Cells

21 pages, 6314 KiB

Open AccessReview

Mechanisms of Activation of Brain’s Drainage during Sleep: The Nightlife of Astrocytes

by Dmitry Postnov, Oxana Semyachkina-Glushkovskaya, Elena Litvinenko, Jürgen Kurths and Thomas Penzel

Cells 2023, 12(22), 2667; https://doi.org/10.3390/cells12222667 - 20 Nov 2023

Cited by 5 | Viewed by 2163

Abstract

The study of functions, mechanisms of generation, and pathways of movement of cerebral fluids has a long history, but the last decade has been especially productive. The proposed glymphatic hypothesis, which suggests a mechanism of the brain waste removal system (BWRS), caused an [...] Read more.

The study of functions, mechanisms of generation, and pathways of movement of cerebral fluids has a long history, but the last decade has been especially productive. The proposed glymphatic hypothesis, which suggests a mechanism of the brain waste removal system (BWRS), caused an active discussion on both the criticism of some of the perspectives and our intensive study of new experimental facts. It was especially found that the intensity of the metabolite clearance changes significantly during the transition between sleep and wakefulness. Interestingly, at the cellular level, a number of aspects of this problem have been focused on, such as astrocytes–glial cells, which, over the past two decades, have been recognized as equal partners of neurons and perform many important functions. In particular, an important role was assigned to astrocytes within the framework of the glymphatic hypothesis. In this review, we return to the “astrocytocentric” view of the BWRS function and the explanation of its activation during sleep from the viewpoint of new findings over the last decade. Our main conclusion is that the BWRS’s action may be analyzed both at the systemic (whole-brain) and at the local (cellular) level. The local level means here that the neuro-glial-vascular unit can also be regarded as the smallest functional unit of sleep, and therefore, the smallest functional unit of the BWRS. Full article

(This article belongs to the Special Issue The Emerging Role of Astrocytes in Health and Neurological Diseases)

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Graphical abstract

Graphical abstract
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The changes in activity of the brain waste removal system (BWRS) during sleep and wakefulness: (a) photo of real-time multiphoton monitoring of BWRS in non-anesthetized mouse under EEG control. (b,c) Representative images of real-time multiphoton microscopy of fluorescein isothiocyanate-dextran (FITCD, green) distribution in perivascular spaces (PVSs) surrounding the cerebral vessels filled with Evans Blue dye (EBD, red) after its injection into the right lateral ventricle in awake (b) and sleeping (c) male mouse under EEG control. During wakefulness, PVSs are not filled with FITCD and appear empty. However, during sleep, PVSs are completely filled with FITCD. (d,e) Representative ex vivo confocal images of FITCD distribution in the brain after its injection into the right lateral ventricle in awake (d) and sleeping (e) mice. The intensity of fluorescent signal from FITCD is higher in sleeping vs. waking brain. (f) Schematic illustration of changes in PVS size during wakefulness and sleep. Full article ">Figure 2
Simplified chart of pathways according to [<a href="#B163-cells-12-02667" class="html-bibr">163</a>]. Glutamate spillover reaches both astrocyte (1) and closely located noradrenaline (NA) varicosite (2). Released NA activates presynaptic <math display="inline"><semantics> <mi>β</mi> </semantics></math>-adrenoreceptors (3) and thus promotes further glutamate release. When astrocyte reaches membrane, NA cooperates with glutamate to activate calcium response via inositol triphosphate (IP3) production (4). Activated astrocyte releases gliotransmitters, including D-serine, which is a co-agonist of N-methyl-D-aspartate (NMDA) receptors at NA varicosite (5). This, in turn, promotes further NE release. Autoreceptors at NA varicosities inhibit its release at low levels but amplify it at high levels and thus serve as neural gain amplifiers (6). Full article ">Figure 3
Extracellular space (ECS) fraction and molecular transport, physical background. (a) The diffusion flux is proportional to the channel cross-section if the concentration difference is constant. However, if the amount of substance is constant, then increasing the volume of the channel will not increase the flow. (b) The flow during advection (transport of particles by liquid) depends on the shape of the cross-section of the channels. (c) The specific choice of the approximating method is determined. Full article ">

20 pages, 4444 KiB

Open AccessArticle

Oral Administration of Rhamnan Sulfate from Monostroma nitidum Suppresses Atherosclerosis in ApoE-Deficient Mice Fed a High-Fat Diet

by Masahiro Terasawa, Liqing Zang, Keiichi Hiramoto, Yasuhito Shimada, Mari Mitsunaka, Ryota Uchida, Kaoru Nishiura, Koichi Matsuda, Norihiro Nishimura and Koji Suzuki

Cells 2023, 12(22), 2666; https://doi.org/10.3390/cells12222666 - 20 Nov 2023

Cited by 2 | Viewed by 2624

Abstract

Oral administration of rhamnan sulfate (RS), derived from the seaweed Monostroma nitidum, markedly suppresses inflammatory damage in the vascular endothelium and organs of lipopolysaccharide-treated mice. This study aimed to analyze whether orally administered RS inhibits the development of atherosclerosis, a chronic inflammation [...] Read more.

Oral administration of rhamnan sulfate (RS), derived from the seaweed Monostroma nitidum, markedly suppresses inflammatory damage in the vascular endothelium and organs of lipopolysaccharide-treated mice. This study aimed to analyze whether orally administered RS inhibits the development of atherosclerosis, a chronic inflammation of the arteries. ApoE-deficient female mice were fed a normal or high-fat diet (HFD) with or without RS for 12 weeks. Immunohistochemical and mRNA analyses of atherosclerosis-related genes were performed. The effect of RS on the migration of RAW264.7 cells was also examined in vitro. RS administration suppressed the increase in blood total cholesterol and triglyceride levels. In the aorta of HFD-fed mice, RS reduced vascular smooth muscle cell proliferation, macrophage accumulation, and elevation of VCAM-1 and inhibited the reduction of Robo4. Increased mRNA levels of Vcam1, Mmp9, and Srebp1 in atherosclerotic areas of HFD-fed mice were also suppressed with RS. Moreover, RS directly inhibited the migration of RAW264.7 cells in vitro. Thus, in HFD-fed ApoE-deficient mice, oral administration of RS ameliorated abnormal lipid metabolism and reduced vascular endothelial inflammation and hyperpermeability, macrophage infiltration and accumulation, and smooth muscle cell proliferation in the arteries leading to atherosclerosis. These results suggest that RS is an effective functional food for the prevention of atherosclerosis. Full article

(This article belongs to the Special Issue Research Advances Related to Cardiovascular System)

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Graphical abstract

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Effects of rhamnan sulfate (RS) on the plasma lipid parameters and sterol regulatory element-binding protein 1 (Srebp1) mRNA expression. Apolipoprotein E-deficient (ApoE−/−) mice were fed either a normal diet (ND) or a high-fat diet (HFD) for 12 weeks. The groups were divided into two groups: one group not treated with RS (open columns) and the other with 0.1% RS (gray columns). Graphical representation of (A) plasma levels of total cholesterol (TCHO), (B) Srebp1 mRNA levels in the aorta quantified using quantitative real-time PCR (qPCR), and (C) plasma levels of triglyceride (TG) in each group of mice. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences between each group using two-way ANOVA (n = 5 or 6 for lipid parameter assays; n = 3 for qPCR). Full article ">Figure 2
Effect of RS on vascular cell adhesion molecule-1 (VCAM-1) expression in the liver and aorta of ApoE−/− mice fed an ND or a HFD supplemented with or without RS. (A) Immunohistochemical analysis of VCAM-1 in the liver and aorta of mice in the ND, ND + RS, HFD, and HFD + RS groups. Scale bar = 100 μm. Graphical representation of the fluorescence intensity (FI) of VCAM-1 in the (B) liver and (C) aorta of mice in the ND and HFD groups. (D) Graphical representation of the Vcam1 mRNA levels in the aorta of mice in the ND and HFD groups quantified using qPCR. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences between each group using two-way ANOVA (n = 4 for immunohistochemistry; n = 3 for qPCR). Full article ">Figure 3
Effects of RS on intercellular adhesion molecule-1 (ICAM-1) expression in the liver and aorta of ApoE−/− mice fed an ND or HFD treated with or without RS. (A) Immunohistochemical analysis of ICAM-1 in the liver and aorta of mice in the ND, ND + RS, HFD, and HFD + RS groups. Scale bar: 100 μm. The columns show the FI of ICAM-1 in the (B) liver and (C) aorta. (D) Icam1 mRNA level in the aorta quantified using qPCR. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. Data are shown as the mean ± SD. (n = 4 for immunohistochemistry; n = 3 for qPCR). Full article ">Figure 4
Effect of RS treatment on macrophage infiltration in the organ tissues of ApoE−/− mice fed either an ND or HFD supplemented with or without RS. (A) Immunohistochemical analysis of F4/80 (marker of macrophage) in the liver and aorta of mice in the ND, ND + RS, HFD, and HFD + RS groups. Scale bar = 100 μm. Graphical representation of FI of F4/80 in the (B) liver and (C) aorta of mice in the ND and HFD groups. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. The data are shown as the mean ± SD. *** p < 0.001 indicates significant differences between each group using two-way ANOVA (n = 4). Full article ">Figure 5
Effect of RS treatment on platelet-derived growth factor receptor (PDGFRβ) expression in the liver and aorta of ApoE−/− mice fed an ND or HFD supplemented with or without RS. (A) Immunohistochemical analysis of PDGFRβ in the liver and aorta of mice in the ND, ND + RS, HFD, and HFD + RS groups. Scale bar = 100 μm. Graphical representation of FI of PDGFRβ in the (B) liver and (C) aorta of mice in the ND and HFD groups. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. The data are shown as mean ± SD. *** p < 0.001 indicates significant differences between each group using two-way ANOVA (n = 4). Full article ">Figure 6
Effect of RS treatment on alpha smooth muscle actin (αSMA) expression in the liver and aorta of ApoE−/− mice fed an ND or HFD supplemented with or without RS. (A) Immunohistochemical analysis of αSMA in the liver and aorta of mice in the ND, ND + RS, HFD, and HFD + RS groups. Scale bar = 100 μm. Graphical representation of FI of αSMA in the (B) liver and (C) aorta of mice in the ND and HFD groups. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. The data are shown as the mean ± SD. *** p < 0.001 indicates significant differences between the respective groups using two-way ANOVA (n = 4). Full article ">Figure 7
Effect of RS treatment on Roundabout 4 (Robo4) expression in the liver and aorta of ApoE−/− mice fed an ND or HFD supplemented with or without RS. (A) Immunohistochemical analysis of Robo4 in the liver and aorta of mice in the ND, ND + RS, HFD, and HFD + RS groups. Scale bar = 100 μm. Graphical representation of FI of Robo4 in the (B) liver and (C) aorta of mice in the ND and HFD groups. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. Data are shown as the mean ± SD. *** p < 0.001 indicates significant differences between the respective groups using two-way ANOVA (n = 4). Full article ">Figure 8
Effect of RS treatment on the mRNA expression of matrix metallopeptidase 2 (Mmp2) and Mmp9 in the aorta of ApoE−/− mice fed either an ND or HFD supplemented with or without RS. Graphical representation of (A) Mmp2 and (B) Mmp9 mRNA levels in the aorta of mice in ND, ND + RS, HFD, and HFD + RS groups, quantified with qPCR. Open and gray columns indicate the RS-untreated and RS-treated groups, respectively. Data are shown as the mean ± SD. * p < 0.05 indicates significant differences between the respective groups using two-way ANOVA (n = 3). Full article ">Figure 9
Effect of RS on the migration of RAW264.7 cells treated with or without RS. RS was added to the upper or lower chamber and incubated for 4 or 24 h. Cells that migrated to the underside of the membrane filter were fixed and stained with Hoechst33342 solution. Photographs (×200) of the representative image of cells stained with Hoechst are shown. Scale bar = 100 μm. The graphs on the left and in the center show the number of migrated cells at 4 and 24 h after placing RS in the upper chamber, respectively. The graph on the right shows the number of migrated cells 24 h after placing RS in the lower chamber. Data are shown as the mean ± SD. *** p < 0.001 indicates significant differences between the respective groups using one-way ANOVA (n = 5). Full article ">Figure 9 Cont.
Effect of RS on the migration of RAW264.7 cells treated with or without RS. RS was added to the upper or lower chamber and incubated for 4 or 24 h. Cells that migrated to the underside of the membrane filter were fixed and stained with Hoechst33342 solution. Photographs (×200) of the representative image of cells stained with Hoechst are shown. Scale bar = 100 μm. The graphs on the left and in the center show the number of migrated cells at 4 and 24 h after placing RS in the upper chamber, respectively. The graph on the right shows the number of migrated cells 24 h after placing RS in the lower chamber. Data are shown as the mean ± SD. *** p < 0.001 indicates significant differences between the respective groups using one-way ANOVA (n = 5). Full article ">

27 pages, 14734 KiB

Open AccessArticle

Fluid and Bubble Flow Detach Adherent Cancer Cells to Form Spheroids on a Random Positioning Machine

by José Luis Cortés-Sánchez, Daniela Melnik, Viviann Sandt, Stefan Kahlert, Shannon Marchal, Ian R. D. Johnson, Marco Calvaruso, Christian Liemersdorf, Simon L. Wuest, Daniela Grimm and Marcus Krüger

Cells 2023, 12(22), 2665; https://doi.org/10.3390/cells12222665 - 20 Nov 2023

Cited by 4 | Viewed by 3344

Abstract

In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable [...] Read more.

In preparing space and microgravity experiments, the utilization of ground-based facilities is common for initial experiments and feasibility studies. One approach to simulating microgravity conditions on Earth is to employ a random positioning machine (RPM) as a rotary bioreactor. Combined with a suitable low-mass model system, such as cell cultures, these devices simulating microgravity have been shown to produce results similar to those obtained in a space experiment under real microgravity conditions. One of these effects observed under real and simulated microgravity is the formation of spheroids from 2D adherent cancer cell cultures. Since real microgravity cannot be generated in a laboratory on Earth, we aimed to determine which forces lead to the detachment of individual FTC-133 thyroid cancer cells and the formation of tumor spheroids during culture with exposure to random positioning modes. To this end, we subdivided the RPM motion into different static and dynamic orientations of cell culture flasks. We focused on the molecular activation of the mechanosignaling pathways previously associated with spheroid formation in microgravity. Our results suggest that RPM-induced spheroid formation is a two-step process. First, the cells need to be detached, induced by the cell culture flask’s rotation and the subsequent fluid flow, as well as the presence of air bubbles. Once the cells are detached and in suspension, random positioning prevents sedimentation, allowing 3D aggregates to form. In a comparative shear stress experiment using defined fluid flow paradigms, transcriptional responses were triggered comparable to exposure of FTC-133 cells to the RPM. In summary, the RPM serves as a simulator of microgravity by randomizing the impact of Earth’s gravity vector especially for suspension (i.e., detached) cells. Simultaneously, it simulates physiological shear forces on the adherent cell layer. The RPM thus offers a unique combination of environmental conditions for in vitro cancer research. Full article

(This article belongs to the Special Issue Cells in Space and on Earth)

