern Poland
by Oimahmad Rahmonov, Robert Krzysztofik, Dorota Środek and Justyna Smolarek-Lach
Biology 2020, 9(7), 164; https://doi.org/10.3390/biology9070164 - 15 Jul 2020
Cited by 33 | Viewed by 4586
Abstract
The study focused on the changes in vegetation and soils on an undeveloped area of coal mine spoil heaps. The process of vegetation changes was evaluated on the basis of historical cartographic materials and fieldwork. Changes of vegetation in nearly 200 years are [...] Read more.
The study focused on the changes in vegetation and soils on an undeveloped area of coal mine spoil heaps. The process of vegetation changes was evaluated on the basis of historical cartographic materials and fieldwork. Changes of vegetation in nearly 200 years are presented herein. The main purpose of this study is to present an analysis of spatio-temporal changes in vegetation and their influence on soil features. The diversity of ecological species in terms of habitat requirements, tendency of hornbeam communities formation, and the relationship between forest communities and soil features was found. The basic soil properties were examined under selected plant communities (pH, Corg, Nt), available forms of elements (P, K, Mg), and as plant nutrients and heavy metal occurrence (Fe, Zn, Mn, Co, Cd, Pb, Sr, Cr, Cu). The soil organic carbon (Corg) content varied from 3.17 ± 0.007% to 17.7 ± 0.21% and significant differences were observed between sites. The highest total nitrogen (Nt) content was recorded in the soils of the sites that were represented by Populus-Betula stands (0.60 ± 0.01%). Soil acidity (pH) varied greatly, ranging from acidic (pH = 4.1) to weakly acidic (pH = 5.9). The highest value for Mgavail (205.43 ± 0.5 mg·kg−1) was noted in the soils under Calamagrostis epegijos (L.) Roth community and for Pavail (184.07 ± 3.77) and Kavail (346.19 ± 2.92 mg·kg−1) under the Quercus-Pinus stand. On all sites, Zn was a dominant element and its concentration ranged from 526.1 to 1060.4 mg·kg−1. Obtained results show how important it is to study the issue of vegetation changes and the formation of the landscape within an industrial city. The described results are important for the management of urban greening issues. Human influence on the disintegration and development of the natural environment is clearly visible. Due to the diversity of former mining areas and their time of creation, the studied area is one of the most important experimental areas for the determination link between vegetation and soil. Full article
(This article belongs to the Section Ecology)
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<p>Distribution of vegetation and soil sampling sites: (1) Artificial birch stand (urban park); (2) Betula pendula stand; (3) <span class="html-italic">Populus-Betula</span> stands, (4) <span class="html-italic">Quercus-Pinus</span> stand; (5) <span class="html-italic">Robinia pseudoacacia</span> stand; (6) <span class="html-italic">Calamagrostietum epigeji</span>; (7) Thickets of <span class="html-italic">R. pseudoacacia</span>; (8) <span class="html-italic">Juncus effusus</span> community; (9) Alder community; (10) <span class="html-italic">Ulmus-Quercus</span> stands; (11) <span class="html-italic">Arctio-Artemisietum vulgaris</span>; (12) <span class="html-italic">Rudbeckio-Solidaginetium</span>; (13) <span class="html-italic">Pinus sylvestris</span> stand; (14) <span class="html-italic">Quercus-Pinus</span> stand; (15) Populus-Alder-Salix stand, (16) Soil sampling places (I–XII). Source: by authors on the base of: OpenStreetMap.org, 2019.</p> Full article ">Figure 2
<p>Changes in forested area, 1833–2019. Source: by authors based on historical maps [<a href="#B50-biology-09-00164" class="html-bibr">50</a>,<a href="#B51-biology-09-00164" class="html-bibr">51</a>,<a href="#B52-biology-09-00164" class="html-bibr">52</a>,<a href="#B53-biology-09-00164" class="html-bibr">53</a>,<a href="#B54-biology-09-00164" class="html-bibr">54</a>,<a href="#B55-biology-09-00164" class="html-bibr">55</a>,<a href="#B58-biology-09-00164" class="html-bibr">58</a>] and contemporary field research in 2019.</p> Full article ">Figure 3
<p>Percentage share of plant life forms in the Niwka postmining area: (H) hemicryptophyte; (M) megaphanerophyte, (N) nanophanerophyte, (T) therophyte; (G) geophytes; (Ch) chamaephytes; (Hy) hydrophytes; (C) herbaceous chamaephyte, (li) liana, and (pp) semiparasite.</p> Full article ">Figure 4
<p>Plant requirement in respect of light values: 2—moderate shade; 3—semi-shade, 4—moderate light and 5—full light.</p> Full article ">Figure 5
<p>Plant requirement with respect to temperature: (1) coldest climatic conditions, (2) moderately cold areas, (3) moderately cool climatic conditions, (4) moderately warm climatic conditions, (5) warmest regions and microhabitats.</p> Full article ">
12 pages, 1554 KiB  
Article
Cytotoxic, Antioxidant, and Enzyme Inhibitory Properties of the Traditional Medicinal Plant Matthiola incana (L.) R. Br.
by Maria Fernanda Taviano, Natalizia Miceli, Rosaria Acquaviva, Giuseppe Antonio Malfa, Salvatore Ragusa, Deborah Giordano, Guillermo Cásedas, Francisco Les and Víctor López
Biology 2020, 9(7), 163; https://doi.org/10.3390/biology9070163 - 13 Jul 2020
Cited by 17 | Viewed by 4415
Abstract
Matthiola incana (L.) R. Br. (Brassicaceae) is widely cultivated for ornamental purposes and utilized as a medicinal plant. In the present work, the hydroalcoholic extract from the aerial parts of this species has been evaluated in different bioassays in order to detect potential [...] Read more.
Matthiola incana (L.) R. Br. (Brassicaceae) is widely cultivated for ornamental purposes and utilized as a medicinal plant. In the present work, the hydroalcoholic extract from the aerial parts of this species has been evaluated in different bioassays in order to detect potential pharmacological applications. The cytotoxic capacity against the human colorectal adenocarcinoma (CaCo-2) and breast cancer (MCF-7) cell lines was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The extract was investigated as a neuroprotective inhibitor of central nervous system (CNS) enzymes such as monoamine oxidase A, tyrosinase, acetylcholinesterase, and as a natural enzyme inhibitor of α-glucosidase and lipase involved in some metabolic disorders such as obesity or type 2 diabetes. The antioxidant ability was also evaluated in an enzymatic system (xanthine/xanthine oxidase assay). Results showed that the M. incana extract displayed moderate to low cytotoxicity vs. CaCo-2 cells. The extract acted as a superoxide radical scavenger and enzymatic inhibitor of monoamine oxidase A, tyrosinase, α-glucosidase, and lipase. The best results were found in the α-glucosidase assay, as M. incana hydroalcoholic extract was able to inhibit the enzyme α-glucosidase up to 100% without significant differences, compared to the antidiabetic drug acarbose. Matthiola incana has been demonstrated to exert different biological properties. These are important in order to consider this species as a source of bioactive compounds. Full article
(This article belongs to the Special Issue Bioactivity of Medicinal Plants and Extracts)
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<p>Cell viability in CaCo2 cells treated for 48 and 72 h with the hydroalcoholic extract of <span class="html-italic">M. incana</span> aerial parts evaluated by MTT assay. Values are the mean ± SD of four experiments in triplicate. * Significant vs. untreated control cells: <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>LDH released in CaCo2 cells untreated and treated for 48 and 72 h with the hydroalcoholic extract of <span class="html-italic">M. incana</span> aerial parts. Values are the mean ± SD of four experiments in triplicate. * Significant vs. untreated control cells: <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 3
<p>Antioxidant activity of the hydroalcoholic extract of <span class="html-italic">M. incana</span> aerial parts evaluated in the xanthine/xanthine oxidase system. IC<sub>50</sub> values were calculated by non-linear regression. All concentrations were tested at least in triplicate, and each point represents mean ± SEM.</p> Full article ">Figure 4
<p>Activity of the hydroalcoholic extract of <span class="html-italic">M. incana</span> aerial parts in enzymes related to central nervous system (CNS) pathologies. IC<sub>50</sub> values were calculated by non-linear regression. All concentrations were tested at least in triplicate, and each point represents mean ± SEM. (<b>A</b>): monoamine oxidase A (MAO-A) inhibition performed by <span class="html-italic">M. incana</span> extract and clorgyline as standard. (<b>B</b>): tyrosinase (TYR) inhibition by <span class="html-italic">M. incana</span> extract and kojic acid as standard.</p> Full article ">Figure 4 Cont.