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Spheroid formation of various adherent human carcinoma cell lines differently oriented with respect to Earth’s gravity vector for 72 h. Rotational movements of the cell culture flasks are indicated with green arrows. (A) Benchtop RPM in an incubator during an experiment with a T25 flask attached. (B) Visible spheroids formed after 3 days of random positioning in a cell culture flask. (C) Standard static cell culture. (D) Random positioning in real random mode. (E) RPM clinorotation mode with horizontal orientation of the T25 flask. (F) RPM clinorotation mode with vertical orientation of the T25 flask. (G) Upside-down static cell culture. (H) RNA expression changes in specific genes in FTC-133 cells that have been described previously to play a role in spheroid formation on the RPM. The plots show the mean ± SD ΔΔCT values of three cell cultures performed in triplicate together with the individual data points. Scale bars: 300 µm. The outlined areas reflect the magnification of the field of view in most images except for larger spheroids formed in FTC-133 and PC-3 cells in RPM and horizontal clinorotation mode. * Independent sample t-test p ≤ 0.05, ** p ≤ 0.01, ns non-significant. Parts of the figure were drawn by using pictures from <a href="http://Biorender.com" target="_blank">Biorender.com</a> and from Servier Medical Art. Full article ">Figure 2
F-actin density and transcription factor localization in the remaining adherent cells in differently oriented and rotated cell culture flasks. (A) Static cell cultures in different orientations: normal cell culture, upright flasks, and inverted flasks. (B) Effect of cell compression on nuclear transport of transcription factors (red circles) via nuclear pores (NP). (C) F-actin density (phalloidin staining) and immunofluorescence of the mechanoresponsive transcription factors (D) YAP1, (E) p38 MAPK, and (F) MRTF-A after 72 h (n = 5 for each condition; one representative picture is shown). Outlines of the nuclei as indicated using DAPI staining (not shown) depicted as dashed lines. (G) Rotating cell cultures: horizontal clinorotation (60°/s), vertical clinorotation (60°/s), and random positioning (average speed 60°/s). (H) F-actin density. (I) YAP1, (J) p38 MAPK, and (K) MRTF-A localization after 72 h (n = 5 for each condition; one representative picture is shown). Outlines of the nuclei are shown as dashed lines. (Below) The relative mean nuclear–cytoplasmic (N/C) ratio of transcription factors was measured for at least 15 cells (5 pictures per condition). Static cell culture was set as reference. The experimental conditions where the spheroid formation was observed are shaded gray. Scale bars: 300 µm. The fluorescence images in this figure were optimized to visualize protein localization and unsuitable for comparative protein level quantification. * Mann–Whitney p ≤ 0.05, ** p ≤ 0.01. Parts of the figure were drawn by using pictures from Servier Medical Art. Full article ">Figure 3
Spheroid formation of FTC-133 cells exposed to the RPM at different angular velocities for 24 h. (A) Illustration of fluid shear within cell culture flasks on the RPM. Time-averaged residual gravity levels and shear forces were calculated at RPM average velocities of (B) 25°/s, (C) 60°/s, and (D) 90°/s. Spheroid formation was examined in FTC-133 cultures that had previously been allowed to adhere to the bottom of the cell culture flask for (E) 24 h or (F) 48 h. The outlined area reflects the magnification of the field of view in most images except for larger spheroids compared at 60°/s after 24 h of normal cell growth followed by 24 h of exposure to the RPM. (G) Expression changes (compared to static cell culture) in gene transcripts known to be responsive to shear stress after 24 h exposure to the RPM operated with different velocities. The plots show the mean ± SD ΔΔCT values of four cell cultures performed in triplicate together with the individual data points. The experimental conditions where spheroid formation was observed are shaded gray. Scale bars: 300 µm. * Independent sample t-test p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns non-significant vs. static control. § Shear stress τ according to Wuest et al. [<a href="#B14-cells-12-02665" class="html-bibr">14</a>]. Parts of the figure were drawn by using pictures from Servier Medical Art. Full article ">Figure 4
Effect of fluid flow on detachment of adherent FTC-133 cells. (A) Setup of the flow experiment. (B) Flow schematics. (C) Confluency of cell cultures over 24 h (n = 3). (D) Snapshots from the brightfield time-lapse recordings of one representative experiment with different flow speeds. (E) Migration traces of single cells in the flow channel perfused with different flow rates. The blue arrow indicates the direction of flow, and the position of cells (n > 40 cells for each condition) after 24 h is indicated by black circles. The red cross (+) indicates the mean value (M) of spatial cell migration (values are given below the plot). D: directness. (F) Cumulative migration distances of cells over 24 h at different flow rates. Scale bars: 300 µm. * Independent sample t-test (C) or Mann–Whitney (F) p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Full article ">Figure 5
Enhancing effect of air bubbles on detachment of adherent FTC-133 cells. (A) Spheroid formation on the RPM depends on the culture flask geometry and the presence of air bubbles during the experiment. (B) Air bubbles were able to counteract the inhibitory effect of dexamethasone on the spheroid formation of FTC-133 cells on the RPM. (C) Setup of the bubble experiment. (D) Confluence of cell cultures over 24 h (n = 3). (E) Snapshots from the time-lapse recordings of one representative experiment with a flow rate of 1 mL/min without and with 1 bubble/min. (F) Migration traces of single cells in the flow channel. The blue arrow indicates the flow direction, and black circles indicate the position of cells after 24 h (n > 40 cells for each condition). The red cross (+) indicates the mean value (M) of spatial cell migration (values are given below the plot). D: directness. (G) Cumulative migration distances of cells over 24 h at a 1 mL/min flow rate with and without air bubbles. (H) Experiment time until initial cell detachment. Scale bars: 300 µm. * Independent sample t-test (D) or Mann–Whitney (G,H) p ≤ 0.05, *** p ≤ 0.001, ns, not significant. Parts of the figure were drawn by using pictures from Servier Medical Art. Full article ">Figure 6
Transcription factor levels in random positioning compared to continuous flow. (A) Fluid motions acting on cells within different cell culture vessels. (B) Actin density and immunofluorescence of the mechanoresponsive transcription factors (C) RelA, (D) p38 MAPK, (E) MRTF-A, (F) β-catenin, and (G) YAP1 after 4 h. Outlines of the nuclei are shown as dashed lines. The small graphs indicate fold changes (FC) in nuclear protein levels compared to static cell cultures (n = 5 for each condition; one representative picture is shown). (Right) The mean nuclear/cytoplasmic (N/C) ratio of transcription factor localizations was measured for five overview images from independent experiments, each showing at least three cells. (H) Gene expression response after 4 h exposure to the RPM (60°/s) and continuous flow (1 mL/min). The plots show the mean ± SD ΔΔCt values of 3–5 independent experiments performed in triplicate. To mimic the channel slide’s low medium and nutrient volume, the RPM experiment was performed once identically to the flow (“+”, dark gray bars) experiment and once with a prior starvation phase (“−”, light gray bars). Scale bars: 300 µm. The fluorescence images in this figure were optimized to visualize protein localization and unsuitable for comparative protein level quantification. * Mann–Whitney (G) or independent sample t-test (H) p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns non-significant vs. static control. Parts of the figure were drawn using pictures from Servier Medical Art. Full article ">Figure 7
Transcription factor effects in response to oscillatory flow compared with continuous flow. (A) Setup for an oscillatory flow experiment (1 mL/min, direction change every 7.5 s). (B) Snapshots of cells in the channel before the start of the experiment and after 24 h of oscillating flow show reduced detachment of cells. (C) Nuclear transport of transcription factors after 4 h oscillatory flow (n = 5 for each condition; one representative picture is shown). (Below) Fold changes in nuclear protein levels compared to unidirectional flow. The mean nuclear/cytoplasmic (N/C) ratio of transcription factor localizations was measured for five overview images from independent experiments, each showing at least three cells. (D) Gene expression response after 4 h exposure to the oscillatory flow (Osc) compared to unidirectional flow (Uni) and to RPM culture (n = 3–5). Scale bars: 300 µm. The fluorescence images in this figure were optimized to visualize protein localization and unsuitable for comparative protein level quantification. * Mann–Whitney (C) or independent sample t-test (D) p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns non-significant. Parts of the figure were drawn using pictures from <a href="http://Biorender.com" target="_blank">Biorender.com</a> (accessed on 30 October 2023). Full article ">Figure 8
(A) Illustration of the hypothesized processes occurring with adherent cells in an initially air-bubble-free cell culture flask on the RPM, finally leading to spheroid formation. We observed that RPM-induced spheroid formation is a two-step process. First, the adherent cells detach due to mechanical stress (e.g., fluid flow, air bubbles). Then, “simulated microgravity” (free-fall) leads to the formation of three-dimensional spheroids by preventing the sedimentation of suspended cells. The small picture indicates the amplifying mechanical effect of air bubbles. (B) The two cell populations (adherent cell layer and suspension cells) of an RPM cell culture of adherent cells are subjected to different mechanical forces. While the suspension cells are held in a mostly stress-free suspension, the cell layer experiences shear forces similar to those in the human lymphatic system. Parts of the figure were drawn by using pictures from <a href="http://Biorender.com" target="_blank">Biorender.com</a> and from Servier Medical Art. Full article ">

20 pages, 3139 KiB

Open AccessReview

Chromophore-Targeting Precision Antimicrobial Phototherapy

by Sebastian Jusuf and Pu-Ting Dong

Cells 2023, 12(22), 2664; https://doi.org/10.3390/cells12222664 - 20 Nov 2023

Cited by 4 | Viewed by 1900

Abstract

Phototherapy, encompassing the utilization of both natural and artificial light, has emerged as a dependable and non-invasive strategy for addressing a diverse range of illnesses, diseases, and infections. This therapeutic approach, primarily known for its efficacy in treating skin infections, such as herpes [...] Read more.

Phototherapy, encompassing the utilization of both natural and artificial light, has emerged as a dependable and non-invasive strategy for addressing a diverse range of illnesses, diseases, and infections. This therapeutic approach, primarily known for its efficacy in treating skin infections, such as herpes and acne lesions, involves the synergistic use of specific light wavelengths and photosensitizers, like methylene blue. Photodynamic therapy, as it is termed, relies on the generation of antimicrobial reactive oxygen species (ROS) through the interaction between light and externally applied photosensitizers. Recent research, however, has highlighted the intrinsic antimicrobial properties of light itself, marking a paradigm shift in focus from exogenous agents to the inherent photosensitivity of molecules found naturally within pathogens. Chemical analyses have identified specific organic molecular structures and systems, including protoporphyrins and conjugated C=C bonds, as pivotal components in molecular photosensitivity. Given the prevalence of these systems in organic life forms, there is an urgent need to investigate the potential impact of phototherapy on individual molecules expressed within pathogens and discern their contributions to the antimicrobial effects of light. This review delves into the recently unveiled key molecular targets of phototherapy, offering insights into their potential downstream implications and therapeutic applications. By shedding light on these fundamental molecular mechanisms, we aim to advance our understanding of phototherapy’s broader therapeutic potential and contribute to the development of innovative treatments for a wide array of microbial infections and diseases. Full article

(This article belongs to the Special Issue Immunopathogenesis of Bacterial Infection)

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21 pages, 697 KiB

Open AccessReview

Liquid Biopsy in Head and Neck Cancer: Its Present State and Future Role in Africa

by Dada Oluwaseyi Temilola, Henry Ademola Adeola, Johan Grobbelaar and Manogari Chetty

Cells 2023, 12(22), 2663; https://doi.org/10.3390/cells12222663 - 20 Nov 2023

Cited by 1 | Viewed by 2099

Abstract

The rising mortality and morbidity rate of head and neck cancer (HNC) in Africa has been attributed to factors such as the poor state of health infrastructures, genetics, and late presentation resulting in the delayed diagnosis of these tumors. If well harnessed, emerging [...] Read more.

The rising mortality and morbidity rate of head and neck cancer (HNC) in Africa has been attributed to factors such as the poor state of health infrastructures, genetics, and late presentation resulting in the delayed diagnosis of these tumors. If well harnessed, emerging molecular and omics diagnostic technologies such as liquid biopsy can potentially play a major role in optimizing the management of HNC in Africa. However, to successfully apply liquid biopsy technology in the management of HNC in Africa, factors such as genetic, socioeconomic, environmental, and cultural acceptability of the technology must be given due consideration. This review outlines the role of circulating molecules such as tumor cells, tumor DNA, tumor RNA, proteins, and exosomes, in liquid biopsy technology for the management of HNC with a focus on studies conducted in Africa. The present state and the potential opportunities for the future use of liquid biopsy technology in the effective management of HNC in resource-limited settings such as Africa is further discussed. Full article

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21 pages, 9758 KiB

Open AccessArticle

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

by Grace Mercedes Freke, Tiago Martins, Rosalind Jane Davies, Tina Beyer, Marian Seda, Emma Peskett, Naila Haq, Avishek Prasai, Georg Otto, Jeshmi Jeyabalan Srikaran, Victor Hernandez, Gaurav D. Diwan, Robert B. Russell, Marius Ueffing, Martina Huranova, Karsten Boldt, Philip L. Beales and Dagan Jenkins

Cells 2023, 12(22), 2662; https://doi.org/10.3390/cells12222662 - 20 Nov 2023

Viewed by 1477

Abstract

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation [...] Read more.

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate. Full article

(This article belongs to the Special Issue Complex Role of Cilium-Generated Signaling)

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(A). Relative levels of the 65 kDa Bbs1 protein. For each cell line, the Bbs1 signal intensity was normalised to that of β-actin. All protein levels (in arbitrary units) are shown relative to the parental WT cell line. Data are for a single experiment. (B). Normalised Bbs1 mRNA expression relative to WT IMCD3 cells. Bars show mean fold change, and error bars show SEM of three independent experiments. (C). Representative cilia staining. Cilia were identified as structures which stained for acetylated α-tubulin (ac-tub) and ARL13B. Nuclei were stained with DAPI. Bbs1ex1.1 was able to form cilia. Scale bars represent 20 μm. (D). Quantification of ciliogenesis, calculated as the percentage of cells which formed a cilium (co-stained for ac-tub and ARL13B with a γ-tub-positive basal body). Coloured bars show the mean percentage of ciliogenesis, and error bars show SEM of three independent experiments. One-way ANOVA was performed after arcsine square root transformation of proportions of cells with cilia. This indicated a statistically significant difference between the mean proportion of ciliogenesis of cell lines (p < 0.0001). Post hoc Bonferroni multiple comparisons tests indicated that all Bbs1 mutant and WTcr clones differed significantly compared to parental WT (p-adj < 0.0001 for all comparisons). Compared to their respective WTcr clones, Bbs1ex8 and Bbs1ex12.4 differed significantly (p-adj < 0.0001 for both comparisons), whereas Bbs1ex1.4 and Bbs1M390R did not (p-adj > 0.9999 for both comparisons). On the graph, adjusted p-values are indicated: * p = 0.01 to 0.05; ** p = 0.001 to 0.01; *** p < 0.001; those in black are relative to WT, and those in colour are relative to the respective WTcr clone. (E). Mean number of nuclei per ciliogenesis assay image. Reduced ciliogenesis in Bbs1ex8, WTcrex12.1, Bbs1M390R and WTcrM390R.1 was not due to decreased cell density. Coloured bars show mean numbers of nuclei per field, and error bars show SEM of three independent experiments. One-way ANOVA indicated a significant difference among means of different cell lines (p < 0.0001). Post hoc Bonferroni multiple comparisons tests indicated that, compared to WT, Bbs1ex8 (p-adj = 0.0038), WTcrex12.1 (p-adj < 0.0001) and WTcrM390R.1 (p-adj = 0.0081) differed significantly, whereas Bbs1ex1.4 (p-adj > 0.9999), WTcrex1 (p-adj > 0.9999), WTcrex8 (p-adj = 0.5386), Bbs1ex12.4 (p-adj > 0.9999) and Bbs1M390R (p-adj > 0.9999) did not. Compared to their respective WTcr clones, Bbs1ex8 (p-adj < 0.0001), Bbs1ex12.4 (p-adj > 0.0001) and Bbs1M390R (p-adj = 0.0133) differed significantly, whereas Bbs1ex1.4 did not (p-adj > 0.9999). On the graph, adjusted p-values are indicated: * p-adj = 0.01 to 0.05; ** p-adj = 0.001 to 0.01; *** p-adj < 0.001; those in black are relative to WT, and those in colour are relative to the respective WTcr clone. (F). Quantification of ciliogenesis, calculated as the percentage of cells which formed a cilium (co-stained for ac-tub and ARL13B). Coloured bars show the mean percentage of ciliogenesis, and error bars show SEM of three independent experiments. A paired t-test was performed after arcsine square root transformation of proportions of cells with cilia. Ciliogenesis in Bbs1ex1.1 did not differ significantly from that in WT (p = 0.0603). (G). Mean number of nuclei per ciliogenesis assay image. Coloured bars show mean numbers of nuclei per field, and error bars show SEM of three independent experiments. A paired t-test showed no statistically significant difference between numbers of nuclei per field of Bbs1ex1.1 and WT (p = 0.1950). Full article ">Figure 2
(A) Representative images of IMCD3 cell clones of the indicated genotypes transduced with YFP-BBS8, either with or without mCherry-BBS1, showing subcellular localisation of these BBSome components in relation to PCM1. Quantification of the proportion of PCM1 immunofluorescence coinciding with the YFP-BBS8 signal. Scale bars are 2 microns. (B) log2 enrichment and peptide intensities comparing affinity purifications for BBS1 wild-type and p.M390R baits versus themselves and a RAF1 control. Full article ">Figure 2 Cont.
(A) Representative images of IMCD3 cell clones of the indicated genotypes transduced with YFP-BBS8, either with or without mCherry-BBS1, showing subcellular localisation of these BBSome components in relation to PCM1. Quantification of the proportion of PCM1 immunofluorescence coinciding with the YFP-BBS8 signal. Scale bars are 2 microns. (B) log2 enrichment and peptide intensities comparing affinity purifications for BBS1 wild-type and p.M390R baits versus themselves and a RAF1 control. Full article ">Figure 3
Representative immuno-staining of ZO-1 and occludin in tight junctions. The F-actin cytoskeleton and nuclei were stained with phalloidin and DAPI, respectively. Z-projections of planes containing tight junctions are displayed. Scale bars represent 20 μm. Qualitative assessment of tight junction formation is summarised on the ZO-1 channel. In Bbs1ex8, localisation of ZO-1 to the peripheries of some cells was seen, but there was a failure of cell peripheries to seal together (white arrowhead). Full article ">Figure 4
Representative immuno-staining of E-cadherin and β-catenin in adherens junctions. The F-actin cytoskeleton and nuclei were stained with phalloidin and DAPI, respectively. Z-projections of planes containing tight junctions are displayed. Scale bars represent 20 μm. Qualitative assessment of tight junction formation is summarised on the β-catenin channel. Full article ">Figure 5
Hierarchical clustering of NES values comparing GSEAs for DEGs observed in 6 IMCD3 Bbs1-/- clones vs. Wtcr controls, or all mutant clones versus all Wtcr controls (“KOs vs. WTs”) (A), and for DEGs identified in p.M390R patient fibroblasts, mouse hypothalamus and BBS7-depleted HeLa cells (B) (‘+’ and ‘-’ indicate statistically significant positive and negative enrichment, respectively. (C,D) Relative expression of selected mesenchymal genes in Bbs1-/- and WtCr clones. Full article ">Figure 5 Cont.
Hierarchical clustering of NES values comparing GSEAs for DEGs observed in 6 IMCD3 Bbs1-/- clones vs. Wtcr controls, or all mutant clones versus all Wtcr controls (“KOs vs. WTs”) (A), and for DEGs identified in p.M390R patient fibroblasts, mouse hypothalamus and BBS7-depleted HeLa cells (B) (‘+’ and ‘-’ indicate statistically significant positive and negative enrichment, respectively. (C,D) Relative expression of selected mesenchymal genes in Bbs1-/- and WtCr clones. Full article ">Figure 6
Mesenchymal gene expression in parental (non-clonal) IMCD3 cells (WT) assessed at the indicated passage numbers (P10-64). Full article ">

21 pages, 2863 KiB

Open AccessArticle

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells

by Corey Kretzmer, Kelsey Reger, Vincent Balassi, Quang Long Pham, Michael Johns, Samuel T. Peters, Amber Petersen, Jana Mahadevan, Jason Gustin, Trissa Borgschulte and David Razafsky

Cells 2023, 12(22), 2661; https://doi.org/10.3390/cells12222661 - 20 Nov 2023

Viewed by 1965

Abstract

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein [...] Read more.