<p>Activity of the hydroalcoholic extract of <span class="html-italic">M. incana</span> aerial parts in enzymes related to central nervous system (CNS) pathologies. IC<sub>50</sub> values were calculated by non-linear regression. All concentrations were tested at least in triplicate, and each point represents mean ± SEM. (<b>A</b>): monoamine oxidase A (MAO-A) inhibition performed by <span class="html-italic">M. incana</span> extract and clorgyline as standard. (<b>B</b>): tyrosinase (TYR) inhibition by <span class="html-italic">M. incana</span> extract and kojic acid as standard.</p> Full article ">Figure 5
<p>Activity of the hydroalcoholic extract of <span class="html-italic">M. incana</span> aerial parts on lipase (<b>A</b>) and α-glucosidase (<b>B</b>). IC<sub>50</sub> values were calculated by non-linear regression. All concentrations were tested at least in triplicate, and each point represents mean ± SEM. A: Lipase inhibition performed by <span class="html-italic">M. incana</span> extract and orlistat as standard. B: α-glucosidase (α-GLU) inhibition by <span class="html-italic">M. incana</span> extract and acarbose as standard.</p> Full article ">
11 pages, 1638 KiB  
Review
Aconitase: To Be or not to Be Inside Plant Glyoxysomes, That Is the Question
by Luigi De Bellis, Andrea Luvisi and Amedeo Alpi
Biology 2020, 9(7), 162; https://doi.org/10.3390/biology9070162 - 12 Jul 2020
Cited by 9 | Viewed by 6298
Abstract
After the discovery in 1967 of plant glyoxysomes, aconitase, one the five enzymes involved in the glyoxylate cycle, was thought to be present in the organelles, and although this was found not to be the case around 25 years ago, it is still [...] Read more.
After the discovery in 1967 of plant glyoxysomes, aconitase, one the five enzymes involved in the glyoxylate cycle, was thought to be present in the organelles, and although this was found not to be the case around 25 years ago, it is still suggested in some textbooks and recent scientific articles. Genetic research (including the study of mutants and transcriptomic analysis) is becoming increasingly important in plant biology, so metabolic pathways must be presented correctly to avoid misinterpretation and the dissemination of bad science. The focus of our study is therefore aconitase, from its first localization inside the glyoxysomes to its relocation. We also examine data concerning the role of the enzyme malate dehydrogenase in the glyoxylate cycle and data of the expression of aconitase genes in Arabidopsis and other selected higher plants. We then propose a new model concerning the interaction between glyoxysomes, mitochondria and cytosol in cotyledons or endosperm during the germination of oil-rich seeds. Full article
(This article belongs to the Section Plant Science)
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<p>The path of carbon in the classical glyoxylate cycle except for aconitase in the cytosol. Black arrows indicate glyoxylate cycle reactions; the five enzymes of the glyoxylate cycle are in green.</p> Full article ">Figure 2
<p>The path of carbon in the glyoxylate cycle after van Roermund et al. [<a href="#B46-biology-09-00162" class="html-bibr">46</a>], Pracharoenwattana et al. [<a href="#B49-biology-09-00162" class="html-bibr">49</a>] and Pracharoenwattana and Smith [<a href="#B5-biology-09-00162" class="html-bibr">5</a>]. Black arrows indicate glyoxylate cycle reactions up to sugars, while light blue arrows indicate the malate/oxaloacetate shuttle transport/reactions; the cytosolic enzymes are given in green, the glyoxysomal enzymes in blue, and the enzymes of the glyoxylate cycle including the cytosolic malate dehydrogenase are underlined. MDH: mitochondrial malate dehydrogenase.</p> Full article ">Figure 3
<p>Expression of Arabidopsis genes plotted with Genevestigator (<a href="https://genevestigator.com" target="_blank">https://genevestigator.com</a>) on Affymetrix Arabidopsis ATH1 Genome Array data. (<b>A</b>) Data expressed in a color coding scaled to the expression potential (=the maximum expression a gene reaches across all experiments); ACO1: aconitase 1 (At4g35830); ACO2: aconitase 2 (At4g26970); ACO3: aconitase 3 (At2g05710); ICL: isocitrate lyase (AT3G21720); MLS: malate synthase (At5g03860). (<b>B</b>) Data expressed as log2 scale; <span style="color:red">●</span>ACO1: aconitase 1 (At4g35830); <span style="color:#0202FE">●</span>ACO2: aconitase 2 (At4g26970); <span style="color:#71FE00">●</span>ACO3: aconitase 3 (At2g05710); <span style="color:#F29211">●</span> ICL: isocitrate lyase (AT3G21720); <span style="color:#9C2070">●</span> MLS: malate synthase (At5g03860). The developmental stages are germinated seed, seedling, young rosette, botting, young flower, developed flower, flowers and siliques, mature siliques, and senescence, respectively.</p> Full article ">Figure 3 Cont.
<p>Expression of Arabidopsis genes plotted with Genevestigator (<a href="https://genevestigator.com" target="_blank">https://genevestigator.com</a>) on Affymetrix Arabidopsis ATH1 Genome Array data. (<b>A</b>) Data expressed in a color coding scaled to the expression potential (=the maximum expression a gene reaches across all experiments); ACO1: aconitase 1 (At4g35830); ACO2: aconitase 2 (At4g26970); ACO3: aconitase 3 (At2g05710); ICL: isocitrate lyase (AT3G21720); MLS: malate synthase (At5g03860). (<b>B</b>) Data expressed as log2 scale; <span style="color:red">●</span>ACO1: aconitase 1 (At4g35830); <span style="color:#0202FE">●</span>ACO2: aconitase 2 (At4g26970); <span style="color:#71FE00">●</span>ACO3: aconitase 3 (At2g05710); <span style="color:#F29211">●</span> ICL: isocitrate lyase (AT3G21720); <span style="color:#9C2070">●</span> MLS: malate synthase (At5g03860). The developmental stages are germinated seed, seedling, young rosette, botting, young flower, developed flower, flowers and siliques, mature siliques, and senescence, respectively.</p> Full article ">
26 pages, 5157 KiB  
Article
Development of an LDL Receptor-Targeted Peptide Susceptible to Facilitate the Brain Access of Diagnostic or Therapeutic Agents
by Séverine André, Lionel Larbanoix, Sébastien Verteneuil, Dimitri Stanicki, Denis Nonclercq, Luce Vander Elst, Sophie Laurent, Robert N. Muller and Carmen Burtea
Biology 2020, 9(7), 161; https://doi.org/10.3390/biology9070161 - 11 Jul 2020
Cited by 13 | Viewed by 4147
Abstract
Blood-brain barrier (BBB) crossing and brain penetration are really challenging for the delivery of therapeutic agents and imaging probes. The development of new crossing strategies is needed, and a wide range of approaches (invasive or not) have been proposed so far. The receptor-mediated [...] Read more.
Blood-brain barrier (BBB) crossing and brain penetration are really challenging for the delivery of therapeutic agents and imaging probes. The development of new crossing strategies is needed, and a wide range of approaches (invasive or not) have been proposed so far. The receptor-mediated transcytosis is an attractive mechanism, allowing the non-invasive penetration of the BBB. Among available targets, the low-density lipoprotein (LDL) receptor (LDLR) shows favorable characteristics mainly because of the lysosome-bypassed pathway of LDL delivery to the brain, allowing an intact discharge of the carried ligand to the brain targets. The phage display technology was employed to identify a dodecapeptide targeted to the extracellular domain of LDLR (ED-LDLR). This peptide was able to bind the ED-LDLR in the presence of natural ligands and dissociated at acidic pH and in the absence of calcium, in a similar manner as the LDL. In vitro, our peptide was endocytosed by endothelial cells through the caveolae-dependent pathway, proper to the LDLR route in BBB, suggesting the prevention of its lysosomal degradation. The in vivo studies performed by magnetic resonance imaging and fluorescent lifetime imaging suggested the brain penetration of this ED-LDLR-targeted peptide. Full article
(This article belongs to the Special Issue Molecular Targets and Targeting in Biomedical Sciences)
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<p>(<b>A</b>,<b>B</b>) Specific binding of the 50 clones isolated from the pool of the 3rd round of panning, determined by the ratio between the binding to the extracellular domain of low-density lipoprotein receptor (ED-LDLR) relative to bovine serum albumin (BSA) (see <a href="#app1-biology-09-00161" class="html-app">Figure S1</a>). (<b>A</b>) Clones 1 to 24. (<b>B</b>) Clones 25 to 50. (<b>C</b>) Frequency of amino acids in the 13 different peptide sequences obtained after DNA sequencing.</p> Full article ">Figure 2
<p>Binding of selected clones to ED-LDLR in various experimental conditions. (<b>A</b>) Apparent dissociation constants (K*<sub>d</sub>) of the 13 representative clones selected from the phage display experiments. Clones with low K*<sub>d</sub>, revealing high affinities and selected for further characterization, are in green. (<b>B</b>–<b>D</b>) K*<sub>d</sub> of the clones 40, 41, and 47 in normal conditions, at acidic pH (pH 6.0, pH 5.0), and in the absence of calcium (Ca<sup>2+</sup> free). (<b>E</b>–<b>G</b>) Ratios between K*<sub>d</sub> in modified conditions and K*<sub>d</sub> of the clones, reflecting the inhibitory effects of these modifications on the clones binding. (<b>H</b>) Percentage of dissociation of the clones from the LDLR.</p> Full article ">Figure 3