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN^® GS^−/− cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients. Full article

(This article belongs to the Section Mitochondria)

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Figure 1
High-throughput screen to enhance recombinant protein production. (A) High-throughput screening process to identify compounds which enhance productivity. (B) Rank order plot of 1280 compounds screened on four model cell lines at 3 µM. Fold change in volumetric titer is reported from static cultures. (C) Rank order plot of 1280 compounds screened on four model cell lines at 30 µM. Fold change in volumetric titer is reported from static cultures. Full article ">Figure 2
Validation of hit compound BrdU in transient production platform. (A) Viability of transiently transfected cells treated with BrdU (black) or untreated (gray). Five independent molecules were transfected into CHOZN® GS−/− cells and viability was measured. Fc Fusion (square), IgG-A (diamond), IgG-B (line), IgG-C (circle), and IgG-D (triangle). (B) Viable cell density (VCD) of transiently transfected cells treated with BrdU (black) or untreated (gray). (C) Volumetric titer of BrdU-treated (black) and untreated (gray) cells was measured starting on day 3 of a transient productivity assay. (D) Fold change in peak volumetric titer of BrdU-treated cultures relative to untreated controls. Average represents the mean peak fold change in the five recombinant proteins tested and indicates consistently higher productivity of BrdU-treated cultures. Full article ">Figure 3
Validation of hit compounds at 30 µM in stably expressing suspension cultures. (A–D) Viability of stable producing cells in a 7-day batch assay. Cells were treated with DMSO only (black), untreated (gray), 30 µM BrdU (purple), or 30 µM Forskolin (pink). IgG-A (A), IgG-B (B), Fc Fusion-A (C), and Fc Fusion-B (D). (E–H) Viable cell density of stable producing cells in a 7-day batch assay. Cells were treated with DMSO only (black), untreated (gray), 30 µM BrdU (purple), or 30 µM Forskolin (pink). IgG-A (E), IgG-B (F), Fc Fusion-A (G), and Fc-fusion-B (H). BrdU and Forskolin both decrease the growth rate of all four clonal cell lines. (I) Fold change in qP of cells treated with either 30 µM BrdU or 30 µM forskolin relative to DMSO-treated controls. Full article ">Figure 4
BrdU reduces cell growth and enhances productivity via alterations in the transcriptome. (A) Viability of clone IgG-B is comparable when treated with either BrdU (purple) or DMSO (black) in duplicate. (B) Viable cell density measurements of two replicates of clone IgG-B treated with BrdU (purple) or DMSO (black). BrdU-treated cultures have a slower growth profile and reach a reduced peak viable cell density. (C) Cell diameter of clone IgG-B treated in duplicate with either BrdU (purple) or DMSO (black). BrdU-treated cultures maintain a larger cell diameter for the duration of the assay. (D–E) Volumetric productivity (D) and qP (E) of clone IgG-B treated in duplicate with either BrdU (purple) or DMSO (black). Both volumetric productivity and qP increased in BrdU-treated cultures. Full article ">Figure 5
BrdU treatment or genetic modification of Ndufa13 leads to enhanced mitochondrial membrane potential and alters ATP production. (A,C) MMP was measured via FACS analysis after staining BrdU- and DMSO-treated cells with TMRM (A). Additionally, MMP was measured on Ndufa13 edited and wildtype clones (C). BrdU treatment as well as genetic modification of Ndufa13 led to increased MMP. (B,D) ATP production rate as well as the mitochondrial and glycolytic contributions to ATP production were measured in BrdU- and DMSO-treated cells (B) and cells with genetic modifications of Ndufa13 as well as identically treated wildtype counterparts (D). (E,F) Oxygen consumption rate (OCR) was measured in BrdU- and DMSO-treated cells (E) and cells with genetic modifications of Ndufa13 as well as identically treated unedited counterparts (F). BrdU treatment as well as modification of the Ndufa13 gene led to an increased OCR. Full article ">Figure 6
Genetic modification of Ndufa13 partially recapitulates the phenotypes of BrdU treatment. (A) The viability of wildtype clones (gray) and Ndufa13 edited clones (white) is similar in a simulated perfusion assay. (B) Peak viable cell density of the population of all unedited clones (gray) and all Ndufa13 edited clones (white) was similar. However, many of the top producing Ndufa13 edited clones had slower doubling times. (C) The peak volumetric productivity of Ndufa13 edited (white) and wildtype (gray) counterparts is similar. When productivity is normalized by cell number, there is a greater probability of Ndufa13 edited clones having a qP >25 pg cell−1 day−1. (D) The peak volumetric productivity and peak cell-specific productivity of Ndufa13 edited clones treated with BrdU (white) and DMSO (gray). Full article ">

35 pages, 8372 KiB

Open AccessReview

Exploration of the Noncoding Genome for Human-Specific Therapeutic Targets—Recent Insights at Molecular and Cellular Level

by Wolfgang Poller, Susmita Sahoo, Roger Hajjar, Ulf Landmesser and Anna M. Krichevsky

Cells 2023, 12(22), 2660; https://doi.org/10.3390/cells12222660 - 20 Nov 2023

Cited by 2 | Viewed by 1864

Abstract

While it is well known that 98–99% of the human genome does not encode proteins, but are nevertheless transcriptionally active and give rise to a broad spectrum of noncoding RNAs [ncRNAs] with complex regulatory and structural functions, specific functions have so far been [...] Read more.

While it is well known that 98–99% of the human genome does not encode proteins, but are nevertheless transcriptionally active and give rise to a broad spectrum of noncoding RNAs [ncRNAs] with complex regulatory and structural functions, specific functions have so far been assigned to only a tiny fraction of all known transcripts. On the other hand, the striking observation of an overwhelmingly growing fraction of ncRNAs, in contrast to an only modest increase in the number of protein-coding genes, during evolution from simple organisms to humans, strongly suggests critical but so far essentially unexplored roles of the noncoding genome for human health and disease pathogenesis. Research into the vast realm of the noncoding genome during the past decades thus lead to a profoundly enhanced appreciation of the multi-level complexity of the human genome. Here, we address a few of the many huge remaining knowledge gaps and consider some newly emerging questions and concepts of research. We attempt to provide an up-to-date assessment of recent insights obtained by molecular and cell biological methods, and by the application of systems biology approaches. Specifically, we discuss current data regarding two topics of high current interest: (1) By which mechanisms could evolutionary recent ncRNAs with critical regulatory functions in a broad spectrum of cell types (neural, immune, cardiovascular) constitute novel therapeutic targets in human diseases? (2) Since noncoding genome evolution is causally linked to brain evolution, and given the profound interactions between brain and immune system, could human-specific brain-expressed ncRNAs play a direct or indirect (immune-mediated) role in human diseases? Synergistic with remarkable recent progress regarding delivery, efficacy, and safety of nucleic acid-based therapies, the ongoing large-scale exploration of the noncoding genome for human-specific therapeutic targets is encouraging to proceed with the development and clinical evaluation of novel therapeutic pathways suggested by these research fields. Full article

(This article belongs to the Special Issue Evolutionary Impact of the Noncoding Genome in Higher Organisms – Recent Insights Obtained from the Molecular and Cellular Level to Systems Biology)

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Figure 1
Evolutionary expansion of the noncoding genome in higher organisms. (A) The basic molecules of life already found in the earliest and most simple organisms are increasingly supplemented, during the evolution to complex species, by molecules needed for correct embryonic development and homeostatic stability of their morphology and functions. (B) Whereas the number of protein-coding genes remains similar from simple to complex species, it is the noncoding part of the genome that increases dramatically with morphological complexity to >98% in humans. (C) Few types of noncoding RNAs arising from the noncoding genome have been phylogenetically mapped in depth. Thus, investigation of microRNA (miRNA) family evolution revealed impressive increases with the advent of vertebrates, and ancient miRNAs families can well be distinguished from those more recently arising. (D) No definitive classification of the huge number of lncRNAs has been established so far. Several basic elements suitable as components for classification are shown, encompassing sequence elements, conserved structural motifs, mechanisms of action, and physiological or disease processes in which the respective lncRNAs are involved (Modified from Poller et al. 2013 [<a href="#B9-cells-12-02660" class="html-bibr">9</a>] by permission of Circ. Res.). Full article ">Figure 2
Endogenous non-coding RNAs as blueprints for RNA therapeutics. Non-coding RNAs may be addressed as therapeutic targets, but an increasing spectrum of endogenous ncRNAs (e.g., siRNAs) are also employed as blueprints for the development of novel therapeutic tools. The spectrum of possible therapeutic targets has vastly expanded beyond proteins, but likewise the therapeutic ‘toolbox’. One current topic is therapeutic RNA interference triggers (siRNAs) originally developed from endogenous siRNAs as blueprints, and made clinically applicable based on sophisticated chemical modifications and coupling to carriers/ligands for tissue targeting. Appreciation of the profound pathogenic impact of diverse small and long ncRNAs has inspired the development of multiple other therapeutic tools engaging these ncRNAs. The tools may be engineered nucleic acids themselves, acting through sequence homologies, or “classical” small molecule drugs designed to interact with e.g., conserved 3D structural motifs in lncRNAs which are not necessarily dependent on strict RNA sequence conservation. Full article ">Figure 3
Multi-level functional integration of extended regions of the human genome, above and beyond individual noncoding RNAs. The NEAT1–MALAT1 genomic region encodes a biologically integrated circuit controlling innate immune sensing and cell–cell interactions. From an evolutionary perspective, the NEAT1–MALAT1 genomic region appears as a highly integrated RNA processing circuitry critically contributing to immune homeostasis. Its components MEN-β, MEN-ε, menRNA, MALAT1, TALAM1, and mascRNA are obviously set for well-balanced interactions with each other. Genetic ablation of any element therefore leads to major dysfunction. Beyond prior work in NEAT1 and MALAT1 knockout mice, a recent cell biological study identified menRNA and mascRNA as novel components of innate immunity with deep impact upon cytokine regulation, immune cell–endothelium interactions, angiogenesis, and macrophage formation and functions. These tRNA-like transcripts appear to be prototypes of a class of ncRNAs distinct from other small transcripts (miRNAs, siRNAs) by biosynthetic pathway (enzymatic excision from lncRNAs) and intracellular kinetics, suggesting a novel link for the apparent relevance of the NEAT1–MALAT1 cluster in cardiovascular and neoplastic diseases. For the long primary transcripts of NEAT1, a function of general cell-biological interest has been identified. They are critical for the formation of paraspeckles which are involved in multiple cellular functions, and possibly also in the broader context of micellization and the formation of biomolecular condensates essential for proper subcellular and nuclear compartmentalization. Obviously, molecules involved in these fundamental processes may deeply impact upon various cellular functions in a context-dependent manner, so that their observed association with diverse diseases is therefore not entirely unexpected. Overall, the NEAT1–MALAT1 genomic region may serve as paradigm of a biological integrated circuit fine-tuning multiple cellular processes covering innate immune sensing and cell–cell interactions. (Modified from Poller et al. 2023 [<a href="#B11-cells-12-02660" class="html-bibr">11</a>] by permission from J. Clin. Med.). Full article ">Figure 4
Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus, and neoplastic transformation in glioma. miR-10b is silenced in normal neuroglial cells of the brain but commonly activated in glioma, where it assumes an essential tumor-promoting role. The entire miR-10b-hosting HOXD locus is activated in glioma via the cis-acting mechanism involving 3D chromatin reorganization and CTCF-cohesin-mediated looping. This mechanism requires two interacting lncRNAs, HOXD-AS2 and LINC01116, one associated with HOXD3/HOXD4/miR-10b promoter and another with the remote enhancer. Knockdown of either lncRNA in glioma cells alters CTCF and cohesin binding, abolishes chromatin looping, inhibits the expression of all genes within HOXD locus, and leads to glioma cell death. Conversely, in cortical astrocytes, enhancer activation is sufficient for HOXD/miR-10b locus reorganization, gene derepression, and neoplastic cell transformation. LINC01116 RNA is essential for this process. Our results demonstrate the interplay of two lncRNAs in the chromatin folding and concordant regulation of miR-10b and multiple HOXD genes normally silenced in astrocytes and triggering the neoplastic glial transformation. (Modified from Deforzh et al. 2022 [<a href="#B88-cells-12-02660" class="html-bibr">88</a>] by permission from Mol. Cell). Full article ">Figure 5
The fundamental clinical efficacy determinants of ASO and siRNA therapeutics. Despite broad diversity of the new nucleic acid-based therapeutic principles and tools, they share key common determinants of clinical efficacy which are critical for possible translational success and need to be closely monitored in any clinical trial. Full article ">Figure 6
Vector-based genetic therapies for protein augmentation or RNAi-mediated target RNA depletion. The ‘classical’ approach of gene transfer for protein augmentation or (in the case of monogenic disorders) protein substitution recently gained clinical impact in the hemophilia field where the missing coagulation factor genes could be successfully and durably transferred to the liver using AAV vectors. In the cardiovascular field, cardiac-targeted gene augmentation (SERCA2a) or ablation (phospholamban) therapies were successful in animal models, but this could not yet be translated to the clinical arena due to as yet insufficient gene transfer efficacy in patients. The opposite approach is post-transcriptional silencing of genes involved in disease pathogenesis. Complementary to chemically synthesized base- and backbone-modified ASOs or siRNAs (<a href="#cells-12-02660-f006" class="html-fig">Figure 6</a>), silencing of any protein-coding or noncoding transcript may be achieved by viral vector-based RNA interference (RNAi). Two fundamentally distinct approaches (lower panel) use synthetic siRNAs, or recombinant shRNAs continuously produced from viral vectors. RNA is inherently unstable and must be modified to achieve sufficient biostability, and delivered via synthetic carriers, to become therapeutically useful. Viral vectors, which may be organ-targeted and regulatable, may circumvent targeting issues by their inherent biological properties, and the RNA stability problem by continuous synthesis in the host cells. Apart from these differences, the same characteristics will be considered when the therapeutic potential of synthetic or recombinant RNA drugs is assessed. AAV indicates adeno-associated virus; ASO, antisense oligonucleotide; LNA, locked nucleic acid; shRNA, short hairpin RNA; siRNA, short interfering RNA; TS, target site. (Modified from Poller et al. 2013 [<a href="#B9-cells-12-02660" class="html-bibr">9</a>] by permission from Circ. Res.). Full article ">Figure 7
Summary and outlook. On the right side, this figure aims to summarize- the molecular and cellular basis of liver targeting of nucleic acid-based drugs, which is already being applied clinically for several disorders. Here, drug delivery is facilitated by the fenestrated endothelium of the liver and the availability of a safe and efficient hepatocyte-specific ligand-receptor system. The left side outlines the multiple challenges arising once targeting to other organs is attempted, focusing upon brain delivery. Targeted and safe delivery of any nucleic acid-based (siRNA, ASO) drug to specific regions of the brain appears as far greater challenge than liver targeting or ex vivo blood stem cell modulation. A remarkable spectrum of brain-targeting approaches encompasses synthetic nanoparticles and viral vector, yet so far, none of these are established with respect to key efficacy requirements (<a href="#cells-12-02660-f005" class="html-fig">Figure 5</a>). AAV vectors encounter high interest for brain-targeted therapies since genetically engineered and surface-modified (pseudotyped) versions of this vector have been extensively studied in other fields of medicine (e.g., hematology, cardiovascular medicine). The blood-brain-barrier (BBB) constitutes a particularly challenging anatomical barrier against nanoparticle or vector based drug delivery. Remarkably, the serotype AAV9 is capable to cross the BBB under certain conditions, raising the possibility of intravascular administration as a non-invasive delivery route of nucleic acid-based drugs to the CNS. Noteably, this same AAV serotype is also able to enter the myocardium across the tight cardiovascular endothelium (impermeable for other AAV serotypes) and was previously employed for cardiac-targeted gene transfer and RNA interference therapy. Regarding the next step of delivery, little is known about differential tropism of currently available AAV variants for distinct brain cell types of specific therapeutic interest. Recent high-throughput screens have identified host proteins essential for AAV delivery in a comprehensive manner and revealed unanticipated complexity and serotype specificity of the entry process. Theoretical predictability of any in vivo effects of vector modifications is therefore limited and experimental validation essential. The figure depicts recent experimental approaches to improve BBB passage and brain cell type-specific delivery. Starting from AAV9 holding promise for trans-BBB therapy, AAV-PHP.eB was engineered by insertion of a 7-amino acid peptide and point mutations of neighboring residues into the AAV9 capsid and enhanced CNS delivery in mice only under certain conditions. Similar challenges with regard to clinical translation, generated by species differences, have been extensively investigated before for another “hard target”, i.e., the heart. Instead of recognizing the glutamate receptor GluA4 through a displayed GluA4-specific DARPin, AAVs deficient in HSPGs attachment resulted in preferential >90% transduction of interneurons. Another highly innovative strategy employs membrane protein-specific nanobodies inserted into a surface loop of the VP1 capsid protein of AAVs. Nanobody-VP1 fusion was applied to AAV1, AAV2, AAV8, and AAV9 and effectively re-directed the target specificity of all these AAV serotypes. Beyond stability in the blood circulation and capability to cross the blood–brain barrier, transgene expression stability or even control is also desirable. Alphaherpesvirus latency-associated promoters (LAPs) enabled stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months. Thus, these LAPs are suitable candidates for AAV-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration. Full article ">