<p>Computational three-dimensional structure (<b>A</b>,<b>F</b>) and spatial conformation (<b>B<span class="html-italic">,</span>G</b>) of peptides LRPep1 (<b>A</b>,<b>B</b>) and LRPep2 (<b>F</b>,<b>G</b>). The three-dimensional structures of peptides were drawn with ACD/ChemSketch 2.0 software. The peptides are represented with disulfide bridges that could occur in oxidizing conditions between the pairs of Cys (in yellow in <b>A</b> and <b>F</b>). Their spatial conformations were obtained with MarvinSketch 19.2 software (2019, <a href="http://www.chemaxon.com" target="_blank">http://www.chemaxon.com</a>). Interaction of LRPep1 (<b>C</b>–<b>E</b>) and LRPep2 (<b>H</b>–<b>J</b>) with ED-LDLR was predicted using the HPEPDOCK program of blind peptide-protein docking (<a href="http://huanglab.phys.hust.edu.cn/hpepdock/" target="_blank">http://huanglab.phys.hust.edu.cn/hpepdock/</a>) [<a href="#B16-biology-09-00161" class="html-bibr">16</a>]. The binding regions of peptides to ED-LDLR are zoomed-in figures <b>D</b>–<b>E</b> (LRPep1) and <b>I</b>–<b>J</b> (LRPep2) to better observe the docking models. LRPep1 and LRPep2 appear in yellow, and ED-LDLR in orange. LRPep, LDL receptor-peptide.</p> Full article ">Figure 4
<p>Endocytosis of the peptides LRPep1-rho and LRPep2-rho (stained in red) in (<b>A</b>) HUVEC and HepaRG cells and (<b>B</b>) ACBRI376 and 1321N1 cells. Nuclei are stained in blue with Hoechst. (<b>C</b>–<b>F</b>) The endocytosis of peptides, as well as the expression of LDLR, was semi-quantitatively evaluated by the measurement of fluorescent labeling using the ImageJ software and was normalized to the number of cells and to the background, giving the relative ratio of fluorescent labeling (RRFL); *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.001. (<b>G</b>) The correlation coefficient between the expression of LDLR and the endocytosis of peptides.</p> Full article ">Figure 5
<p>Colocalization of LRPep2-rho with LDLR on mouse brain slices (<b>A</b>) and ACBRI376 cells (<b>B</b>). LRPep2 appears in red due to the coupled rhodamine, LDLR is stained in green with fluorescein, and nuclei in blue with DAPI. White arrows highlight examples of colocalization areas.</p> Full article ">Figure 6
<p>Semi-quantitative analysis of fluorescent labeling of caveolae and lysosomes when ACBRI376 (<b>A</b>), HepaRG (<b>B</b>), N18(H) (<b>C</b>), and 1321N1 (<b>D</b>) cells were incubated or not with peptide LRPep2 by the measurement of the RRFL. *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>(<b>A</b>) The apparent dissociation constant (K*<sub>d</sub>) of ultrasmall superparamagnetic particles of iron oxide (USPIO)-LRPep2 for LDLR. The inflection point of the curve corresponded to the apparent dissociation constant (K*<sub>d</sub>) of USPIO-LRPep2 for the binding to LDLR, and the strength of the affinity was inversely proportional to the K*<sub>d</sub> value. (<b>B</b>–<b>D</b>) <span class="html-italic">In vivo</span> evaluation of the blood-brain barrier (BBB) crossing ability of USPIO-LRPep2 by MRI. (<b>B</b>) Representative raw coronal MRI images of the brains (bregma: −1.64 mm) of NMRI (Naval Medical Research Institute) mice acquired with the rapid acquisition with relaxation enhancement (RARE) protocol (spatial resolution = 48 µm) before (pre-contrast) and 52 min after injection of USPIO derivatives (post-contrast). Accumulation of USPIO derivatives (negative contrast agents) led to the darkening of the brain tissue. (<b>C</b>) Color overlay images, allowing to better visualize the negative contrast produced by USPIO, which is directly proportional to the red and black pixels. (<b>D</b>) Analysis of the percentage change of signal-to-noise ratio (Δ%SNR) produced by USPIO on MRI images of the brain, measured by the ImageJ software. A decreased Δ%SNR corresponded to the negative contrast generated by USPIO derivatives. *: <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 8
<p>Biodistribution of USPIO-LRPep2 and USPIO-NSP at 55 min post-injection. (<b>A</b>) R<sub>2</sub><sup>Norm</sup> (=1/T<sub>2</sub><sup>Sample</sup> − 1/T<sub>2</sub><sup>Control</sup>) for each USPIO derivative in the kidney, the spleen, and the liver. * <span class="html-italic">p</span> &lt; 0.05. (<b>B</b>,<b>C</b>) Iron concentration (µmol/L) in urine and plasma, respectively. (<b>D</b>) R<sub>2(1)</sub><sup>Norm</sup> for each USPIO derivative in the brain. (<b>E</b>) Iron concentration (µmol/g of dried tissue) in brains after digestion in acidic conditions. (<b>F</b>) Correlation coefficients between the R<sub>2(1)</sub><sup>Norm</sup> of USPIO derivatives in the brains and their concentrations in the blood.</p> Full article ">Figure 9
<p>Perls’-DAB brown staining of USPIO derivatives in mouse brains collected at 55 min post-injection. Mice injected with PBS were used as a negative control. USPIO derivatives are stained in brown by the DAB.</p> Full article ">Figure 10
<p>(<b>A</b>) FLI images of the brains of nude mice before (pre-iv) and after injection of CF770-LRPep2 or CF770 (post-iv). (<b>B</b>) Analysis of the fluorescence observed on FLI images, measured by the M3Vision software in the brain area, and normalized to the pre-iv signal. * <span class="html-italic">p</span> &lt; 0.05. (<b>C</b>) FLI images of mouse brains ex vivo. (<b>D</b>) Analysis of the fluorescence of the brains ex vivo normalized to the brain signal of a non-injected mouse. § <span class="html-italic">p</span> = 0.05.</p> Full article ">
24 pages, 1631 KiB  
Review
Insight into the Role of Dental Pulp Stem Cells in Regenerative Therapy
by Shinichiro Yoshida, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii and Hidefumi Maeda
Biology 2020, 9(7), 160; https://doi.org/10.3390/biology9070160 - 9 Jul 2020
Cited by 49 | Viewed by 11768
Abstract
Mesenchymal stem cells (MSCs) have the capacity for self-renewal and multilineage differentiation potential, and are considered a promising cell population for cell-based therapy and tissue regeneration. MSCs are isolated from various organs including dental pulp, which originates from cranial neural crest-derived ectomesenchyme. Recently, [...] Read more.
Mesenchymal stem cells (MSCs) have the capacity for self-renewal and multilineage differentiation potential, and are considered a promising cell population for cell-based therapy and tissue regeneration. MSCs are isolated from various organs including dental pulp, which originates from cranial neural crest-derived ectomesenchyme. Recently, dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) have been isolated from dental pulp tissue of adult permanent teeth and deciduous teeth, respectively. Because of their MSC-like characteristics such as high growth capacity, multipotency, expression of MSC-related markers, and immunomodulatory effects, they are suggested to be an important cell source for tissue regeneration. Here, we review the features of these cells, their potential to regenerate damaged tissues, and the recently acquired understanding of their potential for clinical application in regenerative medicine. Full article
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<p>Isolation of dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs). DPSCs and SHEDs isolated from pulp tissue of permanent teeth and deciduous teeth, respectively, have the potential to be cell sources in regenerative medicine.</p> Full article ">Figure 2
<p>Self-renewal mechanisms of stem cells. Stem cells are defined by their ability to make new stem cells (self-renewal). Self-renewal of stem cells is divided into two processes: Generation of daughter cells or of cells fated to differentiate.</p> Full article ">Figure 3
<p>Multipotency of DPSCs and SHEDs. DPSCs and SHEDs can differentiate into multiple lineages such as osteoblasts, odontoblasts, adipocytes, chondrocytes, neural cells, endotheliocytes, myocytes, hepatocytes, and pancreatic cells under appropriate culture conditions. In addition, DPSCs can also differentiate into corneal epithelial cells and cardiomyocytes.</p> Full article ">Figure 4
<p>Schema of the effects of Semaphorin 3A (Sema3A) on DPSCs. In case of pulp exposure, DPSCs migrate toward the exposure site, then they proliferate and differentiate into odontoblasts to form reparative dentin. Sema3A can act as a bioactive agent to facilitate these regenerative processes. #, sealing material; *, Sema3A. The original data was published in [<a href="#B40-biology-09-00160" class="html-bibr">40</a>].</p> Full article ">
15 pages, 4839 KiB  
Article
Assessment of Cytocompatibility and Anti-Inflammatory (Inter)Actions of Genipin-Crosslinked Chitosan Powders
by Simona Dimida, Matteo Santin, Tiziano Verri, Amilcare Barca and Christian Demitri
Biology 2020, 9(7), 159; https://doi.org/10.3390/biology9070159 - 8 Jul 2020
Cited by 4 | Viewed by 3161
Abstract
Chitosan is a polysaccharide commonly used, together with its derivatives, in the preparation of hydrogel formulations, scaffolds and films for tissue engineering applications. Chitosan can be used as such, but it is commonly stabilized by means of chemical crosslinkers. Genipin is one of [...] Read more.