17 pages, 4612 KiB

Open AccessArticle

Neuroprotective Action of Tacrolimus before and after Onset of Neonatal Hypoxic–Ischaemic Brain Injury in Rats

by Madeleine J. Smith, Tayla Penny, Yen Pham, Amy E. Sutherland, Graham Jenkin, Michael C. Fahey, Madison C. B. Paton, Megan Finch-Edmondson, Suzanne L. Miller and Courtney A. McDonald

Cells 2023, 12(22), 2659; https://doi.org/10.3390/cells12222659 - 20 Nov 2023

Cited by 1 | Viewed by 1348

Abstract

(1) Background: Neonatal brain injury can lead to permanent neurodevelopmental impairments. Notably, suppressing inflammatory pathways may reduce damage. To determine the role of neuroinflammation in the progression of neonatal brain injury, we investigated the effect of treating neonatal rat pups with the immunosuppressant [...] Read more.

(1) Background: Neonatal brain injury can lead to permanent neurodevelopmental impairments. Notably, suppressing inflammatory pathways may reduce damage. To determine the role of neuroinflammation in the progression of neonatal brain injury, we investigated the effect of treating neonatal rat pups with the immunosuppressant tacrolimus at two time points: before and after hypoxic–ischaemic (HI)-induced injury. (2) Methods: To induce HI injury, postnatal day (PND) 10 rat pups underwent single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). Pups received daily tacrolimus (or a vehicle) starting either 3 days before HI on PND 7 (pre-HI), or 12 h after HI (post-HI). Four doses were tested: 0.025, 0.05, 0.1 or 0.25 mg/kg/day. Pups were euthanised at PND 17 or PND 50. (3) Results: All tacrolimus doses administered pre-HI significantly reduced brain infarct size and neuronal loss, increased the number of resting microglia and reduced cellular apoptosis (p < 0.05 compared to control). In contrast, only the highest dose of tacrolimus administered post-HI (0.25 mg/kg/day) reduced brain infarct size (p < 0.05). All doses of tacrolimus reduced pup weight compared to the controls. (4) Conclusions: Tacrolimus administration 3 days pre-HI was neuroprotective, likely mediated through neuroinflammatory and cell death pathways. Tacrolimus post-HI may have limited capacity to reduce brain injury, with higher doses increasing rat pup mortality. This work highlights the benefits of targeting neuroinflammation during the acute injurious period. More specific targeting of neuroinflammation, e.g., via T-cells, warrants further investigation. Full article

(This article belongs to the Special Issue New Advances in Neuroinflammation)

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21 pages, 2428 KiB

Open AccessArticle

LGR5 Expression Predicting Poor Prognosis Is Negatively Correlated with WNT5A in Colon Cancer

by Lubna M. Mehdawi, Souvik Ghatak, Payel Chakraborty, Anita Sjölander and Tommy Andersson

Cells 2023, 12(22), 2658; https://doi.org/10.3390/cells12222658 - 20 Nov 2023

Cited by 2 | Viewed by 2110

Abstract

WNT/β-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair β-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of [...] Read more.

WNT/β-catenin signaling is essential for colon cancer development and progression. WNT5A (ligand of non-canonical WNT signaling) and its mimicking peptide Foxy5 impair β-catenin signaling in colon cancer cells via unknown mechanisms. Therefore, we investigated whether and how WNT5A signaling affects two promoters of β-catenin signaling: the LGR5 receptor and its ligand RSPO3, as well as β-catenin activity and its target gene VEGFA. Protein and gene expression in colon cancer cohorts were analyzed by immunohistochemistry and qRT-PCR, respectively. Three colon cancer cell lines were used for in vitro and one cell line for in vivo experiments and results were analyzed by Western blotting, RT-PCR, clonogenic and sphere formation assays, immunofluorescence, and immunohistochemistry. Expression of WNT5A (a tumor suppressor) negatively correlated with that of LGR5/RSPO3 (tumor promoters) in colon cancer cohorts. Experimentally, WNT5A signaling suppressed β-catenin activity, LGR5, RSPO3, and VEGFA expression, and colony and spheroid formations. Since β-catenin signaling promotes colon cancer stemness, we explored how WNT5A expression is related to that of the cancer stem cell marker DCLK1. DCLK1 expression was negatively correlated with WNT5A expression in colon cancer cohorts and was experimentally reduced by WNT5A signaling. Thus, WNT5A and Foxy5 decrease LGR5/RSPO3 expression and β-catenin activity. This inhibits stemness and VEGFA expression, suggesting novel treatment strategies for the drug candidate Foxy5 in the handling of colon cancer patients. Full article

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Figure 1
Expression of WNT5A and LGR5 proteins in colon cancer tissue and their relation to the survival of these patients. (A) Representative IHC staining images of the WNT5A protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. (B) Representative IHC staining images of the LGR5 protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. (C) Representative IHC staining images of low and high WNT5A protein expression in colon cancer tissue. (D) Multivariate overall five-year survival analysis for low-risk (high WNT5A expression) and high-risk (low WNT5A expression) patients when adjusted for sex, LNM and TNM stage (<a href="#app1-cells-12-02658" class="html-app">Table S1</a>). (E) Representative IHC staining images of low and high LGR5 protein expression in colon cancer tissue. (F) Multivariate overall five-year survival analysis for low-risk (low LGR5 expression) and high-risk (high LGR5 expression) patients when adjusted for the prognostic factors sex and TNM stage (<a href="#app1-cells-12-02658" class="html-app">Table S1</a>). The results in Panels A and B are shown as the mean ± SEM; *** p < 0.001, analyzed with Wilcoxon’s match paired Student’s t test. Full article ">Figure 2
mRNA expression levels of WNT5A and LGR5 in colon cancer tissue and their correlations with the survival of colon cancer patients based on data from publicly available cohorts. Using the TCGA-COAD cohort, we performed univariate five-year overall survival analysis for (A) WNT5A and (B) LGR5. (C) Five-year overall survival analysis of 4 possible groups based on the combinations of WNT5A (low and high) and LGR5 (low and high) mRNA expressions. (D) Spearman’s correlation test between WNT5A and LGR5 mRNA expressions using the publicly available GSE44076 cohort. Full article ">Figure 3
Effects of Foxy5 on colony formation and LGR5 expression in vitro and in vivo. (A) HCT-116 colon cancer cells were grown to form colonospheres to optimize the expression of LGR5. Thereafter, the cells were treated with either vehicle or 100 µM Foxy5 for 48 h. The depicted images of the colonospheres were taken after these treatments using inverted phase-contrast microscopy without (upper images) or with ImageJ analysis (lower images). (B) The effects of Foxy5 treatment on LGR5 expression in these colonospheres were analyzed by Western blotting and the results are presented in the graph. (C) HT-29 colon cancer cells treated in vitro with vehicle or 100 µM Foxy5 for 24 h, after which LGR5 mRNA expression was analyzed. (D) Representative images of colony formation of HCT-116 cells stimulated with vehicle (0.1% BSA in water) or with 400 ng/mL rWNT5A and with vehicle (NaCl) or 100 µM Foxy5 for 24 h are shown. The results in panels (B–D) are presented in the graphs as the mean ± SEM for at least three separate experiments. (E) LGR5 mRNA expression in HT-29 colon cancer xenografts was evaluated by qRT-PCR and the results are presented in the graph. (F) Representative IHC images of LGR5 protein expression in the HT-29 cell xenografts from mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in panels (E,F) are presented as the mean ± SEM, and 5 xenograft tissues from 5 different mice were analyzed per group of animals. * p < 0.05, ** p < 0.01 and *** p < 0.001, analyzed with Mann-Whitney unpaired Student’s t test. Full article ">Figure 4
Effects of Foxy5 on LGR5 and β-catenin in colon cancer cells and correlation between β-catenin and WNT5A transcript expressions. SW480 colon cancer cells were treated with either vehicle or 100 µM Foxy5 for 24 h. (A) Representative immunofluorescence images of LGR5 and active β-catenin in SW480 cells treated with vehicle (left panels) or Foxy5 (right panels). The scale bars represent 10 µm in the upper panel and 5 µm in the lower panel. The mean fluorescence intensity (MFI) of (B) LGR5, (C) total active β-catenin, and (D) nuclear active β-catenin are outlined in their respective graphs and shown as the mean ± SEM for at least three independent experiments. * p < 0.05 and ** p < 0.01, analyzed with Mann-Whitney unpaired Student’s t test. (E) Spearman’s correlation test reveals a correlation between the expression of CTNNB1 and WNT5A transcripts based on data from the GSE44076 cohort. Full article ">Figure 5
The expression of the LGR5 ligand RSPO3 correlates positively with LGR5 expression and negatively with WNT5A expression in colon cancer tissue and is downregulated by Foxy5. (A) Representative IHC staining images of the RSPO3 protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. The results are shown as the mean ± SEM; * p < 0.05, analyzed with Wilcoxon match-paired Student’s t test. (B) Spearman’s correlation test between LGR5 and RSPO3 protein expressions in the Malmö-CC cohort. (C) Spearman’s correlation test between LGR5 and RSPO3 mRNA expressions using the publicly available GSE44076 cohort. (D) Spearman’s correlation test between RSPO3 and WNT5A mRNA expressions using the publicly available GSE44076 cohort. (E) RSPO3 mRNA expression in HT-29 colon cancer cells stimulated in vitro with or without 100 µM Foxy5 for 24 h. The result in the graph is shown as the mean ± SEM for at least three independent experiments. (F) RSPO3 mRNA levels in HT-29 colon cancer tissues from xenograft mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in the graph are shown as the mean ± SEM. (G) Representative IHC images of RSPO3 protein expression in the HT-29 cell xenografts from mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in Panels (F,G) are presented in the graph as the mean ± SEM, and 5 xenograft tissues from 5 different mice were analyzed per group of animals. * p < 0.05, ** p < 0.01, analyzed with Mann-Whitney unpaired Student’s t test. Full article ">Figure 6
The expression of the β-catenin target VEGFA predicts a poor prognosis, correlates with the expression of WNT5A and β-catenin at the transcript level in colon cancer tissue, and is downregulated by WNT5A signaling. (A) Using the TCGA-COAD cohort, we performed univariate five-year overall survival analysis for VEGFA. (B) Five-year overall survival analysis of 4 possible patient groups from the TCGA-COAD cohort based on the combinations of VEGFA (low and high) and WNT5A (low and high) mRNA expressions. Spearman’s correlation tests are shown between (C) VEGFA and WNT5A mRNA expression levels and (D) VEGFA and β-catenin mRNA (CTNNB1). Both correlations were made based on data from the publicly available GSE44076 cohort. (E) This panel outlines VEGFA mRNA expression in HT-29 colon cancer cells stimulated in vitro with or without 400 ng/mL rWNT5A or 100 µM Foxy5 for 24 h. (F) Representative Western blot and the accumulated densitometric VEGFA protein expression in HCT-116 colon cancer cells stimulated in vitro with or without 400 ng/mL rWNT5A or 100 µM Foxy5 for 24 h. The results in the graphs are shown as the mean ± SEM for at least three independent experiments. (G) VEGFA mRNA levels in HT-29 colon cancer tissues from xenograft mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. (H) Representative IHC images of VEGFA protein expression in the HT-29 colon cancer cell xenografts from mice treated with vehicle or Foxy5 (2 µg/g) every other day for a 14-day period. The results in panels (G,H) are presented in the graphs as the mean ± SEM (5 xenograft tissues from 5 different mice were analyzed per group of animals); * p < 0.05, ** p < 0.01 and *** p < 0.001, analyzed with Mann-Whitney unpaired Student’s t test. Full article ">Figure 7
The expression of the colon cancer stem cell marker DCLK1 predicts a poor prognosis, correlates with the expression of WNT5A in colon cancer tissue, and is downregulated by Foxy5. (A) Representative IHC staining images of the DCLK1 protein in normal mucosa and matched colon cancer tissue and their mean IRS values are presented in the graph. The results are given as the mean ± SEM; *** p < 0.001, analyzed with Wilcoxon match-paired Student’s t test. (B) Representative IHC staining images of low and high DCLK1 protein expression in colon cancer tissue. (C) Univariate five-year overall survival analysis of patients with either low or high DCLK1 expression in their tumors. (D) Multivariate five-year overall survival analysis for low-risk (low DCLK1 expression) and high-risk (high DCLK1 expression) patients when adjusted for sex, LNM and TNM stage (<a href="#app1-cells-12-02658" class="html-app">Table S1</a>). (E) Using the TCGA-COAD cohort we performed univariate five-year overall survival analysis for DCLK1. (F) Five-year overall survival analysis of four possible groups from the TCGA-COAD cohort based on the combinations of DCLK1 (low and high) and WNT5A (low and high) mRNA expressions. (G) Spearman’s correlation test between DCLK1 and WNT5A mRNA expressions based on data from the publicly available GSE44076 cohort. (H) HCT-116 colon cancer cells were grown to form colonospheres, and then the cells were treated with either vehicle or 100 µM Foxy5. (I) DCLK1 mRNA expression in HT-29 colon cancer cells stimulated in vitro with or without 100 µM Foxy5. The effects of Foxy5 treatment on DCLK1 expression were analyzed by Western blotting (panel H) or RT-qPCR (panel I) and the results are presented in the graphs as the mean ± SEM for at least three independent experiments; ** p < 0.01, analyzed with Mann-Whitney unpaired Student’s t test. Full article ">

17 pages, 2223 KiB

Open AccessArticle

The Posttraumatic Increase of the Adhesion GPCR EMR2/ADGRE2 on Circulating Neutrophils Is Not Related to Injury Severity

by Leyu Zheng, Moujie Rang, Carolin Fuchs, Annette Keß, Mandy Wunsch, Julia Hentschel, Cheng-Chih Hsiao, Christian Kleber, Georg Osterhoff and Gabriela Aust

Cells 2023, 12(22), 2657; https://doi.org/10.3390/cells12222657 - 20 Nov 2023

Viewed by 1373

Abstract

Trauma triggers a rapid innate immune response to aid the clearance of damaged/necrotic cells and their released damage-associated molecular pattern (DAMP). Here, we monitored the expression of EMR2/ADGRE2, involved in the functional regulation of innate immune cells, on circulating neutrophils in [...] Read more.