Chitosan is a polysaccharide commonly used, together with its derivatives, in the preparation of hydrogel formulations, scaffolds and films for tissue engineering applications. Chitosan can be used as such, but it is commonly stabilized by means of chemical crosslinkers. Genipin is one of the crosslinkers that has been considered that is a crystalline powder extracted from the fruit of Gardenia jasminoides and processed to obtain an aglycon compound. Genipin is gaining interest in biological applications because of its natural origin and anti-inflammatory actions. In this paper, the ability of chitosan-based materials crosslinked with genipin to exert anti-inflammation properties in applications such as bone regeneration was studied. Powders obtained from chitosan–genipin scaffolds have been tested in order to mimic the natural degradation processes occurring during biomaterials implantation in vivo. The results from osteoblast-like cells showed that specific combinations of chitosan and genipin stimulate high permissiveness towards cells, with higher performance than the pure chitosan. In parallel, evidences from monocyte-like cells showed that the crosslinker, genipin, seems to promote slowing of the monocyte-macrophage transition at morphological level. This suggests a sort of modularity of pro-inflammatory versus anti-inflammatory behavior of our chitosan-based biomaterials. Being both the cell types exposed to microscale powders, as an added value our results bring information on the cell–material interactions in the degradative dynamics of chitosan scaffold structures during the physiological resorption processes. Full article
(This article belongs to the Special Issue Physiological and Pathophysiological Responses to Biomaterials)
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<p>SEM morphological surface analysis of fragments from CS (<b>A</b>), GNp (<b>B</b>), GN1p (<b>C</b>) and GN2p (<b>D</b>) powders, 500× magnification.</p> Full article ">Figure 2
<p>Analysis of the viability of MG63 cells in the presence of the CS, GN1p and GN2p powders. MTT assays were performed on MG63 cells cultured in the presence of serial dilutions (1.5, 0.15 and 0.015 mg/mL) of (<b>A</b>) CS powder, (<b>B</b>) GN1p and (<b>C</b>) GN2p at different time points (24, 48 and 72 h). Data are reported as mean values from <span class="html-italic">n</span> = 6 biological replicates and are expressed as % viability (± S.E.M.) with respect to the untreated control cells (Ctr) at each time point. Statistical analysis was assessed by a Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 3
<p>Analysis of viability of MG63 cells in the presence of serial dilutions of free genipin (gn). MTT assays were performed on MG63 cells cultured in the presence of gn solutions at different time points (24, 48 and 72 h). Data are reported as mean values from <span class="html-italic">n</span> = 6 biological replicates and are expressed as % viability (± S.E.M.) with respect to the untreated control cells (Ctr) at each time point. Statistical analysis: Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 4
<p>Evaluation of cell–material interactions by imaging of MG63 cells cultured in the presence of CS and GN1p powders. HE staining and fluorescence imaging of MG63 cells grown in the presence of GN1p (<b>A</b>–<b>D</b>) and CS powder (<b>E</b>,<b>F</b>). Representative pictures after 72 h growth in the presence of 1.5 mg/mL material powders. (<b>A</b>) Bright-field image of the cells growing on a surface area surrounding a GN1p fragment. (<b>B</b>) Fluorescence image (red fluorescence) of the same fragment. (<b>C</b>,<b>D</b>) Additional images showing cell adhesion on GN1p fragments with heterogeneous shapes; pictures show high efficiency of the cell spreading onto the fragments regardless size and morphology. (<b>E</b>,<b>F</b>) Bright-field and fluorescence images of cells in the presence of a CS powder fragment (stereoscope magnification 10×; scale bar 300 µm; Nikon red fluo filter (mcherry) ex. 562/40 em. 641/75). In B, representative 300 µm × 300 µm squares are reported to depict the selection of the areas for the cell counts. In the table below: cell numbers from three different counts per biological replicate (3 different replicates) were performed; data are reported as mean values (±S.E.M.); statistical analysis: Student’s <span class="html-italic">t</span>-test; different/equal letters indicate statistically different/equal values.</p> Full article ">Figure 5
<p>Morphological analysis of THP-1 cells grown in the presence of the material powders or genipin and treated with 100 nM PMA for 48 h. (<b>A</b>) Positive control of THP-1 cells treated by the only PMA inflammatory stimulus. (<b>B</b>) PMA-treated cells + CS powder (0.15 mg/mL). (<b>C</b>) PMA-treated cells + GN1p (0.15 mg/mL); the representative picture is comparable to data not shown for the GNp and GN2p treatments. (<b>D</b>) PMA-treated cells + gn (0.022 µg/mL). MPM: monocytes pre-macrophage cells; AI: adhered stage I, cells showing low morphological complexity, with <span class="html-italic">n</span> = 1 pseudopodial elongation; AII: adhered stage II, adhered cells with <span class="html-italic">n</span> ≥ 2 pseudopodial elongations and advanced morphological complexity (Bright field images, 10× magnification, scale bar: 50 µm).</p> Full article ">Figure 6
<p>Plotting of the cell counts for the MPM, A I and A II stages. For each treatment condition, the table below reports each percentage (±S.E.M.) with respect to the total cell count (100%). Cell counts were performed in 4 different areas of the culture plate, of 2 biological replicates. Statistical analysis: Student’s <span class="html-italic">t</span>-test; different/equal letters indicate statistically different/equal values (<span class="html-italic">p</span> &lt; 0.05). MPM: monocytes pre-macrophage cells; AI: adhered stage I, cells showing low morphological complexity, with <span class="html-italic">n</span> = 1 pseudopodial elongation; AII: adhered stage II, adhered cells with <span class="html-italic">n</span> ≥ 2 pseudopodial elongations and advanced morphological complexity.</p> Full article ">
19 pages, 2620 KiB  
Article
Understanding the Morpho-Anatomical, Physiological, and Functional Response of Sweet Basil to Isosmotic Nitrate to Chloride Ratios
by Giandomenico Corrado, Luigi Formisano, Veronica De Micco, Antonio Pannico, Maria Giordano, Christophe El-Nakhel, Pasquale Chiaiese, Raffaele Sacchi and Youssef Rouphael
Biology 2020, 9(7), 158; https://doi.org/10.3390/biology9070158 - 8 Jul 2020
Cited by 13 | Viewed by 3495
Abstract
Sweet basil (Ocimum basilicum L.) is a leafy green with a short-production cycle that is emerging as a model species among aromatic plants. Modulating the mineral composition of the nutrient solution has proved to be a valuable tool to uncover the mechanisms [...] Read more.
Sweet basil (Ocimum basilicum L.) is a leafy green with a short-production cycle that is emerging as a model species among aromatic plants. Modulating the mineral composition of the nutrient solution has proved to be a valuable tool to uncover the mechanisms and responses that higher plants adopt in relation to the availability of mineral nutrients. The aim of this work was to examine the effects on basil of four isosmotic nutrient solutions with different nitrate to chloride ratios. These two anions share uptake and transport mechanisms in plants and are often considered antagonist. To this goal, we analyzed morpho-anatomical and physiological parameters as well as quality-related traits, such as the antioxidant capacity, the leaf color, the mineral composition, and the aromatic profile in relation to the nutrient ratios. Moreover, using a full factorial design, we analyzed leaves in two consecutive harvests. The data indicated a broad, multifaceted plant response to the different nutritional ratios, with almost all the recorded parameters involved. Overall, the effects on basil can be explained by considering an interdependent combination of the nitrate and chloride roles in plant nutrition and physiology. Our work revealed the extent of the modification that can be achieved in basil through the modification of the nutrient solution. It also provided indications for more nutrient efficient growing conditions, because a moderate increase in chloride limits the expected negative impact of a sub-optimal nitrate fertilization. Full article
(This article belongs to the Section Plant Science)
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<p>Principal Component Analysis of the basil response. Each condition is represented by a circle, colored according to the different NO<sub>3</sub><sup>−</sup>:Cl<sup>−</sup> ratios (20:80, 40:60, 60:40, 80:20). For each cut (CT1 and CT2), the plot displays in different colors the confidence ellipse around the mean point (not shown). The confidence level was 0.95. Color legend is reported on the right-hand side.</p> Full article ">Figure 2
<p>Main effect of the NO<sub>3</sub><sup>−</sup>:Cl<sup>−</sup> ratio in the nutrient solution in the two harvests (CT1, azure; CT2, salmon) on fresh yield of basil. Different letters indicate significant differences according to Duncan’s test (<span class="html-italic">p</span> = 0.05). All data are expressed as mean standard error, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 3
<p>Main effect of the NO<sub>3</sub><sup>−</sup>:Cl<sup>−</sup> ratios in the nutrient solution in the two harvest on nitrate content of basil (CT1: azure; CT2: salmon). Different letters indicate significant differences according to Duncan’s test (<span class="html-italic">p</span> = 0.05). All data are expressed as mean standard error, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 4
<p>Transmitted light (<b>a</b>–<b>e</b>) and epi-fluorescence (<b>f</b>–<b>h</b>) microscopy views of cross sections (<b>a</b>–<b>e</b>) and epidermal peels (<b>f</b>–<b>h</b>) of basil leaves subjected to the four different NO<sub>3</sub><sup>−</sup>:Cl<sup>−</sup> ratios in the nutrient solution (<b>a</b>, 80:20; <b>b</b>, 40:60; <b>c</b>, 60:40; <b>d</b>, 20:80). Details of glandular trichomes and stomata: (<b>e</b>,<b>f</b>,<b>g</b>) (long arrows) show peltate glands; (<b>f</b>,<b>h</b>) (short arrows) show capitate glands; long arrow in (<b>h</b>) shows a stomata. Scale bars are 100 mm in (<b>a</b>–<b>d</b>), 50 mm in (<b>e</b>–<b>h</b>).</p> Full article ">
18 pages, 2648 KiB  
Article
Role of B Cell Lymphoma 2 in the Regulation of Liver Fibrosis in miR-122 Knockout Mice
by Kun-Yu Teng, Juan M. Barajas, Peng Hu, Samson T. Jacob and Kalpana Ghoshal
Biology 2020, 9(7), 157; https://doi.org/10.3390/biology9070157 - 8 Jul 2020
Cited by 10 | Viewed by 3591
Abstract
MicroRNA-122 (miR-122) has been identified as a marker of various liver injuries, including hepatitis- virus-infection-, alcoholic-, and non-alcoholic steatohepatitis (NASH)-induced liver fibrosis. Here, we report that the extracellular miR-122 from hepatic cells can be delivered to hepatic stellate cells (HSCs) to modulate their [...] Read more.
MicroRNA-122 (miR-122) has been identified as a marker of various liver injuries, including hepatitis- virus-infection-, alcoholic-, and non-alcoholic steatohepatitis (NASH)-induced liver fibrosis. Here, we report that the extracellular miR-122 from hepatic cells can be delivered to hepatic stellate cells (HSCs) to modulate their proliferation and gene expression. Our published Argonaute crosslinking immunoprecipitation (Ago-CLIP) data identified several pro-fibrotic genes, including Ctgf, as miR-122 targets in mice livers. However, treating Ctgf as a therapeutic target failed to rescue the fibrosis developed in the miR-122 knockout livers. Alternatively, we compared the published datasets of human cirrhotic livers and miR-122 KO livers, which revealed upregulation of BCL2, suggesting its potential role in regulating fibrosis. Notably, ectopic miR-122 expression inhibited BCL2 expression in human HSC (LX-2) cells). Publicly available ChIP-seq data in human hepatocellular cancer (HepG2) cells and mice livers suggested miR-122 could regulate BCL2 expression indirectly through c-MYC, which was confirmed by siRNA-mediated depletion of c-MYC in Hepatocellular Carcinoma (HCC) cell lines. Importantly, Venetoclax, a potent BCL2 inhibitor approved for the treatment of leukemia, showed promising anti-fibrotic effects in miR-122 knockout mice. Collectively, our data demonstrate that miR-122 suppresses liver fibrosis and implicates anti-fibrotic potential of Venetoclax. Full article
(This article belongs to the Section Biochemistry and Molecular Biology)
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<p>Anti-fibrotic role of miR-122 in both mouse and human fibrotic livers, and delivery of functional miR-122 into LX-2 cells through extracellular vesicles. (<b>A</b>) The miR-122 expression in the normal liver (<span class="html-italic">n</span> = 21) and non-tumor cirrhotic liver tissues (<span class="html-italic">n</span> = 90). (<b>B</b>) Ectopic expression of miR-122 suppresses LX-2 growth (<span class="html-italic">n</span> = 3). (<b>C</b>) The expression of fibrotic genes in the miR-122-tranfected LX-2 cells (<span class="html-italic">n</span> = 3). (<b>D</b>) Hydroxyproline (Hy:Pro) level in the CCl4-injected wild-type (WT) and miR-122 KO mice livers (<span class="html-italic">n</span> = 5). (<b>E</b>) Liver histology of the CCl4-treated WT and miR-122 KO mice (scale bar: 100 µm). (<b>F</b>) A schematic representation of the co-culture experiment involving LX-2 cells and control (empty vector-transfected) or miR-122 expression-vector-transfected HCC cells. (<b>G</b>) Mature miR-122 was measured by RT-qPCR in the purified extracellular vesicles from the co-cultured media shown in <a href="#biology-09-00157-f001" class="html-fig">Figure 1</a>F (<span class="html-italic">n</span> = 3). (<b>H</b>) RT-qPCR data in LX-2 cells co-cultured with the lentivirus-carrying empty vector-infected HCC (PLC/PRF5 (Vec-PLC and HCC-LM3 (Vec-LM3) or miR-122 expression-vector-infected HCC (PLC/PRF5 (122-PLC) and HCC-LM3 (122-LM3)) cell lines. Note: * <span class="html-italic">p</span> &lt; 0.05, Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 2
<p>Argonaute-crosslinking immunoprecipitation (Ago-CLIP) analysis identified several pro-fibrotic targets of miR-122. (<b>A</b>) Examples of effects of Ago-CLIP-identified miR-122 seed sequence on its targets, Ctgf and Pdgfrβ. Note that the miR-122-dependent binding peaks are presented (highlighted in light blue) in the WT but not in KO liver. The arrows on the mRNA sequence indicate the directions of gene transcription. The solid and broken lines denote exons and introns, respectively. (<b>B</b>) The miR-122 binding site on the Ctgf 5′UTR and Pdgfrβ CDS. (<b>C</b>) Reciprocal expression of Ctgf and miR-122 in the primary KO mouse hepatocytes transfected with miR-122 mimic or NC mimic (KO hepatocytes) and WT hepatocytes transfected with anti-sense miR-122 or anti-sense NC (<span class="html-italic">n</span> = 3). NC-S: negative control for miR-122 sense; 122-S: miR-122 sense; NC-AS: negative control for miR-122 anti-sense; 122-AS: miR-122 anti-sense. (<b>D</b>,<b>E</b>) The miR-122 wild-type (WT) and mutant sequences in the 5′UTR of Ctgf cloned into psi-Check2 vector (<b>D</b>); each construct was co-transfected with either scramble RNA (NC-S) or miR-122 mimic (122-S) (<span class="html-italic">n</span> = 3) and luciferase (Renilla and firefly) activities were measured after 48 h (E). Data are presented as the fold change of the <span class="html-italic">Renilla</span> to firefly luciferase. Note: * <span class="html-italic">p</span> &lt; 0.05, Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 3
<p>BCL2 is upregulated in both human and mouse fibrotic livers. (<b>A</b>) Overexpressed pro-fibrotic genes in the cirrhosis patients (red) overlay with those identified in the miR-122 KO mouse liver (blue). The <span class="html-italic">p</span>-value &lt; = 0.001 was the cut off. (<b>B</b>) BCL2 expression in HBV-infection-induced cirrhotic liver tissues. Normal group: both inflammation and fibrosis scores = 0 (<span class="html-italic">n</span> = 26); cirrhosis group: fibrosis scores &gt; = 1 (<span class="html-italic">n</span> = 81). (<b>C</b>) BCL2 expression in normal liver (<span class="html-italic">n</span> = 19) and HCV-infection-induced cirrhotic liver tissues (<span class="html-italic">n</span> = 41). (<b>D</b>) Correlations between miR-122 and BCL2 expression (Pearson’s correlation; intercept = 18.179; slope = -0.62463) in The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC) with fibrotic livers (<span class="html-italic">n</span> = 292). (<b>E</b>) BCL2 expression determined from the RNA-seq data of wild-type (WT, <span class="html-italic">n</span> = 5) and miR-122 KO (<span class="html-italic">n</span> = 4) mice livers. (<b>F</b>) BCL2 expression determined from the microarray data of the wild-type (WT, <span class="html-italic">n</span> = 4) and miR-122 LKO (<span class="html-italic">n</span> = 4) mice livers. (<b>G</b>) BCL2 protein level in the liver lysates of WT (<span class="html-italic">n</span> = 3) and miR-122 liver-specific knockout (LKO) mouse (<span class="html-italic">n</span> = 3) at the age of 15 weeks. BCL2 levels were normalized to that of GAPDH and the value was indicated. Quantification was done by ImageJ (<a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a>). Note: * <span class="html-italic">p</span> &lt; 0.05, Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 4
<p>The miR-122 represses <span class="html-italic">BCL2</span> transcription through c-MYC. (<b>A</b>) The expression of <span class="html-italic">BCL2</span> mRNA (<b>A</b>) and hnRNA (<b>B</b>) in the miR-122-transfected LX-2 cells (<span class="html-italic">n</span> = 3). (<b>C</b>) ChIP-seq data of RNA polymerase II (POL II), H3K4me3, and c-MYC in the proximal promoter region of <span class="html-italic">BCL2</span> gene in the human HCC cell line HepG2 (accession numbers GSM822291 and GSM733737) and in the mouse liver (<b>D</b>) (accession number GSE76078). (<b>E</b>) BCL2 protein levels in the LX-2 cells transfected with either negative control RNA (Si-NC) or c-MYC siRNA (si-c-MYC). Vinculin was used as a loading control. (<b>F</b>) The expression of <span class="html-italic">c-MYC</span> mRNA, <span class="html-italic">BCL2</span> mRNA, and <span class="html-italic">BCL2</span> hnRNA (primary transcript) in the c-MYC siRNA-transfected LX-2 cells (<span class="html-italic">n</span> = 3). Note: * <span class="html-italic">p</span> &lt; 0.05, Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 5
<p>Venetoclax, a BCL2 selective inhibitor, suppresses hepatic stellate cell (HSC) activation. (<b>A</b>) Representative microscopic pictures (scale bar: 25 µm) of LX-2 cells treated with Venetoclax (10 µM) at 0 and 72 h (200× magnification). (<b>B</b>) Survival of LX-2 cell treated with different concentrations of Venetoclax for 72 h was measured by CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay (<span class="html-italic">n</span> = 3). Survival at 0 h was arbitrarily assigned a value of 1 and the survival in drug-treated cells compared to the vehicle-treated cells at each drug concentrations were plotted. (<b>C</b>) A schematic of Venetoclax treatment of miR-122 KO mice that spontaneously developed massive liver fibrosis at 32 weeks. (<b>D</b>) Representative Sirius red- stained pictures of 32-week-old KO liver (400× magnification; scale bar: 25 µm). (<b>E</b>) H&amp;E and Sirius red staining of the vehicle and Venetoclax-treated mice liver sections (scale bar, 250 µm; inset, 25 µm). (<b>F</b>) Quantification of the H&amp;E and Sirius red staining data in <a href="#biology-09-00157-f005" class="html-fig">Figure 5</a>E. Quantification was done by ImageJ (<a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a>) of three randomly chosen fields (100× magnification) per animal (<span class="html-italic">n</span> = 5). (<b>G</b>) <span class="html-italic">Col1a1</span> and <span class="html-italic">Acta2</span> mRNAs in the liver were measured by RT-qPCR (<span class="html-italic">n</span> = 5). (<b>H</b>) Hydroxyproline levels were measured in the vehicle and Venetoclax-treated mice liver tissue extracts. Hydroxyproline contents (µg) were normalized to the respective liver tissue weights (g) used in the assay (<span class="html-italic">n</span> = 5). Note: * <span class="html-italic">p</span> &lt; 0.05, Student’s <span class="html-italic">t</span>-test.</p> Full article ">Figure 6
<p>A model depicting the anti-fibrotic role of miR-122 in the liver. In healthy livers, miR-122 could be delivered to HSCs to inhibit the expression of fibrogenic BCL2 through suppressing c-MYC. When the miR-122 level goes down, c-MYC is de-repressed in the HSCs. Elevation of c-MYC enhances BCL2 transcription and increases the anti-apoptotic capability of HSCs.</p> Full article ">
13 pages, 1419 KiB  
Article
CoVid-19 Pandemic Trend Modeling and Analysis to Support Resilience Decision-Making
by Romney B. Duffey and Enrico Zio
Biology 2020, 9(7), 156; https://doi.org/10.3390/biology9070156 - 7 Jul 2020
Cited by 10 | Viewed by 4138
Abstract
Policy decision-making for system resilience to a hazard requires the estimation and prediction of the trends of growth and decline of the impacts of the hazard. With focus on the recent worldwide spread of CoVid-19, we take the infection rate as the relevant [...] Read more.