Trauma triggers a rapid innate immune response to aid the clearance of damaged/necrotic cells and their released damage-associated molecular pattern (DAMP). Here, we monitored the expression of EMR2/ADGRE2, involved in the functional regulation of innate immune cells, on circulating neutrophils in very severely and moderately/severely injured patients up to 240 h after trauma. Notably, neutrophilic EMR2 showed a uniform, injury severity- and type of injury-independent posttraumatic course in all patients. The percentage of EMR2⁺ neutrophils and their EMR2 level increased and peaked 48 h after trauma. Afterwards, they declined and normalized in some, but not all, patients. Circulating EMR2⁺ compared to EMR2⁻ neutrophils express less CD62L and more CD11c, a sign of activation. Neutrophilic EMR2 regulation was verified in vitro. Remarkably, it increased, depending on extracellular calcium, in controls as well. Cytokines, enhanced in patients immediately after trauma, and sera of patients did not further affect this neutrophilic EMR2 increase, whereas apoptosis induction disrupted it. Likely the damaged/necrotic cells/DAMPs, unavoidable during neutrophil culture, stimulate the neutrophilic EMR2 increase. In summary, the rapidly increased absolute number of neutrophils, especially present in very severely injured patients, together with upregulated neutrophilic EMR2, may expand our in vivo capacity to react to and finally clear damaged/necrotic cells/DAMPs after trauma. Full article

(This article belongs to the Special Issue Structures, Regulation, Signaling, and Physiological Functions of Adhesion G-Protein Coupled Receptors)

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Posttraumatic course of leukocyte blood counts.(a) Correlation of the percentage of subsets within all leukocytes (set 100%) quantified via flow cytometry (flow) or an automatic blood cell analyzer (auto); n = 32 patients, n= 186 time points; Spearman’s correlation coefficient r and p values are shown. (b–d) Absolute numbers of circulating leukocytes (b), mature (c), and immature neutrophils (d) in patients 1–240 h after trauma, determined using an automatic analyzer. Comparison between patient groups ISS < 25 (9–24) and ISS ≥ 25, t-test (leukocytes, mature neutrophils), U-test (immature neutrophils); comparison between consecutive time points in one patient group: ANOVA; only significant changes related to the previous time point are shown; * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 2
Posttraumatic course of EMR2 in circulating neutrophils. (a,b) Percentage of EMR2+ mature (CD16high) neutrophils (a) and median fluorescence intensity (MFI) of EMR2 (b) in these cells. (c) Time course of EMR2 expression on neutrophils of one typical patient. The percentage of EMR2+ cells is indicated in the graph; the MFI of EMR2+ cells (at 1 and 8 h isotype control) is shown in the upper right corner. (d,e) Percentage of CD16low EMR2 neutrophils (d) and their EMR2 MFI (e). (a–e) Comparison between patient groups ISS < 25 (9–24) and ISS ≥ 25: t-test; comparison between consecutive time points in one patient group: ANOVA, only significant changes related to the previous time point are shown; * p < 0.05, ** p < 0.01, *** p < 0.001. (f) The expression of ADGRE2, encoding EMR2, during the maturation of myeloid cells. RNA sequencing data were derived from [<a href="#B30-cells-12-02657" class="html-bibr">30</a>]. ADGRE2 is upregulated during the maturation of neutrophils. ADGRE1, encoding EMR1, is present only in circulating eosinophils [<a href="#B19-cells-12-02657" class="html-bibr">19</a>]. Thus, it decreased after the immature stage. FPKM, fragments per kilobase per million mapped reads; PMNC, peripheral blood polymorphonuclear cells; imm, immature; seg, segmented neutrophils. (g) Quantitation of ADGRE2 via qRT-PCR in circulating leukocytes in the posttraumatic course; n = 5 injured patients (1/5 patient only 1–48 h), n = 4 uninjured volunteers (1/4 only 1–48 h); mean ± SEM. Full article ">Figure 3
EMR2− and EMR2+ neutrophils differ in the expression of activation-related CD62L and CD11c. Leukocytes of polytrauma patients (n = 6, 24 h after trauma) were left untreated or stimulated with 10 ng/mL TNFα for 20 min, mAb-stained, and analyzed via flow cytometry. (a) The EMR2- and EMR2+ neutrophils were separately analyzed for (b) CD62L, CD11b, and CD11c; mean ± SEM, paired t-test, ** p < 0.011. Full article ">Figure 4
Expression of EMR2 on circulating neutrophils after trauma and its correlation to clinic. (a,b) Correlation of the percentage of EMR2+ neutrophils and the expression level of EMR2 on these cells to clinicopathological parameters 240 h after trauma. In the correlation matrix (a), square color indicates the magnitude of correlation; only significant correlations (Spearman’s) are shown. In (b), the significant correlations of the expression level of EMR2 on neutrophils to CRP, IL-6, PCT, and days of hospitalization are visualized. (c) Posttraumatic course of CRP, comparison between patient groups ISS < 25 (9–24) and ISS ≥ 25, t-test; comparison between consecutive time points in one patient group: ANOVA, only significant changes related to the previous time point are shown; * p < 0.05, ** p < 0.01, *** p < 0.001. (d) Circulating CRP levels and expression levels of EMR2 on neutrophils in patients 240 h after trauma. Patients who underwent surgery (n = 16) within 120–240 h after trauma were compared to patients not operated on in this period; unpaired t-test, ** p < 0.01). Full article ">Figure 5
Regulation of EMR2 expression on neutrophils in vitro. (a–d) Ca2+-dependent increase in EMR2 expression on neutrophils during culture. Peripheral leukocytes of uninjured volunteers were cultured in medium/10% FCS (control, ctr) for the indicated time, some EDTA and BAPTA-AM was added. EMR2 was analyzed on mature neutrophils using flow cytometry. (a) EMR2 (MFI) on neutrophils; n = 3–14 volunteers/time point, * p < 0.05, *** p < 0.001, unpaired t-test. (b) Leukocytes were incubated in medium with 10% FCS (ctr), serum, or plasma of either patients or volunteers for 17 h. Patients serum and plasma were taken 24 h after injury. One typical experiment is shown. The percentage of EMR2+ neutrophils after 17 h is indicated in the graph, the EMR2 MFI is shown in the upper right corner. (c,d) EDTA, but not BAPTA-AM, prevented the increase in EMR2 expression on neutrophils in vitro. (c) Leukocytes of volunteers (n = 8) were incubated in medium/10% FCS (ctr) for 17 h without or with 1 mM EDTA or 1 μM BAPTA-AM; *** p < 0.001, paired t-test. (d) One typical experiment is shown. The percentage of EMR2+ neutrophils after 17 h is indicated in the graph, MFI of EMR2+ cells is shown in the upper right corner. (e,f) EMR2 expression on apoptotic neutrophils. Leukocytes were either cultured in serum-free medium or in medium/10% FCS (ctr) and with staurosporin or TNFα/cycloheximid for 17 h. (e) EMR2 MFI was quantified on neutrophils using flow cytometry; n = 4–6 volunteers/condition, Mann–Whitney U-test, ** p < 0.01. (f) One typical experiment is shown. Upper panel: scatter analysis of all cells (pre-gating to single cells only). The percentage of neutrophils and dead cells/cells debris (low forward scatter, FSC-A) is indicated. Middle and lower panel: the percentage of neutrophils expressing EMR2 and CD16 was quantified and indicated at/or in the gate; MFI of EMR2+ neutrophils is shown in the upper right corner. Full article ">

16 pages, 1589 KiB

Open AccessReview

Histopathological Markers for Target Therapies in Primary Cutaneous Lymphomas

by Benedetta Sonego, Adalberto Ibatici, Giulia Rivoli, Emanuele Angelucci, Simona Sola and Cesare Massone

Cells 2023, 12(22), 2656; https://doi.org/10.3390/cells12222656 - 20 Nov 2023

Cited by 1 | Viewed by 1570

Abstract

In recent years, targeted (biological) therapies have become available also for primary cutaneous T-cell lymphomas (PCTCLs) including anti-CD30 (brentuximab vedotin) in mycosis fungoides, primary cutaneous anaplastic large T-cell lymphoma, lymphomatoid papulosis; anti-CCR4 (mogamulizumab) in Sezary syndrome; anti-CD123 (tagraxofusp) in blastic plasmocytoid cell neoplasm. [...] Read more.

In recent years, targeted (biological) therapies have become available also for primary cutaneous T-cell lymphomas (PCTCLs) including anti-CD30 (brentuximab vedotin) in mycosis fungoides, primary cutaneous anaplastic large T-cell lymphoma, lymphomatoid papulosis; anti-CCR4 (mogamulizumab) in Sezary syndrome; anti-CD123 (tagraxofusp) in blastic plasmocytoid cell neoplasm. Moreover, anti-PD1 (nivolumab), anti-PDL1 (pembrolizumab, atezolizumab), anti-CD52 (alemtuzumab), anti-KIR3DL2-CD158k (lacutamab), and anti-CD70 (cusatuzumab) have been tested or are under investigations in phase II trials. The expression of these epitopes on neoplastic cells in skin biopsies or blood samples plays a central role in the management of PCTCL patients. This narrative review aims to provide readers with an update on the latest advances in the newest therapeutic options for PCTCLs. Full article

(This article belongs to the Special Issue Molecular Biomarkers and Signaling Pathways for Skin Cancer Screening, Diagnosis and Therapy)

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33 pages, 2457 KiB

Open AccessReview

Molecular Mechanisms Associated with Antifungal Resistance in Pathogenic Candida Species

by Karolina M. Czajka, Krishnan Venkataraman, Danielle Brabant-Kirwan, Stacey A. Santi, Chris Verschoor, Vasu D. Appanna, Ravi Singh, Deborah P. Saunders and Sujeenthar Tharmalingam

Cells 2023, 12(22), 2655; https://doi.org/10.3390/cells12222655 - 19 Nov 2023

Cited by 20 | Viewed by 7212

Abstract

Candidiasis is a highly pervasive infection posing major health risks, especially for immunocompromised populations. Pathogenic Candida species have evolved intrinsic and acquired resistance to a variety of antifungal medications. The primary goal of this literature review is to summarize the molecular mechanisms associated [...] Read more.

Candidiasis is a highly pervasive infection posing major health risks, especially for immunocompromised populations. Pathogenic Candida species have evolved intrinsic and acquired resistance to a variety of antifungal medications. The primary goal of this literature review is to summarize the molecular mechanisms associated with antifungal resistance in Candida species. Resistance can be conferred via gain-of-function mutations in target pathway genes or their transcriptional regulators. Therefore, an overview of the known gene mutations is presented for the following antifungals: azoles (fluconazole, voriconazole, posaconazole and itraconazole), echinocandins (caspofungin, anidulafungin and micafungin), polyenes (amphotericin B and nystatin) and 5-fluorocytosine (5-FC). The following mutation hot spots were identified: (1) ergosterol biosynthesis pathway mutations (ERG11 and UPC2), resulting in azole resistance; (2) overexpression of the efflux pumps, promoting azole resistance (transcription factor genes: tac1 and mrr1; transporter genes: CDR1, CDR2, MDR1, PDR16 and SNQ2); (3) cell wall biosynthesis mutations (FKS1, FKS2 and PDR1), conferring resistance to echinocandins; (4) mutations of nucleic acid synthesis/repair genes (FCY1, FCY2 and FUR1), resulting in 5-FC resistance; and (5) biofilm production, promoting general antifungal resistance. This review also provides a summary of standardized inhibitory breakpoints obtained from international guidelines for prominent Candida species. Notably, N. glabrata, P. kudriavzevii and C. auris demonstrate fluconazole resistance. Full article

(This article belongs to the Special Issue Fungal Infections and Resistance)

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19 pages, 844 KiB

Open AccessReview

Gut Microbiota, Inflammatory Bowel Disease, and Cancer: The Role of Guardians of Innate Immunity

by Vincenzo Giambra, Danilo Pagliari, Pierluigi Rio, Beatrice Totti, Chiara Di Nunzio, Annalisa Bosi, Cristina Giaroni, Antonio Gasbarrini, Giovanni Gambassi and Rossella Cianci

Cells 2023, 12(22), 2654; https://doi.org/10.3390/cells12222654 - 19 Nov 2023

Cited by 5 | Viewed by 3379

Abstract

Inflammatory bowel diseases (IBDs) are characterized by a persistent low-grade inflammation that leads to an increased risk of colorectal cancer (CRC) development. Several factors are implicated in this pathogenetic pathway, such as innate and adaptive immunity, gut microbiota, environment, and xenobiotics. At the [...] Read more.

Inflammatory bowel diseases (IBDs) are characterized by a persistent low-grade inflammation that leads to an increased risk of colorectal cancer (CRC) development. Several factors are implicated in this pathogenetic pathway, such as innate and adaptive immunity, gut microbiota, environment, and xenobiotics. At the gut mucosa level, a complex interplay between the immune system and gut microbiota occurs; a disequilibrium between these two factors leads to an alteration in the gut permeability, called ‘leaky gut’. Subsequently, an activation of several inflammatory pathways and an alteration of gut microbiota composition with a proliferation of pro-inflammatory bacteria, known as ‘pathobionts’, take place, leading to a further increase in inflammation. This narrative review provides an overview on the principal Pattern Recognition Receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs), focusing on their recognition mechanisms, signaling pathways, and contributions to immune responses. We also report the genetic polymorphisms of TLRs and dysregulation of NLR signaling pathways that can influence immune regulation and contribute to the development and progression of inflammatory disease and cancer. Full article

(This article belongs to the Special Issue Gut Microbiota and Inflammatory Bowel Disease)

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13 pages, 1332 KiB

Open AccessReview

Effects of Everolimus in Modulating the Host Immune Responses against Mycobacterium tuberculosis Infection

by Anmol Raien, Sofia Davis, Michelle Zhang, David Zitser, Michelle Lin, Graysen Pitcher, Krishna Bhalodia, Selvakumar Subbian and Vishwanath Venketaraman

Cells 2023, 12(22), 2653; https://doi.org/10.3390/cells12222653 - 18 Nov 2023

Cited by 1 | Viewed by 1948

Abstract

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (P13K/AKT/mTOR) pathway plays a key role in tuberculosis (TB) pathogenesis and infection. While the activity levels of this pathway during active infection are still debated, manipulating this pathway shows potential benefit for host-directed therapies. Some [...] Read more.

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (P13K/AKT/mTOR) pathway plays a key role in tuberculosis (TB) pathogenesis and infection. While the activity levels of this pathway during active infection are still debated, manipulating this pathway shows potential benefit for host-directed therapies. Some studies indicate that pathway inhibitors may have potential for TB treatment through upregulation of autophagy, while other studies do not encourage the use of these inhibitors due to possible host tissue destruction by Mycobacterium tuberculosis (M. tb) and increased infection risk. Investigating further clinical trials and their use of pathway inhibitors is necessary in order to ascertain their potential for TB treatment. This paper is particularly focused on the drug everolimus, an mTOR inhibitor. One of the first clinical trials sponsored by the Aurum Institute showed potential benefit in using everolimus as an adjunctive therapy for tuberculosis. Infection with tuberculosis is associated with a metabolic shift from oxidative phosphorylation towards glycolysis. The everolimus arm in the clinical trial showed further reduction than the control for both maximal and peak glycolytic activity. Compared with control, those receiving everolimus demonstrated increased lung function through forced expiratory volume in 1 s (FEV1) measurements, suggesting that everolimus may mitigate inflammation contributing to lung damage. Full article

(This article belongs to the Special Issue PI3K/AKT/mTOR Signaling Network in Human Health and Diseases 2.0)

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28 pages, 2248 KiB

Open AccessReview

The Neuropharmacological Evaluation of Seaweed: A Potential Therapeutic Source

by Khoshnur Jannat, Rengasamy Balakrishnan, Jun-Hyuk Han, Ye-Ji Yu, Ga-Won Kim and Dong-Kug Choi

Cells 2023, 12(22), 2652; https://doi.org/10.3390/cells12222652 - 18 Nov 2023

Cited by 2 | Viewed by 2816

Abstract

The most common neurodegenerative diseases (NDDs), such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), are the seventh leading cause of mortality and morbidity in developed countries. Clinical observations of NDD patients are characterized by a progressive loss of neurons in the brain [...] Read more.

The most common neurodegenerative diseases (NDDs), such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), are the seventh leading cause of mortality and morbidity in developed countries. Clinical observations of NDD patients are characterized by a progressive loss of neurons in the brain along with memory decline. The common pathological hallmarks of NDDs include oxidative stress, the dysregulation of calcium, protein aggregation, a defective protein clearance system, mitochondrial dysfunction, neuroinflammation, neuronal apoptosis, and damage to cholinergic neurons. Therefore, managing this pathology requires screening drugs with different pathological targets, and suitable drugs for slowing the progression or prevention of NDDs remain to be discovered. Among the pharmacological strategies used to manage NDDs, natural drugs represent a promising therapeutic strategy. This review discusses the neuroprotective potential of seaweed and its bioactive compounds, and safety issues, which may provide several beneficial insights that warrant further investigation. Full article

(This article belongs to the Section Plant, Algae and Fungi Cell Biology)

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Hallmarks of neurodegenerative diseases and neuroprotective potential target of seaweeds. These include environmental risk factors, metabolic stress associated with mitochondrial dysfunction as well as oxidative stress, genetic contribution, misfolded protein aggregation, and neuroinflammation. Full article ">Figure 2
Structures of some chemical compounds of seaweeds. Full article ">Figure 3
Pathophysiology of Alzheimer’s disease and Parkinson’s disease and the protective effect of seaweed extract and their bioactive compounds: 1. seaweed extracts, k-carrageenan, and fucoidan; 2, 3. seaweed extract, k-carrageenan, fucoidan, alginate, fucoxanthin, and taurine; 4. extract of K. malesians and E. cava, fucoidan, fucoxanthin, and carotenoid; 5. fucoidan; 6. fucoidan; 7. U. lactuca extract, taurine; 8. polymannuronic acid, fucoidan, phlorotannin, and fatty acids; 9. Sargassum, E. cava, B. bifurcate, C. tomentosum extract, and fucoidan; 10. seaweed extract, fucoidan, dieckol, fucoxanthin, and DHA; 11. Sargassum, P. pavonica, B. bifurcata, extract, dieckol, and eleganolone; 12. phycoerythrin; 13. phycoerythrin, dieckol; 14. E. cava, P. gymnospora, E. prolifera, C. tomentosum, H. valentae extracts, fucoidan, fucoxanthin, and phlorotannin; 15. U. lactuca, I. foliacea, E. cava, E. prolifera extracts, fucosterol, fucoxanthin, and fucoidan; 16. E. prolifera, P. pavonica, S. fusiforme, A. nodosum, E. radiata, G. gracilis extract, taurine, dieckol, fucoxanthin, and alginate; 17. sargassum extract, fucoxanthin, fatty acid, polymannuronic acid, k-carrageenan, fucoidan, and eleganolone. Full article ">

14 pages, 9113 KiB

Open AccessArticle

Ciliary Ultrastructure Assessed by Transmission Electron Microscopy in Adults with Bronchiectasis and Suspected Primary Ciliary Dyskinesia but Inconclusive Genotype

by Ben O. Staar, Jan Hegermann, Bernd Auber, Raphael Ewen, Sandra von Hardenberg, Ruth Olmer, Isabell Pink, Jessica Rademacher, Martin Wetzke and Felix C. Ringshausen

Cells 2023, 12(22), 2651; https://doi.org/10.3390/cells12222651 - 18 Nov 2023

Cited by 1 | Viewed by 1822

Abstract

Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain [...] Read more.

Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain significance in approximately 30% of tested individuals. We aimed to assess genotype–phenotype correlations in adults with bronchiectasis, clinical suspicion of PCD, and inconclusive whole-exome sequencing results using transmission electron microscopy (TEM) and ciliary image averaging by the PCD Detect software. We recruited 16 patients with VUS in CCDC39, CCDC40, CCDC103, DNAH5, DNAH5/CCDC40, DNAH8/HYDIN, DNAH11, and DNAI1 as well as variants in the PCD candidate genes DNAH1, DNAH7, NEK10, and NME5. We found normal ciliary ultrastructure in eight patients with VUS in CCDC39, DNAH1, DNAH7, DNAH8/HYDIN, DNAH11, and DNAI1. In six patients with VUS in CCDC40, CCDC103, DNAH5, and DNAI1, we identified a corresponding ultrastructural hallmark defect. In one patient with homozygous variant in NME5, we detected a central complex defect supporting clinical relevance. Using TEM as a targeted approach, we established important genotype–phenotype correlations and definite PCD in a considerable proportion of patients. Overall, the PCD Detect software proved feasible in support of TEM. Full article

(This article belongs to the Special Issue The Role of Cilia in Health and Diseases)

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Microtubular disorganization and inner dynein arm (MTD + IDA) defects in three patients with VUS in CCDC40. Top: TEM images (greyscale); below: averaged cutout images from the PCD Detect software (colored), representing one microtubule doublet corresponding to the pair oriented downward in TEM. Negative control shows normal ciliary ultrastructure with 9 + 2 arrangement and both dynein arms present (arrowheads). In contrast, arrangement of nine microtubule doublets and a central pair is disorganized and IDAs are absent in the depicted patient samples (arrows). Color scale: Black (TEM) and blue (PCD Detect) as well as white (TEM) and yellow (PCD Detect) indicate high and low electron-density, respectively. Scale bars: 100 nm in TEM images and 10 nm in PCD Detect images. Abbreviations: IDA = inner dynein arm; MTD = microtubular disorganization; TEM = transmission electron microscopy; VUS = variant of unknown significance. Full article ">Figure 2
Outer dynein arm (ODA) defects in three patients with VUS in CCDC103, DNAH5, and DNAI1. Ciliary cross-sections in TEM images (greyscale images in upper row) and cutout images of microtubular doublets and ODA (if present) averaged with PCD Detect (colored images in lower row). ODA (white filled arrows) are absent in TEM images of three patients with VUS in CCDC103, DNAH5, and DNAI1, as well as the positive control (DNAH5). PCD Detect images of the positive control and Patient 8 show a partial loss of ODA with a remaining shortened ODA projection (black filled arrows), whereas Patients 1 and 4 show a complete loss of ODA (white filled arrows). Color scale: Black (TEM) and blue (PCD Detect) as well as white (TEM) and yellow (PCD Detect) indicate high and low electron-density, respectively. Scale bars: 100 nm in TEM images and 10 nm in PCD Detect images. Abbreviations: ODA = outer dynein arm; TEM = transmission electron microscopy; VUS = variant of unknown significance. Full article ">Figure 3
Central Complex (CC) defect in a patient with a likely pathogenic homozygous variant in the PCD candidate gene NME5. TEM images show examples of ciliary sections with 9 + 0 structure (A), normal 9 + 2 structure (B), 8 + 0 structure (C), 8 + 1 structure (D), and 9 + 4 structure (E). Scale bar: 100 nm. Abbreviation: TEM = transmission electron microscopy. Full article ">Figure 4
Normal ciliary ultrastructure in four patients with VUS in CCDC39, DNAH11, DNAI1, and DNAH7. TEM images of four patients with VUS show normal 9 + 2 structure and both dynein arms present (arrowheads) in conventional TEM imaging (greyscale images in upper row) and PCD Detect imaging (colored images in lower row). Color scale: Black (TEM) and blue (PCD Detect) as well as white (TEM) and yellow (PCD Detect) indicate high and low electron-density, respectively. Scale bars: 100 nm in TEM images and 10 nm in PCD Detect images. Abbreviations: TEM = transmission electron microscopy; VUS = variant of unknown significance. Full article ">

15 pages, 2238 KiB

Open AccessArticle

Sustained Nrf2 Overexpression-Induced Metabolic Deregulation Can Be Attenuated by Modulating Insulin/Insulin-like Growth Factor Signaling

by Sentiljana Gumeni, Maria Lamprou, Zoi Evangelakou, Maria S. Manola and Ioannis P. Trougakos

Cells 2023, 12(22), 2650; https://doi.org/10.3390/cells12222650 - 18 Nov 2023

Viewed by 1568

Abstract

The modulation of insulin/insulin-like growth factor signaling (IIS) is associated with altered nutritional and metabolic states. The Drosophila genome encodes eight insulin-like peptides, whose activity is regulated by a group of secreted factors, including Ecdysone-inducible gene L2 (ImpL2), which acts as [...] Read more.

The modulation of insulin/insulin-like growth factor signaling (IIS) is associated with altered nutritional and metabolic states. The Drosophila genome encodes eight insulin-like peptides, whose activity is regulated by a group of secreted factors, including Ecdysone-inducible gene L2 (ImpL2), which acts as a potent IIS inhibitor. We recently reported that cncC (cncC/Nrf2), the fly ortholog of Nrf2, is a positive transcriptional regulator of ImpL2, as part of a negative feedback loop aiming to suppress cncC/Nrf2 activity. This finding correlated with our observation that sustained cncC/Nrf2 overexpression/activation (cncC^OE; a condition that signals organismal stress) deregulates IIS, causing hyperglycemia, the exhaustion of energy stores in flies’ tissues, and accelerated aging. Here, we extend these studies in Drosophila by assaying the functional implication of ImpL2 in cncC^OE-mediated metabolic deregulation. We found that ImpL2 knockdown (KD) in cncC^OE flies partially reactivated IIS, attenuated hyperglycemia and restored tissue energetics. Moreover, ImpL2 KD largely suppressed cncC^OE-mediated premature aging. In support, pharmacological treatment of cncC^OE flies with Metformin, a first-line medication for type 2 diabetes, restored (dose-dependently) IIS functionality and extended cncC^OE flies’ longevity. These findings exemplify the effect of chronic stress in predisposition to diabetic phenotypes, indicating the potential prophylactic role of maintaining normal IIS functionality. Full article

(This article belongs to the Collection Insulin-Like Growth Factors in Development, Cancers and Aging)

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ImpL2 downregulation in Drosophila activates IIS. (a) Relative mRNA expression of ImpL2, dIlp2, InR, sgg, Akt, GlyS, and Atgl/bmm genes after ubiquitous downregulation of ImpL2. (b) Immunoblot analyses and relative immunoblotting quantification (n = 3) of tissue protein samples probed with antibodies against Akt, p-Akt, Gsk3, and p-Gsk3 after ubiquitous KD of ImpL2. (c) Confocal imaging of GLY (red, indicated by the arrows) immunofluorescence staining in larvae muscle tissues. Nuclei were stained with DAPI (blue). (d) Relative content of GLU and TREH in flies’ somatic tissues after ImpL2 KD (e) Confocal imaging of mitochondria (Atp5a) and fat body (Bodipy staining) of adult flies after ImpL2 KD. (f) Lipid droplets quantification (number/area) of the shown genotypes. Flies were exposed to 320 μM RU486 for 7 days. Gene expression (a) was plotted vs. control set to 1 (RpL32/rp49 gene was used as reference). Actin (b) probing was used to demonstrate equal protein loading. Bars, ±SD; n = 3, * p < 0.05. Full article ">Figure 2
KD of ImpL2 increases proteasome subunits expression and peptidases activity; it also mildly decreases flies’ longevity. (a) Immunoblot analyses and relative immunoblotting quantification (n = 3) of tissue protein samples probed with antibodies against proteasomal subunits p54/Rpn10, p42/Rpn7, 26S-α, and Rpn6 in flies’ tissues after ImpL2 KD. (b) Chymotrypsin (CT-L)- and caspase (C-L)-like activities of 26S proteasome after ImpL2 KD. (c) ROS levels in flies’ somatic tissues after ImpL2 KD. (d) Longevity curves of ImpL2 KD flies vs. control flies (log-rank, Mantel–Cox tests: non-induced flies vs. ImpL2RNAi-induced flies p < 0.0001). In (a–c), flies were exposed to 320 μM RU486 for 7 days. Gapdh probing (a) was used to demonstrate equal protein loading. Bars, ±SD; n = 3, * p < 0.05. Full article ">Figure 3
Concomitant ImpL2 KD in cncCOE transgenic flies does not affect cncCOE-mediated UPP activation. (a) Representative (n = 3) immunoblot analyses of tissue protein samples from the shown genotypes probed with antibodies against proteasomal subunits 26S-α, p42/Rpn7, and Rpn6. (b) Chymotrypsin (CT-L)- and caspase (C-L)-like 26S proteasome activities. (c) ROS levels of the shown genotypes. In (a–c), flies were exposed to 320 μM RU486 for 7 days. Gapdh probing (a) was used to demonstrate equal protein loading. Bars, ±SD; n = 3, * p < 0.05. Full article ">Figure 4
ImpL2 KD partially reverts sustained cncCOE-mediated toxic phenotypes. (a) Relative mRNA expression of ImpL2, dIlp2, dIlp3, Akt, InR, and sgg/Gsk3 genes after ubiquitous KD of ImpL2 in cncCOE flies. (b) Immunoblot analyses of tissue protein samples probed with antibodies against p-Akt, Akt, Gsk3, and p-Gsk3 after ubiquitous ImpL2 KD in cncCOE flies. (c) Confocal imaging of muscle mitochondria (Atp5a) and (d) of adult flies’ fat body lipid droplets (Bodipy). Nuclei were stained with DAPI (blue). (e) Lipid droplet quantification (number/area) of the indicated genotypes. (f) Relative content of GLU and TREH in somatic tissues of cncCOE or cncCOE, ImpL2RNAi adult flies. (g) Longevity curves of flies with the shown genotypes (log-rank, Mantel–Cox tests: cncCOE RU486+ vs. cncCOE, ImpL2RNAi RU486+ p < 0.000; cncCOE RU486+ vs. cncCOE, ImpL2RNAi, InRRNAi RU486+ p < 0.000; cncCOE, ImpL2RNAi RU486+ vs. cncCOE, ImpL2RNAi; InRRNAi RU486+ p < 0.000). Gene expression (a) was plotted vs. control set to 1 (RpL32/rp49 gene was used as reference). Actin probing (b) was used to demonstrate equal protein loading. Flies in (a–f) were exposed to 320 μM RU486 for 7 days. Bars, ±SD; n = 3, * p < 0.05; ** p < 0.01. Full article ">Figure 5
Met treatment partially suppresses the metabolic phenotypes induced in flies by sustained cncCOE. (a) Relative mRNA expression of ImpL2, dIlp2, dIlp3, Akt, InR, and sgg/Gsk3 genes after treatment with Met (metformin) at the indicated concentrations. (b) Confocal imaging of fat body (Bodipy) and muscle mitochondria (Atp5a) of adult flies of the described genotypes after Met treatment. (c) Lipid droplet quantification (number/area) of the shown genotypes. (d) Relative content of GLU and TREH levels (vs. non-treated cncCOE) in somatic tissues of cncCOE flies after Met treatment. (e) Longevity curves of cncCOE adult flies after treatment with Met at the indicated concentrations (log-rank, Mantel–Cox tests: cncCOE vs. cncCOE 1 mM Met p < 0.007; cncCOE vs. cncCOE 2 mM Met p < 0.001; cncCOE vs. cncCOE 5 mM Met p < 0.000). In (a–d), flies were exposed to 320 μM RU486 for 7 days. In (a–d), Met treatment was applied for 7 days, starting from day 1 of cncC transgene induction. Gene expression (a) was plotted vs. control set to 1 (RpL32/rp49 gene was used as reference). Bars, ±SD; n ≥ 3, * p < 0.05; ** p < 0.01. Full article ">

21 pages, 1042 KiB

Open AccessReview

From CGRP to PACAP, VIP, and Beyond: Unraveling the Next Chapters in Migraine Treatment

by Masaru Tanaka, Ágnes Szabó, Tamás Körtési, Délia Szok, János Tajti and László Vécsei

Cells 2023, 12(22), 2649; https://doi.org/10.3390/cells12222649 - 17 Nov 2023

Cited by 27 | Viewed by 7408

Abstract

Migraine is a neurovascular disorder that can be debilitating for individuals and society. Current research focuses on finding effective analgesics and management strategies for migraines by targeting specific receptors and neuropeptides. Nonetheless, newly approved calcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) have a [...] Read more.

Migraine is a neurovascular disorder that can be debilitating for individuals and society. Current research focuses on finding effective analgesics and management strategies for migraines by targeting specific receptors and neuropeptides. Nonetheless, newly approved calcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) have a 50% responder rate ranging from 27 to 71.0%, whereas CGRP receptor inhibitors have a 50% responder rate ranging from 56 to 71%. To address the need for novel therapeutic targets, researchers are exploring the potential of another secretin family peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), as a ground-breaking treatment avenue for migraine. Preclinical models have revealed how PACAP affects the trigeminal system, which is implicated in headache disorders. Clinical studies have demonstrated the significance of PACAP in migraine pathophysiology; however, a few clinical trials remain inconclusive: the pituitary adenylate cyclase-activating peptide 1 receptor mAb, AMG 301 showed no benefit for migraine prevention, while the PACAP ligand mAb, Lu AG09222 significantly reduced the number of monthly migraine days over placebo in a phase 2 clinical trial. Meanwhile, another secretin family peptide vasoactive intestinal peptide (VIP) is gaining interest as a potential new target. In light of recent advances in PACAP research, we emphasize the potential of PACAP as a promising target for migraine treatment, highlighting the significance of exploring PACAP as a member of the antimigraine armamentarium, especially for patients who do not respond to or contraindicated to anti-CGRP therapies. By updating our knowledge of PACAP and its unique contribution to migraine pathophysiology, we can pave the way for reinforcing PACAP and other secretin peptides, including VIP, as a novel treatment option for migraines. Full article

(This article belongs to the Special Issue Migraine Neuroscience: From Experimental Models to Target Therapy)

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13 pages, 684 KiB

Open AccessReview

Mechanisms of Endothelial Cell Membrane Repair: Progress and Perspectives

by Duoduo Zha, Shizhen Wang, Paula Monaghan-Nichols, Yisong Qian, Venkatesh Sampath and Mingui Fu

Cells 2023, 12(22), 2648; https://doi.org/10.3390/cells12222648 - 17 Nov 2023

Cited by 1 | Viewed by 2374

Abstract

Endothelial cells are the crucial inner lining of blood vessels, which are pivotal in vascular homeostasis and integrity. However, these cells are perpetually subjected to a myriad of mechanical, chemical, and biological stresses that can compromise their plasma membranes. A sophisticated repair system [...] Read more.