Policy decision-making for system resilience to a hazard requires the estimation and prediction of the trends of growth and decline of the impacts of the hazard. With focus on the recent worldwide spread of CoVid-19, we take the infection rate as the relevant metric whose trend of evolution to follow for verifying the effectiveness of the countermeasures applied. By comparison with the theories of growth and recovery in coupled socio-medical systems, we find that the data for many countries show infection rate trends that are exponential in form. In particular, the recovery trajectory is universal in trend and consistent with the learning theory, which allows for predictions useful in the assistance of decision-making of emergency recovery actions. The findings are validated by extensive data and comparison to medical pandemic models. Full article
(This article belongs to the Special Issue Coronavirus Disease 2019 (COVID-19))
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<p>Typical infection rate trend illustrating the initial growth of the peak, <span class="html-italic">R<sub>M</sub></span>, and desired decline to some attainable minimum, <span class="html-italic">R<sub>m</sub></span> (CoVid-19 data for Italy).</p> Full article ">Figure 2
<p>The universal recovery curve.</p> Full article ">Figure 3
<p>Comparison of the universal recovery curve (URC) to the model predictions of required hospital beds by IHME.</p> Full article ">Figure 4
<p>These approximately 7-day periodic peaks and troughs in the infection rate show clear minima occurring every weekend and broad peaks in the week (Source: data for New York City accessed at <a href="http://www1.nyc.gov/site/doh/covid/covid-19-data" target="_blank">www1.nyc.gov/site/doh/covid/covid-19-data</a>).</p> Full article ">
25 pages, 2405 KiB  
Article
The Importance of Protein Phosphorylation for Signaling and Metabolism in Response to Diel Light Cycling and Nutrient Availability in a Marine Diatom
by Maxine H. Tan, Sarah R. Smith, Kim K. Hixson, Justin Tan, James K. McCarthy, Adam B. Kustka and Andrew E. Allen
Biology 2020, 9(7), 155; https://doi.org/10.3390/biology9070155 - 6 Jul 2020
Cited by 5 | Viewed by 4154
Abstract
Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and [...] Read more.
Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms. Full article
(This article belongs to the Special Issue The Molecular Life of Diatoms: From Genes to Ecosystems)
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<p>Summary of the phosphoproteome of <span class="html-italic">P. tricornutum</span>. (<b>a</b>) Proportions of detected phosphoproteins in each KOG class (* indicate significantly enriched groups, ^ indicate significantly depleted groups). Proteins related to signal transduction mechanisms and RNA processing/modification were significantly enriched for phosphorylation. (<b>b</b>) The majority of phosphoproteins (76%) had one or two phosphorylation sites. Highly phosphorylated phosphoproteins (&gt;5 p-sites) was 5.6% of the total phosphoproteins. (<b>c</b>) This study detected 2759 p-sites on 1502 proteins, most of which were not previously detected by Chen et al. [<a href="#B30-biology-09-00155" class="html-bibr">30</a>] in <span class="html-italic">P. tricornutum</span>. (<b>d</b>) PCA shows that phosphorylation in <span class="html-italic">P. tricornutum</span> is affected by nutrient availability and culturing environment. (<b>e</b>) Unique phosphopeptides detected during low (<span class="html-italic">n</span> = 76), medium (<span class="html-italic">n</span> = 50) and high (<span class="html-italic">n</span> = 101) Fe treatments or in Nshort experiments (<span class="html-italic">n</span> = 712) are likely candidates controlling cellular response and environmental sensing. Constitutively phosphorylated phosphoproteins (<span class="html-italic">n</span> = 80) are likely required for core cellular functions.</p> Full article ">Figure 2
<p>Division of phosphoproteome into conditional subsets. (<b>a</b>) Patterns of conditional phosphorylation compared to total protein expression as a response of <span class="html-italic">P. tricornutum</span> to low Fe (LowFe-P), diel light cycling (Diel-P), and N availability (Nshort-P) consisted of 10%, 15% and 59% of the phosphoproteome respectively. (<b>b</b>) Breakdown of LowFe-P subset by pattern. Diel-P Fe treatments were averaged by timepoint to highlight the phosphorylation changes due to the diel cycle. Similarly, to illustrate the response to cellular N status, Nshort-P nitrogen treatments were averaged and log2FC was calculated between timepoints. Normalized phosphopeptide abundances were hierarchically clustered.</p> Full article ">Figure 3
<p>Phosphorylation of carbon metabolism gene products suggests a role for phospho-regulation in directing carbon into fatty acid biosynthesis. Phosphoproteins were identified to be inactivated at 6 AM but reactivated at 10 AM (green, bold), inactivated at both 6 AM and 10 AM (pink, bold) or undetermined (black, bold). Diel profiles normalized across the time course (z score) indicate phospho-regulation as the change in phosphorylation (blue line) is not proportional to the change in total protein abundance (orange line) between timepoints. Chloroplast phosphoglycerate kinase (PGK; Phatr3_J29157); chloroplast glyceraldehyde 3-phosphate dehydrogenase (GAPC; Phatr3_J22122); chloroplast pyruvate orthophosphate dikinase (PPDK; Phatr3_J21988); cytoplasmic acetyl-CoA carboxylase (ACC; Phatr3_J55209), mitochondrial NAD-dependent malate dehydrogenase (MDH; Phatr3_J42398). Refer to <a href="#app1-biology-09-00155" class="html-app">Table S7</a> for a summary of the possible effect of phosphorylation on these phosphoproteins.</p> Full article ">Figure 4
<p>Phosphoproteins which are impacted by cellular nitrogen status. Transcripts (grey) [<a href="#B18-biology-09-00155" class="html-bibr">18</a>], protein (orange) and phosphoprotein (blue line) levels are depicted. Phosphoproteins were classified into N replete (pink), N deplete (blue), or N transition (black) clusters by hierarchical clustering. Significant fold change (log2 FC &gt; 1) in phosphopeptide abundance between timepoints are indicated by an asterisk on the graphs. Phosphofructokinase (PFK; Phatr3_EG02209), nitrate/nitrite transporter (NRT2; Phatr3_J2171), ubiquitin carboxyl-terminal hydrolase (UCH, Phatr3_J42729), ubiquitin E3 ligase (UbE3; Phatr3_EG01549), Tyr-tRNA ligase (Phatr3_EG02310), heat shock protein 90 (HSP90; Phatr3_J55230), Ca2+/calmodulin-dependent protein kinase (CaMK; Phatr3_EG02294), calcium dependent protein kinase (CDPK; Phatr3_J25067), serine/threonine kinase (MARK3; Phatr3_J8773), inositol hexakisphosphate/diphosphoinositol-pentakisphosphate kinase (InsP6/PP-IP5K; Phatr3_J46684), 40S small ribosomal subunit 20 (40S20, Phatr3_J51291), translation initiation factor eIF2 gamma (eIF2y; Phatr3_J42307), gamma-aminobutyric acid type B receptor (GABA; Phatr3_Jdraft1756), phosphoenolpyruvate carboxykinase (PEPCK; Phatr3_EG02232), NAD-glutamate dehydrogenase (GDH2; Phatr3_J45239), manganese superoxide dismutase (MnSOD; Phatr3_J42832), cytochrome C oxidase subunit 6b (COX6B; Phatr3_J11016), ammonium-dependent carbamoyl-phosphate synthetase (unCPS; Phatr3_J24195), transcription initiation factor TFIID subunit BDF1 (Phatr3_J44399), CCR-associated factor 1 (CAF1; Phatr3_J9576), histone deacetylase (Phatr3_J13057), centromere (Phatr3_J43019), sister chromatid cohesion protein (Phatr3_Jdraft1590), cytoplasmic glutamine-dependent carbamoyl phosphate synthetase (pgCPS; Phatr3_EG01947), ammonium transporter (AMT1; Phatr3_J27877), urea symporter (DUR3; Phatr3_J20424). Refer to <a href="#app1-biology-09-00155" class="html-app">Table S7</a> for a summary of the possible effect of phosphorylation on these phosphoproteins.</p> Full article ">
11 pages, 11964 KiB  
Article
Protective Effects of Bee Venom against Endotoxemia-Related Acute Kidney Injury in Mice
by Jung-Yeon Kim, Sun-Jae Lee, Young-In Maeng, Jaechan Leem and Kwan-Kyu Park
Biology 2020, 9(7), 154; https://doi.org/10.3390/biology9070154 - 6 Jul 2020
Cited by 21 | Viewed by 3983
Abstract
Sepsis-associated acute kidney injury (AKI) is a leading cause of death in hospitalized patients worldwide. Despite decades of effort, there is no effective treatment for preventing the serious medical condition. Bee venom has long been used to treat a variety of inflammatory diseases. [...] Read more.
Sepsis-associated acute kidney injury (AKI) is a leading cause of death in hospitalized patients worldwide. Despite decades of effort, there is no effective treatment for preventing the serious medical condition. Bee venom has long been used to treat a variety of inflammatory diseases. However, whether bee venom has protective effects against lipopolysaccharide (LPS)-induced AKI has not been explored. The aim of this study was to evaluate the effects of bee venom on LPS-induced AKI. The administration of bee venom alleviated renal dysfunction and structural injury in LPS-treated mice. Increased renal levels of tubular injury markers after LPS treatment were also suppressed by bee venom. Mechanistically, bee venom significantly reduced plasma and tissue levels of inflammatory cytokines and immune cell infiltration into damaged kidneys. In addition, mice treated with bee venom exhibited reduced renal expression of lipid peroxidation markers after LPS injection. Moreover, bee venom attenuated tubular cell apoptosis in the kidneys of LPS-treated mice. In conclusion, these results suggest that bee venom attenuates LPS-induced renal dysfunction and structural injury via the suppression of inflammation, oxidative stress, and tubular cell apoptosis, and might be a useful therapeutic option for preventing endotoxemia-related AKI. Full article
(This article belongs to the Section Medical Biology)
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<p>Bee venom alleviated renal dysfunction in mice treated with lipopolysaccharide (LPS). Mice received a single intraperitoneal injection of bee venom (100 μg/kg body weight) 1 h before injection of LPS (10 mg/kg body weight). (<b>A</b>) Plasma creatinine. (<b>B</b>) Plasma blood urea nitrogen (BUN). ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. NS, not significant.</p> Full article ">Figure 2
<p>Bee venom ameliorated histological abnormalities in LPS-treated mice. (<b>A</b>) Hematoxylin and eosin (H&amp;E) and periodic acid Schiff (PAS) staining of kidneys sections. Bar = 100 μm. (<b>B</b>) Tubular injury score. (<b>C</b>) Lotus tetragonolobus lectin (LTL) staining of kidneys sections. Bar = 50 μm. (<b>D</b>) Percentage of LTL-positive area per field. <span class="html-italic">n</span> = 8 per group. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. NS, not significant.</p> Full article ">Figure 3
<p>Bee venom attenuated LPS-induced tubular injury. (<b>A</b>) Immunohistochemical (IHC) staining of kidney sections using antibodies against neutrophil gelatinase-associated lipocalin (NGAL) or kidney injury molecule-1 (Kim-1). Bar = 100 μm. (<b>B</b>) Percentage of NGAL-positive area. (<b>C</b>) Percentage of Kim-1-positive area. (<b>D</b>) Representative images of Western blotting of NGAL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in kidneys. <span class="html-italic">n</span> = 8 per group. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. NS, not significant.</p> Full article ">Figure 4
<p>Bee venom reduced plasma and tissue levels of inflammatory cytokines in LPS-treated mice. (<b>A</b>) Plasma tumor necrosis factor-α (TNF-α). (<b>B</b>) Plasma interleukin-6 (IL-6). (<b>C</b>) Representative images of Western blotting of TNF-α, IL-6, and GAPDH in kidneys. <span class="html-italic">n</span> = 8 per group. * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Bee venom prevented immune cell infiltration into damaged kidneys in LPS-treated mice. (<b>A</b>) IHC staining of kidney sections using antibodies against Mac-2 or CD4. Bar = 100 μm. (<b>B</b>) Number of Mac-2-positive cells. (<b>C</b>) Number of CD4-positive cells. <span class="html-italic">n</span> = 8 per group. * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Bee venom attenuated oxidative stress in LPS-treated mice. (<b>A</b>) IHC staining of kidney sections using an antibody against 4-hydroxynonenal (4-HNE). Bar = 100 μm. (<b>B</b>) Percentage of 4-HNE-positive area per field. (<b>C</b>) Renal levels of malondialdehyde (MDA). <span class="html-italic">n</span> = 8 per group. *** <span class="html-italic">p</span> &lt; 0.001. NS, not significant.</p> Full article ">Figure 7
<p>Bee venom reduced tubular cell apoptosis in LPS-treated mice. (<b>A</b>) Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining in kidneys. Bar = 100 μm. (<b>B</b>) Number of TUNEL-positive cells. (<b>C</b>) Representative images of Western blotting of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase-1 (PARP-1), p53, and GAPDH in kidneys. <span class="html-italic">n</span> = 8 per group. *** <span class="html-italic">p</span> &lt; 0.001. NS, not significant.</p> Full article ">
20 pages, 1500 KiB  
Article
A First Insight into North American Plant Pathogenic Fungi Armillaria sinapina Transcriptome
by Narimene Fradj, Nicolas de Montigny, Natacha Mérindol, Fatima Awwad, Yacine Boumghar, Hugo Germain and Isabel Desgagné-Penix
Biology 2020, 9(7), 153; https://doi.org/10.3390/biology9070153 - 4 Jul 2020
Cited by 7 | Viewed by 3592
Abstract
Armillaria sinapina, a fungal pathogen of primary timber species of North American forests, causes white root rot disease that ultimately kills the trees. A more detailed understanding of the molecular mechanisms underlying this illness will support future developments on disease resistance and [...] Read more.