Endothelial cells are the crucial inner lining of blood vessels, which are pivotal in vascular homeostasis and integrity. However, these cells are perpetually subjected to a myriad of mechanical, chemical, and biological stresses that can compromise their plasma membranes. A sophisticated repair system involving key molecules, such as calcium, annexins, dysferlin, and MG53, is essential for maintaining endothelial viability. These components orchestrate complex mechanisms, including exocytosis and endocytosis, to repair membrane disruptions. Dysfunctions in this repair machinery, often exacerbated by aging, are linked to endothelial cell death, subsequently contributing to the onset of atherosclerosis and the progression of cardiovascular diseases (CVD) and stroke, major causes of mortality in the United States. Thus, identifying the core machinery for endothelial cell membrane repair is critically important for understanding the pathogenesis of CVD and stroke and developing novel therapeutic strategies for combating CVD and stroke. This review summarizes the recent advances in understanding the mechanisms of endothelial cell membrane repair. The future directions of this research area are also highlighted. Full article

(This article belongs to the Collection Endothelial Cell Dysfunction and Multi-organ Injury: From Molecular Mechanisms to Therapeutic Interventions)

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2 pages, 175 KiB

Open AccessEditorial

Cardiovascular Biomarkers: Current Status and Future Directions

by Mark G. Filipovic and Markus M. Luedi

Cells 2023, 12(22), 2647; https://doi.org/10.3390/cells12222647 - 17 Nov 2023

Viewed by 1712

Abstract

Cardiovascular disease (CVD) remains a global health concern of paramount significance, claiming millions of lives each year [...] Full article

(This article belongs to the Special Issue Cardiovascular Biomarkers: Current Status and Future Directions)

26 pages, 7248 KiB

Open AccessArticle

Elucidation of GPR55-Associated Signaling behind THC and LPI Reducing Effects on Ki67-Immunoreactive Nuclei in Patient-Derived Glioblastoma Cells

by Marc Richard Kolbe, Tim Hohmann, Urszula Hohmann, Erik Maronde, Ralph Golbik, Julian Prell, Jörg Illert, Christian Strauss and Faramarz Dehghani

Cells 2023, 12(22), 2646; https://doi.org/10.3390/cells12222646 - 17 Nov 2023

Viewed by 1642

Abstract

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids [...] Read more.

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB₁ and CB₂ receptors, THC modulates cellular responses among others via GPR55. Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67⁺ nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gβγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids. Full article

(This article belongs to the Collection Novel Insights into Cannabinoid Receptors, Molecular Targets, and Therapeutic Potentials)

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18 pages, 3061 KiB

Open AccessArticle

Assessment of Primary Human Liver Cancer Cells by Artificial Intelligence-Assisted Raman Spectroscopy

by Concetta Esposito, Mohammed Janneh, Sara Spaziani, Vincenzo Calcagno, Mario Luca Bernardi, Martina Iammarino, Chiara Verdone, Maria Tagliamonte, Luigi Buonaguro, Marco Pisco, Lerina Aversano and Andrea Cusano

Cells 2023, 12(22), 2645; https://doi.org/10.3390/cells12222645 - 17 Nov 2023

Cited by 1 | Viewed by 1729

Abstract

We investigated the possibility of using Raman spectroscopy assisted by artificial intelligence methods to identify liver cancer cells and distinguish them from their Non-Tumor counterpart. To this aim, primary liver cells (40 Tumor and 40 Non-Tumor cells) obtained from resected hepatocellular carcinoma (HCC) [...] Read more.

We investigated the possibility of using Raman spectroscopy assisted by artificial intelligence methods to identify liver cancer cells and distinguish them from their Non-Tumor counterpart. To this aim, primary liver cells (40 Tumor and 40 Non-Tumor cells) obtained from resected hepatocellular carcinoma (HCC) tumor tissue and the adjacent non-tumor area (negative control) were analyzed by Raman micro-spectroscopy. Preliminarily, the cells were analyzed morphologically and spectrally. Then, three machine learning approaches, including multivariate models and neural networks, were simultaneously investigated and successfully used to analyze the cells’ Raman data. The results clearly demonstrate the effectiveness of artificial intelligence (AI)-assisted Raman spectroscopy for Tumor cell classification and prediction with an accuracy of nearly 90% of correct predictions on a single spectrum. Full article

(This article belongs to the Collection Computational Imaging for Biophotonics and Biomedicine)

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(a) Raman spectra preprocessing steps. Architecture of the classifier based on (b) LDA and Tuned-LDA model, (c) PCA-LDA model, and (d) Convolutional and Recurrent Neural Networks trained using sliding window on Raman Spectra. (e) Schematic of the machine learning models used for the blind predictions. Full article ">Figure 2
Bright-field microscope images of uncultured human Non-Tumor (a–c) and Tumor cells (d–f) fixed on CaF2 slides (100× magnification; scale bar 2 µm). (g) Dimensional analysis of all Non-Tumor and Tumor cells. Full article ">Figure 3
(Top): Averaged Raman spectra of Tumor (red line) and Non-Tumor (black line, offset 10%) cells in the FP region. Solid lines show the average over 180 spectra ± standard deviation (shaded areas). (Bottom): Difference between averaged Raman spectra (black line). The horizontal solid line corresponds to 0 intensity, the two horizontal dashed lines correspond to a threshold of ±0.025 intensity. Highlighted in yellow, red, and blue are the Raman bands associated with lipids, nucleic acids, and proteins, respectively. In orange, the Raman peaks associated with lipids/proteins are shown. Full article ">Figure 4
PCA of the Raman spectra. PCA 2D data plot distribution of spectra of uncultured Tumor and Non-Tumor cells based on the first 3 PC components: (a) PC1 vs. PC2 and (b) PC1 vs. PC3. The ellipses account for a confidential level of 95% of the data. Full article ">Figure 5
Learning curve as function of the number of training examples for (a) LDA model and (b) LDA model after Hyper-parameters tuned the optimization methods. Full article ">Figure 6
The loss function trend over epochs, during training, for CNN-LSTM models of 22 and 56 layers with different learning rates. Full article ">

14 pages, 5620 KiB

Open AccessArticle

SMYD3 Modulates AMPK-mTOR Signaling Balance in Cancer Cell Response to DNA Damage

by Martina Lepore Signorile, Paola Sanese, Elisabetta Di Nicola, Candida Fasano, Giovanna Forte, Katia De Marco, Vittoria Disciglio, Marialaura Latrofa, Antonino Pantaleo, Greta Varchi, Alberto Del Rio, Valentina Grossi and Cristiano Simone

Cells 2023, 12(22), 2644; https://doi.org/10.3390/cells12222644 - 17 Nov 2023

Cited by 5 | Viewed by 1618

Abstract

Cells respond to DNA damage by activating a complex array of signaling networks, which include the AMPK and mTOR pathways. After DNA double-strand breakage, ATM, a core component of the DNA repair system, activates the AMPK-TSC2 pathway, leading to the inhibition of the [...] Read more.

Cells respond to DNA damage by activating a complex array of signaling networks, which include the AMPK and mTOR pathways. After DNA double-strand breakage, ATM, a core component of the DNA repair system, activates the AMPK-TSC2 pathway, leading to the inhibition of the mTOR cascade. Recently, we showed that both AMPK and mTOR interact with SMYD3, a methyltransferase involved in DNA damage response. In this study, through extensive molecular characterization of gastrointestinal and breast cancer cells, we found that SMYD3 is part of a multiprotein complex that is involved in DNA damage response and also comprises AMPK and mTOR. In particular, upon exposure to the double-strand break-inducing agent neocarzinostatin, SMYD3 pharmacological inhibition suppressed AMPK cascade activation and thereby promoted the mTOR pathway, which reveals the central role played by SMYD3 in the modulation of AMPK-mTOR signaling balance during cancer cell response to DNA double-strand breaks. Moreover, we found that SMYD3 can methylate AMPK at the evolutionarily conserved residues Lys411 and Lys424. Overall, our data revealed that SMYD3 can act as a bridge between the AMPK and mTOR pathways upon neocarzinostatin-induced DNA damage in gastrointestinal and breast cancer cells. Full article

(This article belongs to the Special Issue DNA Damage and DNA Repair: What’s New in Biology and Cancer Therapeutics?)

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Effects of NCS on the AMPK/mTOR pathways. (a) Immunoblotting showing the levels of proteins involved in the AMPK and mTOR pathways in HCT-116, MDA-MB-231, and AGS cells treated for 24 h with NCS (5 nM). (b) Immunoblotting showing the levels of proteins involved in the AMPK and mTOR pathways in SMYD3-KO cells (HCT-116 and MDA-MB-231) treated for 24 h with NCS (5 nM). H2AX phosphorylation (γH2AX) was analyzed as a control of the induced-DNA damage, VINCULIN was used as a loading control. Full article ">Figure 2
Effects of NCS on the AMPK/mTOR pathways after pre-treatment with EM127. (a–c) Immunoblotting showing the levels of proteins involved in the AMPK and mTOR pathways in HCT-116 (a), AGS (b) and MDA-MB-231 (c) cells pre-treated with the SMYD3 inhibitor EM127 (5 μM) for 24 h and then treated with NCS (5 nM) for 24 h. VINCULIN was used as a loading control. Full article ">Figure 3
Role of SMYD3 in the modulation of AMPK and mTOR signaling pathways. (a) Immunoblotting showing the levels of proteins involved in the AMPK pathway in HCT-116 cells pre-treated or not with EM127 (5 μM) for 24 h and then treated with NCS (5 nM) for 24 h and/or AICAR (5 mM) for 24 h. (b) Immunoblotting showing the levels of proteins involved in the mTOR pathway in HCT-116 cells pre-treated or not with EM127 (5μM) for 24 h and then treated with NCS (5 nM) for 24 h and/or rapamycin (100 nM) for 4 h. VINCULIN was used as a loading control. Full article ">Figure 4
Functional interaction between SMYD3, AMPK, and mTOR. Co-immunoprecipitation of endogenous SMYD3, AMPK, or mTOR in HCT-116 and MDA-MB-231 cells treated or not with NCS (5 nM) for 24 h. Anti-IgGs were used as a negative control. Full article ">Figure 5
SMYD3 methylates AMPK. (a) In vitro methylation assay showing AMPK (AAPK2) methylation by SMYD3. H4 was used as a SMYD3 control substrate. * p < 0.05 vs active SMYD3 (b) In silico methylation prediction analysis. Three in silico prediction servers were used to identify AMPK consensus methylation sites: GPS-MSP, Methyl Sight, and Musite Deep. (c) MS/MS spectrum of SQSK411PYDIMAEVYR and AMK424QLDFEWK, two peptides obtained by double proteolytic digestion of SMYD3-methylated AMPK with the endoproteinases trypsin and Glu-C. (d) Multiple sequence alignment of human AAPK2 and homologous proteins from other species. UniProt IDs are indicated on the left. Lysines 411 and 424 (red boxes) are located in highly conserved regions. CAEEL: C. elegans, PONAB: Pongo abelii. Full article ">

14 pages, 3554 KiB

Open AccessArticle

How to Differentiate between Resistant and Susceptible Wheat Cultivars for Leaf Rust Fungi Using Antioxidant Enzymes and Histological and Molecular Studies?

by Reda I. Omara, Omar Abdullah Alkhateeb, Ahmed Hassan Abdou, Gabr A. El-Kot, Atef A. Shahin, Heba I. Saad-El-Din, Rady Abdelghany, Wasimah B. AL-Shammari, Muayad Albadrani, Yaser Hafez and Khaled Abdelaal

Cells 2023, 12(22), 2643; https://doi.org/10.3390/cells12222643 - 17 Nov 2023

Cited by 3 | Viewed by 1422

Abstract

Eight wheat cultivars, Sakha-94, Giza-171, Sids-1, Sids-12, Sids-13, Shandweel-1, Misr-1, and Misr-2, were evaluated for leaf rust at the seedling and adult stages in the 2021 and 2022 seasons. Biochemical, histological, and genetic analyses were performed to determine the link between cultivars that [...] Read more.

Eight wheat cultivars, Sakha-94, Giza-171, Sids-1, Sids-12, Sids-13, Shandweel-1, Misr-1, and Misr-2, were evaluated for leaf rust at the seedling and adult stages in the 2021 and 2022 seasons. Biochemical, histological, and genetic analyses were performed to determine the link between cultivars that were either sensitive or resistant to the disease. Misr-2 and Giza-171 cultivars had the highest levels of resistance to leaf rust races in 2021 (LTCGT, STSJT, and TTTST) and 2022 (MBGJT, TTTKS, and TTTTT) at the seedling stage. However, at the adult stage, Sakha-94, Giza-171, Misr-1, and Misr-2 cultivars had the highest levels of resistance; consequently, they had the lowest final disease severity and the lowest values of AUDPC. The correlation between the seedling reaction and adult reaction was non-significant, with values of 0.4401 and 0.4793 in the 2021 and 2022 seasons, respectively. Throughout the biochemical, histological, and genetic analyses, it was observed that catalase, peroxidase, and polyphenol oxidase activities significantly increased in the resistant cultivars. The discoloration of superoxide (O₂-) and hydrogen peroxide (H₂O₂) significantly decreased in resistant and moderately resistant wheat cultivars (Sakha-94, Giza-171, Misr-1, and Misr-2); higher hydrogen peroxide (H₂O₂) and superoxide (O₂-) levels were recorded for the susceptible cultivars compared to the resistant cultivars. Molecular markers proved that the Lr50 gene was detected in the resistant cultivars. Puccinia triticina infections negatively affected most histological characteristics of flag leaves, especially in susceptible cultivars. The thickness of the blade (µ), the thickness of the upper and lower epidermis (UE and LE), the thickness of mesophyll tissue (MT), and bundle length and width in the midrib were decreased in susceptible cultivars such as Sids-1, Sids-13, and Shandwel-1 compared with resistant cultivars. Full article

(This article belongs to the Special Issue Antioxidants in Redox Homeostasis of Plant Development)

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17 pages, 34243 KiB

Open AccessArticle

The MYST Family Histone Acetyltransferase SasC Governs Diverse Biological Processes in Aspergillus fumigatus

by Jae-Yoon Kwon, Young-Ho Choi, Min-Woo Lee, Jae-Hyuk Yu and Kwang-Soo Shin

Cells 2023, 12(22), 2642; https://doi.org/10.3390/cells12222642 - 16 Nov 2023

Cited by 2 | Viewed by 1252

Abstract

The conserved MYST proteins form the largest family of histone acetyltransferases (HATs) that acetylate lysines within the N-terminal tails of histone, enabling active gene transcription. Here, we have investigated the biological and regulatory functions of the MYST family HAT SasC in the opportunistic [...] Read more.

The conserved MYST proteins form the largest family of histone acetyltransferases (HATs) that acetylate lysines within the N-terminal tails of histone, enabling active gene transcription. Here, we have investigated the biological and regulatory functions of the MYST family HAT SasC in the opportunistic human pathogenic fungus Aspergillus fumigatus using a series of genetic, biochemical, pathogenic, and transcriptomic analyses. The deletion (Δ) of sasC results in a drastically reduced colony growth, asexual development, spore germination, response to stresses, and the fungal virulence. Genome-wide expression analyses have revealed that the ΔsasC mutant showed 2402 significant differentially expressed genes: 1147 upregulated and 1255 downregulated. The representative upregulated gene resulting from ΔsasC is hacA, predicted to encode a bZIP transcription factor, whereas the UV-endonuclease UVE-1 was significantly downregulated by ΔsasC. Furthermore, our Western blot analyses suggest that SasC likely catalyzes the acetylation of H3K9, K3K14, and H3K29 in A. fumigatus. In conclusion, SasC is associated with diverse biological processes and can be a potential target for controlling pathogenic fungi. Full article

(This article belongs to the Section Plant, Algae and Fungi Cell Biology)

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23 pages, 1256 KiB

Open AccessReview

Examining the Role of a Functional Deficiency of Iron in Lysosomal Storage Disorders with Translational Relevance to Alzheimer’s Disease

by Steven M. LeVine

Cells 2023, 12(22), 2641; https://doi.org/10.3390/cells12222641 - 16 Nov 2023

Cited by 3 | Viewed by 2492

Abstract

The recently presented Azalea Hypothesis for Alzheimer’s disease asserts that iron becomes sequestered, leading to a functional iron deficiency that contributes to neurodegeneration. Iron sequestration can occur by iron being bound to protein aggregates, such as amyloid β and tau, iron-rich structures not [...] Read more.

The recently presented Azalea Hypothesis for Alzheimer’s disease asserts that iron becomes sequestered, leading to a functional iron deficiency that contributes to neurodegeneration. Iron sequestration can occur by iron being bound to protein aggregates, such as amyloid β and tau, iron-rich structures not undergoing recycling (e.g., due to disrupted ferritinophagy and impaired mitophagy), and diminished delivery of iron from the lysosome to the cytosol. Reduced iron availability for biochemical reactions causes cells to respond to acquire additional iron, resulting in an elevation in the total iron level within affected brain regions. As the amount of unavailable iron increases, the level of available iron decreases until eventually it is unable to meet cellular demands, which leads to a functional iron deficiency. Normally, the lysosome plays an integral role in cellular iron homeostasis by facilitating both the delivery of iron to the cytosol (e.g., after endocytosis of the iron–transferrin–transferrin receptor complex) and the cellular recycling of iron. During a lysosomal storage disorder, an enzyme deficiency causes undigested substrates to accumulate, causing a sequelae of pathogenic events that may include cellular iron dyshomeostasis. Thus, a functional deficiency of iron may be a pathogenic mechanism occurring within several lysosomal storage diseases and Alzheimer’s disease. Full article

(This article belongs to the Section Cellular Pathology)

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15 pages, 3633 KiB

Open AccessArticle

Proline and Proline Analogues Improve Development of Mouse Preimplantation Embryos by Protecting Them against Oxidative Stress

by Madeleine L. M. Hardy, Dheerja Lakhiani, Michael B. Morris and Margot L. Day

Cells 2023, 12(22), 2640; https://doi.org/10.3390/cells12222640 - 16 Nov 2023

Cited by 1 | Viewed by 1426

Abstract

The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo development, increasing the percentage that develop to the blastocyst stage and hatch. Staining of 2-cell and 4-cell embryos with [...] Read more.