Armillaria sinapina, a fungal pathogen of primary timber species of North American forests, causes white root rot disease that ultimately kills the trees. A more detailed understanding of the molecular mechanisms underlying this illness will support future developments on disease resistance and management, as well as in the decomposition of cellulosic material for further use. In this study, RNA-Seq technology was used to compare the transcriptome profiles of A. sinapina fungal culture grown in yeast malt broth medium supplemented or not with betulin, a natural compound of the terpenoid group found in abundance in white birch bark. This was done to identify enzyme transcripts involved in the metabolism (redox reaction) of betulin into betulinic acid, a potent anticancer drug. De novo assembly and characterization of A. sinapina transcriptome was performed using Illumina technology. A total of 170,592,464 reads were generated, then 273,561 transcripts were characterized. Approximately, 53% of transcripts could be identified using public databases with several metabolic pathways represented. A total of 11 transcripts involved in terpenoid biosynthesis were identified. In addition, 25 gene transcripts that could play a significant role in lignin degradation were uncovered, as well as several redox enzymes of the cytochromes P450 family. To our knowledge, this research is the first transcriptomic study carried out on A. sinapina. Full article
(This article belongs to the Section Plant Science)
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<p>Sequence length distribution of transcripts of <span class="html-italic">A. sinapina</span> after Trinity de novo assembly (<b>A</b>; N50 = 3523bp) and after BLAST annotation and filtered annotated components (<b>B</b>; N50 = 5081bp).</p> Full article ">Figure 2
<p>Gene Ontology (GO) terms of 35 functional groups of expressed transcripts from <span class="html-italic">Armillaria sinapina.</span></p> Full article ">Figure 3
<p>Functional classification of <span class="html-italic">Armillaria sinapina</span> transcriptome using the Cluster of orthologous groups (CoG).</p> Full article ">Figure 4
<p>Annotation of the <span class="html-italic">Armillaria sinapina</span> putative proteins using the Kyoto encyclopedia of genes and genomes (KEGG).</p> Full article ">Figure 5
<p>Proposed biosynthetic pathway leading to terpenoids in <span class="html-italic">A. sinapina</span>. Enzymes, for which corresponding gene transcripts were identified in this study, are shown in bold black, whereas the ones not found in our transcriptome are shown in bold grey. A broken arrow represents more than one biochemical reaction. Abbreviations: HMG-CoA—3-hydroxy-3-methylglutaryl-CoA; MVA—mevalonic acid; MVAP—MVA phosphate; MVAPP—MVA diphosphate; IPP—isopentenyl diphosphate; DMAPP—dimethylallyl diphosphate; GPP—geranyl diphosphate; FPP—farnesyl diphosphate; THIK—3-ketoacyl-CoA thiolase; HMGL—HMG lyase; HMGR—HMG-CoA reductase; MVK—MVA kinase; PMK—MVAP kinase; PMD—MVAPP decarboxylase; IPI—IPP isomerase; GPS—GPP synthase; FPS—FPP synthase; MUS—muurolene synthase; TRI4—trichodiene oxygenase; PRS—protoilludene synthase; SQS—squalene synthase; SQE—squalene epoxidase; AS—amyrin synthase; AO—amyrin oxidase; LAS—lanosterol synthase; LUS—lupeol synthase.</p> Full article ">
18 pages, 1766 KiB  
Review
Regulation of the Mammalian SWI/SNF Family of Chromatin Remodeling Enzymes by Phosphorylation during Myogenesis
by Teresita Padilla-Benavides, Pablo Reyes-Gutierrez and Anthony N. Imbalzano
Biology 2020, 9(7), 152; https://doi.org/10.3390/biology9070152 - 3 Jul 2020
Cited by 3 | Viewed by 5586
Abstract
Myogenesis is the biological process by which skeletal muscle tissue forms. Regulation of myogenesis involves a variety of conventional, epigenetic, and epigenomic mechanisms that control chromatin remodeling, DNA methylation, histone modification, and activation of transcription factors. Chromatin remodeling enzymes utilize ATP hydrolysis to [...] Read more.
Myogenesis is the biological process by which skeletal muscle tissue forms. Regulation of myogenesis involves a variety of conventional, epigenetic, and epigenomic mechanisms that control chromatin remodeling, DNA methylation, histone modification, and activation of transcription factors. Chromatin remodeling enzymes utilize ATP hydrolysis to alter nucleosome structure and/or positioning. The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) family of chromatin remodeling enzymes is essential for myogenesis. Here we review diverse and novel mechanisms of regulation of mSWI/SNF enzymes by kinases and phosphatases. The integration of classic signaling pathways with chromatin remodeling enzyme function impacts myoblast viability and proliferation as well as differentiation. Regulated processes include the assembly of the mSWI/SNF enzyme complex, choice of subunits to be incorporated into the complex, and sub-nuclear localization of enzyme subunits. Together these processes influence the chromatin remodeling and gene expression events that control myoblast function and the induction of tissue-specific genes during differentiation. Full article
(This article belongs to the Special Issue ATP-dependent Chromatin Remodeler)
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<p>P38 has pleiotropic effects during skeletal muscle differentiation. Phosphorylation of transcription factors (TFs) (E-proteins, Mef2) and the mSWI/SNF subunit BAF60c by p38 enables expression of myogenic genes and differentiation. P38-dependent phosphorylation of Ezh2 downregulates the expression of <span class="html-italic">Pax7</span>. P38 also induces cell cycle withdrawal by activating c-Jun N-terminal kinase (JNK) and Cyclin D1.</p> Full article ">Figure 2
<p>Casein kinase 2 (CK2) modulates mSWI/SNF activity in proliferating myoblasts. CK2-dependent phosphorylation of Brahma related gene 1 (BRG1) regulates the sub-nuclear localization and the subunit composition of the mSWI/SNF complex, viability and cell cycle progression, and the ability to remodel promoter chromatin and promote gene expression that allows myoblast proliferation. The diagram shows the presence or absence of phosphorylation of BRG1 for illustrative purposes. The exact number of phosphorylation sites in the presence of the different mSWI/SNF subunits has not been determined.</p> Full article ">Figure 3
<p>Antagonistic roles of PKCβ<sub>1</sub> and calcineurin in myogenesis. PKCβ<sub>1</sub> interacts with and phosphorylates BRG1 to block differentiation. Upon myogenic stimulus, calcineurin dephosphorylates BRG1 and the TF nuclear factor of activated T-cell (NFAT), enabling chromatin remodeling at myogenic loci, myogenic gene expression, and skeletal muscle differentiation.</p> Full article ">Figure 4
<p>Kinases and phosphatases that modulate the chromatin remodeling activity of the mSWI/SNF complex during myogenesis. Schematic representation of post-natal myogenesis and indications of when specific kinases and phosphatases that control the activity of the mSWI/SNF complex act.</p> Full article ">
9 pages, 3517 KiB  
Article
A Selective PPARγ Modulator Reduces Hepatic Fibrosis
by Benita L. McVicker, Frederick G. Hamel, Ronda L. Simpson and Robert G. Bennett
Biology 2020, 9(7), 151; https://doi.org/10.3390/biology9070151 - 2 Jul 2020
Cited by 6 | Viewed by 2803
Abstract
Hepatic fibrosis is the accumulation of excess collagen as a result of chronic liver injury. If left unabated, hepatic fibrosis can lead to the disruption of the liver architecture, portal hypertension, and increased risk of progression to cirrhosis and hepatocellular carcinoma. The thiazolidinedione [...] Read more.
Hepatic fibrosis is the accumulation of excess collagen as a result of chronic liver injury. If left unabated, hepatic fibrosis can lead to the disruption of the liver architecture, portal hypertension, and increased risk of progression to cirrhosis and hepatocellular carcinoma. The thiazolidinedione class of antidiabetic drugs, through their target peroxisome proliferator-activated receptor γ (PPARγ), have protective effects against liver fibrosis, and can inhibit the profibrotic activity of hepatic stellate cells, the major collagen-producing liver cells. However, these drugs have been ineffective in the treatment of established fibrosis, possibly due to side effects such as increased weight and adiposity. Recently, selective PPARγ modulators that lack these side effects have been identified, but their role in treating fibrosis has not been studied. In this study, we tested the effectiveness of one of these selective modulators, SR1664, in the mouse carbon tetrachloride model of established hepatic fibrosis. Treatment with SR1664 reduced the total and type 1 collagen content without increasing body weight. The abundance of activated hepatic stellate cells was also significantly decreased. Finally, SR1664 inhibited the profibrotic phenotype of hepatic stellate cells. In summary, a selective PPARγ modulator was effective in the reduction of established hepatic fibrosis and the activated phenotype of hepatic stellate cells. This may represent a new treatment approach for hepatic fibrosis. Full article
(This article belongs to the Special Issue Non-alcoholic Liver Injury)
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<p>Serum determinations and histology from control or fibrotic mice treated with SR1664 or vehicle. (<b>A</b>) Serum adiponectin levels; (<b>B</b>,<b>C</b>) Serum ALT and AST levels; (<b>D</b>) Total body weight; (<b>E</b>) Liver weight expressed as percent total body weight; (<b>F</b>) Hematoxylin and eosin staining of liver sections. Bar = 200 µm; mean ± S.E.M; <span class="html-italic">n</span> = 6; * <span class="html-italic">p</span> &lt; 0.05 by ANOVA; ns: not significant.</p> Full article ">Figure 2
<p>Collagen deposition in liver from control or fibrotic mice treated with SR1664 or vehicle. (<b>A</b>) Sirius red stain of total collagen (top panel) or immunohistochemistry for type 1 collagen (bottom panel); bar = 200 µm. (<b>B</b>) Quantification of Sirius red staining intensity.(<b>C</b>) Quantification of type 1 collagen staining. (<b>D</b>) Liver tissue hydroxyproline content. Mean ± S.E.M.; <span class="html-italic">n</span> = 6; * <span class="html-italic">p</span> &lt; 0.05 by ANOVA; ns: not significant.</p> Full article ">Figure 3
<p>Smooth muscle α-actin (SMA) content in liver from control or fibrotic mice treated with SR1664 or vehicle. Immunohistochemistry for SMA (left panel); quantification of SMA staining intensity (right panel); bar = 200 µm; mean ± S.E.M.; <span class="html-italic">n</span> = 6; * <span class="html-italic">p</span> &lt; 0.05 by ANOVA.</p> Full article ">Figure 4
<p>SR1664 had direct effects on hepatic stellate cells. (<b>A</b>) Gene expression in LX-2 cells treated with SR1664 or vehicle; (<b>B</b>) Western blot showing levels of SMA or GAPDH in LX-2 cells treated with vehicle or SR1664; (<b>C</b>) LX-2 cell proliferation in response to PDGF in the presence or absence of SR1664; mean ± S.E.M.; <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> &lt; 0.05 compared with vehicle-treated cells; # <span class="html-italic">p</span> &lt; 0.05 compared with vehicle or PDGF-treated cells. Significance determined by Student’s <span class="html-italic">t</span>-test (A) or ANOVA (C).</p> Full article ">
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