The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo development, increasing the percentage that develop to the blastocyst stage and hatch. Staining of 2-cell and 4-cell embryos with tetramethylrhodamine methyl ester and 2′,7′-dichlorofluorescein diacetate showed that the culture of embryos in the presence of Pro, or either of these analogues, reduced mitochondrial activity and reactive oxygen species (ROS), respectively, indicating potential mechanisms by which embryo development is improved. Inhibition of the Pro metabolism enzyme, proline oxidase, by tetrahydro-2-furoic-acid prevented these reductions and concomitantly prevented the improved development. The ways in which Pro, PA and L4T reduce mitochondrial activity and ROS appear to differ, despite their structural similarity. Specifically, the results are consistent with Pro reducing ROS by reducing mitochondrial activity while PA and L4T may be acting as ROS scavengers. All three may work to reduce ROS by contributing to the GSH pool. Overall, our results indicate that reduction in mitochondrial activity and oxidative stress are potential mechanisms by which Pro and its analogues act to improve pre-implantation embryo development. Full article

(This article belongs to the Special Issue Molecular and Clinical Advances in Understanding Early Embryo Development—Volume II)

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Figure 1

Figure 1
Chemical structures of Pro and Pro analogues, L-tetrahydro-2-furoic acid (THFA), L-pipecolic acid (PA) and L-4-Thiazolidine carboxylic acid (L4T). Full article ">Figure 2
Preimplantation embryo development in vitro in the presence and absence of Pro and Pro analogues. Zygotes were cultured ±400 µM of Pro, THFA, THFA + Pro, PA, or L4T for 5 days with development scored daily. Graphs show development to the (A) ≥5-cell, (B) compacted, (C) blastocyst and (D) hatched blastocyst stages. Bars represent mean ± SEM, obtained from a minimum of three independent experiments containing at least 12 embryos per condition per experiment. Data were compared by one-way ANOVA with a Tukey’s multiple comparisons test. Bars with a different letter are significantly different (p < 0.05). Full article ">Figure 3
Rate of uptake of radiolabelled Pro in the presence of excess unlabelled Pro or THFA. Embryos were incubated in 1 μM L-[3H]-Pro in the absence (control) or presence of 5 mM Pro or 400 μM THFA for 100 min. Uptake at the (A) 2-cell and (B) 4-cell stages was quantified using scintillation counting. Data represent the mean rate of uptake ± SEM from three independent experiments with 12 embryos per treatment performed in triplicate in each experiment. Data were analysed by one-way ANOVA with Tukey’s multiple comparisons test. Bars with a different letter are significantly different (p < 0.05). Full article ">Figure 4
The effect of Pro and Pro analogues on the mitochondrial activity and ROS content in 2-cell embryos. Zygotes were cultured in ModHTF in the presence or absence of 400 µM Pro, THFA, THFA+ Pro, PA, or L4T for 24 h to the 2-cell stage. Embryos were loaded with 10 µM DCFDA for ROS and 100 nM TMRM for mitochondrial activity and imaged using confocal microscopy. (A) Representative image of embryos in each condition. Scale bar = 20 µm. Cell fluorescence for (B) TMRM and (C) DCFDA. Control embryos were stained with DCFDA and TMRM and transferred to a 10 µL drop of medium containing (D) 5 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) or (E) 60 µM H2O2 prior to imaging. Bars represent mean ± SEM of 25–78 embryos obtained from at least three independent experiments. Data were analysed using a one-way ANOVA with a Tukey’s multiple comparisons test. Bars with different letters are significantly different (p < 0.05) and *** represents (p < 0.001). Full article ">Figure 5
The effect of Pro and Pro analogues on the mitochondrial activity and ROS content in 4-cell embryos. Zygotes were cultured in ModHTF in the presence or absence of 400 µM Pro, THFA, THFA+ Pro, PA, or L4T for 48 h to the 4-cell stage. Embryos were loaded with 10 µM DCFDA for ROS and 100 nM TMRM for mitochondrial activity and imaged using confocal microscopy. (A) Representative image of embryos in each condition. Scale bar = 20 µm. Cell fluorescence for (B) TMRM and (C) DCFDA. Control embryos were stained with DCFDA and TMRM and transferred to a 10 µL drop of medium containing (D) 5 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) or (E) 60 µM H2O2 before imaging. Bars represent mean ± SEM of 29–65 embryos obtained from at least three independent experiments. Data were analysed using a one-way ANOVA with a Tukey’s multiple comparisons test. Bars not sharing the same letter are significantly different (p < 0.05) and *** represents (p < 0.001). Full article ">

16 pages, 5929 KiB

Open AccessArticle

A High-Throughput, High-Containment Human Primary Epithelial Airway Organ-on-Chip Platform for SARS-CoV-2 Therapeutic Screening

by Christine R. Fisher, Felix Mba Medie, Rebeccah J. Luu, Robert B. Gaibler, Thomas J. Mulhern, Caitlin R. Miller, Chelsea J. Zhang, Logan D. Rubio, Elizabeth E. Marr, Vidhya Vijayakumar, Elizabeth P. Gabriel, Landys Lopez Quezada, Chun-Hui Zhang, Karen S. Anderson, William L. Jorgensen, Jehan W. Alladina, Benjamin D. Medoff, Jeffrey T. Borenstein and Ashley L. Gard

Cells 2023, 12(22), 2639; https://doi.org/10.3390/cells12222639 - 16 Nov 2023

Cited by 3 | Viewed by 2071

Abstract

COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack [...] Read more.

COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack of human-relevant preclinical models compatible with therapeutic screening on the native virus, which requires a high-containment environment. Here, we report SARS-CoV-2 infection and robust viral replication in PREDICT96-ALI, a high-throughput, human primary cell-based organ-on-chip platform. We evaluate unique infection kinetic profiles across lung tissue from three human donors by immunofluorescence, RT-qPCR, and plaque assays over a 6-day infection period. Enabled by the 96 devices/plate throughput of PREDICT96-ALI, we also investigate the efficacy of Remdesivir and MPro61 in a proof-of-concept antiviral study. Both compounds exhibit an antiviral effect against SARS-CoV-2 in the platform. This demonstration of SARS-CoV-2 infection and antiviral dosing in a high-throughput organ-on-chip platform presents a critical capability for disease modeling and therapeutic screening applications in a human physiology-relevant in vitro system. Full article

(This article belongs to the Collection Advances in 3D Cell Culture)

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Full article ">Figure 1
PREDICT96-ALI lung model and SARS-CoV-2 Infection. (A) PREDICT96-ALI system consists of a customized 96-well culture plate and a corresponding microfluidic pump system that can independently drive media flow on the apical well and/or the underlying basal channel. The apical well contains the differentiated airway tissue at the air–liquid interface while the PREDICT96 pump system recirculates media via the basal inlet and outlet port. (B) Normal human bronchial epithelial cells or small airway epithelial cells collected from human donors are seeded on the membrane of the apical well. After an initial submerged proliferation and differentiation phase, ALI is induced on the apical well where cells are further matured for 3–4 weeks with circulating custom ALI-media in the basal channel. (C) Mature tissue is infected with SARS-CoV-2 infection for 2 h; unbound viruses are removed and ALI is reintroduced. Virus are harvested at 2, 4, and 6 days post infection (dpi). After 6 days of infection, tissue can be fixed and imaged or processed for other downstream applications. Full article ">Figure 2
Donor cells derived from research bronchoscopy and differentiated in PREDICT96-ALI exhibit the structure and composition of the mature human airway. (A) Representative max projection immunofluorescence (IF) images demonstrating the presence of basal (CK5), goblet (Muc5AC), and ciliated (acetylated-tubulin or AceTub) cells in healthy, uninfected pseudostratified PREDICT96-ALI airway tissue derived from donor DH02 and grown for 4 weeks at air liquid interface (ALI), taken at 40× magnification and shown with a 50 µm scale bar. The inset images have a 20 µm scale bar. Tissues were counter-stained for dsDNA with Hoechst stain. (B) Club Cell Secretory Protein (CCSP) detection in PREDICT-ALI airway experiments containing donor DH01 at 4 weeks ALI. Media samples collected from the basal microfluidic chamber of each PREDICT96-ALI tissue exhibit detectable levels of CCSP (mean ± SEM of n = 6). Tissue media samples randomly selected from two independent, representative experiments. (C) Relative fold change in expression of mRNA transcripts from differentiated airway tissues in PREDICT96-ALI derived from donor DH01. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect transcripts, using GAPDH as a reference gene and normalizing it to undifferentiated cells (mean fold change expression ± SEM). n = 12 healthy tissue replicates selected at random from two representative PREDICT96-ALI experiments. (D) Confirmation that epithelial cells isolated from donors DH01 and DH02 are SARS-CoV-2 negative, as determined by RT-qPCR detecting two regions in the SARS-CoV-2 nucleocapsid (N) gene, N1 and N2. RNaseP probed as a tissue sample control. Limit of quantification (LOQ) indicates copy number corresponding to Ct values of ≥40 cycles. Samples that did not meet the minimum signal intensity (undetermined) after 40 PCR cycles are excluded. Data presented as mean Log10 viral RNA copies/mL ± SEM (n = 2). Full article ">Figure 3
SARS-CoV-2-inoculated PREDICT96-ALI airway tissues from multiple donors yield replicative virus by RT-qPCR and plaque assay. (A–C) RT-qPCR analyses for SARS-CoV-2 nucleoprotein RNA copies in the apical wash of PREDICT96-ALI airway tissue at 0, 2, 4, and 6 days post infection (d.p.i.). Panels represent unique donors: (A) donor DH01 (n = 12 tissue replicates per time-point and condition); (B) donor DH02 (n = 12 tissue replicates for mock, n = 18 for 2.5 MOI); (C) donor 04401 (n = 12 tissue replicates for mock, n = 24 for 2.5 MOI). Fold increase in average RNA titer between days 0 and 6 indicated for each donor. An independent experiment with donor 04401 taken to 12 d.p.i. indicated that infection peaked at 6 d.p.i (<a href="#app1-cells-12-02639" class="html-app">Figure S1</a>). LOQ (dotted line): Log10 viral RNA (copies/mL) corresponding to cycle threshold values ≥ 37. (D–F) Plaque assays detecting replicative virus from apical wash samples of PREDICT96-ALI airway tissue at 0, 2, 4, and 6 d.p.i. Panels represent unique donors: (D) donor DH01; (E) donor DH02; (F) donor 04401. n = 3 tissue replicates for mock-infected conditions; n = 6 tissue replicates per time-point in the infected condition, selected after exhibiting the highest viral RNA titers as determined by RT-qPCR. LOQ (dotted line): ≤33.3 PFU/mL. Samples for RT-qPCR and plaque assays came from the same experiments, except for donor 04401, in which RT-qPCR and plaque assay data came from independent experiments. SARS-CoV-2 (USA-WA1/2020) at passage 4 was used to infect the tissue in panels (A,E); passage 5 was used to infect all other panels. All data are shown as mean ± SEM. Statistical significance determined via a two-way analysis of variance (ANOVA) with Dunnett’s test for multiple comparisons (comparing to 0 d.p.i.): * p ≤ 0.05; **** p ≤ 0.0001. Full article ">Figure 4
PREDICT96-ALI airway tissue exhibits heterogeneous SARS-CoV-2 viral foci and co-localized staining with differentiated airway epithelial cell types. (A) Max projection IF images depicting positive staining of the SARS-CoV-2 N (red) and dsDNA (blue) within PREDICT96-ALI airway tissue from donor 04401 (donor DH01 in <a href="#app1-cells-12-02639" class="html-app">Figure S2</a>). Tissues were fixed with 4% paraformaldehyde at 6 d.p.i. following inoculation with SARS-CoV-2. Images are 40× magnification with a 50 µm scale bar. (B) Mean Fluorescent Intensity (MFI) image quantification of SARS-CoV-2 infection in PREDICT96-ALI airway tissues associated with panel (A). Images were taken at 10× magnification and analyzed for MFI using ImageJ Fiji. Statistical significance determined via a one-way ANOVA with Dunnett’s test for multiple comparisons (** p ≤ 0.01). n = 6 for mock, 0.025 and 0.25 MOI; n = 12 for 2.5 MOI. Data also correspond to fluorescence images in <a href="#app1-cells-12-02639" class="html-app">Figure S3</a>. (C,D) Representative max projection IF image of SARS-CoV-2 N (red) and (C) ciliated cells (acetylated α-tubulin, green) or (D) goblet cells (MUC5AC, green) and dsDNA (blue) within PREDICT96-ALI airway tissue from donor 04401 at 6 d.p.i. following inoculation with SARS-CoV-2 at 2.5 MOI. Images were taken at 40× magnification and are shown with 50 µm scale bars. The insets have 20 µm scale bars. Full article ">Figure 5
Assessment of viral replication in tissues from donor 04401 with and without antiviral interventions. Viral replication is measured by RT-qPCR for SARS-CoV-2 N copies in the apical wash of PREDICT96-ALI airway tissue from donor 04401 at 0, 2, 4, and 6 days post infection (d.p.i.). Remdesivir, Mpro-61, or DMSO (vehicle) were dosed at 10 μM into the basal media of tissues infected with SARS-CoV-2 1 h after infection and again on days 2 and 4 p.i. Tissues were infected with either 2.5 MOI (A) or 0.25 MOI (B). Data shown in mean ± SEM, and average fold-change over 0 dpi is noted. N = 4 per condition. Statistical significance determined by a two-way ANOVA with Dunnett’s test for multiple comparisons (comparing to 0 d.p.i.): ns p > 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Full article ">

20 pages, 4408 KiB

Open AccessArticle

High-Level Production of scFv-Fc Antibody Using an Artificial Promoter System with Transcriptional Positive Feedback Loop of Transactivator in CHO Cells

by Binbin Ying, Yoshinori Kawabe, Feiyang Zheng, Yuki Amamoto and Masamichi Kamihira

Cells 2023, 12(22), 2638; https://doi.org/10.3390/cells12222638 - 16 Nov 2023

Cited by 1 | Viewed by 2528

Abstract

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established [...] Read more.

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems. Full article

(This article belongs to the Collection Advances in Cell Culture and Tissue Engineering)

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Full article ">Figure 1
Generation of scFv-Fc-producer CHO cells with the high transgene expression system using an artificial transcription factor. (a) Vector constructs (PB/TRE_aTF/TRE_GFP-2A-Puro, pDonor/scFv-Fc-2A-Puro, PB/TRE_scFv-Fc/Hyg). (b) Recombinant CHO cells with an artificial gene expression system (CHO/aTF_GFP, CHO/aTF_scFv-Fc1, CHO/aTF_scFv-Fc2). The copy numbers of the introduced genes were measured via quantitative PCR using Taqman probes. PTRE, artificial synthetic promoter comprising of TRE sequence and CMV minimal promoter; aTF, tetR-VP48 fusion protein; GFP, copepod Pontellina plumata-derived green fluorescent protein (copGFP); T2A, 2A self-cleaving peptides derived from Thosea asigna virus; Puro, puromycin resistance gene; scFv-Fc, anti-prion single-chain antibody fused with Fc-region of human IgG1; F2A, Furin self-cleaving peptides fused with 2A self-cleaving peptides derived from porcine teschovirus-1; PTK, thymidine kinase promoter; ITR, inverted terminal repeat; Hyg, hygromycin resistance gene. Full article ">Figure 2
Batch culture of CHO cells with the artificial gene expression system in serum-containing medium. CHO-K1, CHO/aTF_scFv-Fc1, or CHO/aTF_scFv-Fc2 cells were seeded with 1 mL/well in a 24-well plate at a cell density of 1.0 × 105 cells/mL, and cultured in F12 medium with 10% FCS for 6 days. (a) Cell proliferation. (b) scFv-Fc concentration. (c) scFv-Fc specific production rate. (d) Glucose concentration. (e) Lactate concentration. CHO-K1 cells (beige square, 37 °C; gray square, 33 °C); CHO/aTF_scFv-Fc1 (red diamond, 37 °C; blue diamond, 33 °C); CHO/aTF_scFv-Fc2 (pink triangle, 37 °C; light blue triangle, 33 °C). Data are expressed as mean ± SD (n = 3). Full article ">Figure 3
Batch culture of CHO/aTF_scFv-Fc2 cells using serum-free medium. CHO/aTF_scFv-Fc2 cells, which had been adapted and suspended in a serum-free medium, were seeded with 1 mL/well in a 24-well plate at a cell density of 1.0 × 106 cells/mL, and cultured for 6 days. (a) Cell proliferation. (b) scFv-Fc concentration. (c) scFv-Fc specific production rate. (d) Glucose concentration. (e) Lactate concentration. Pink triangles, 37 °C; light blue triangles, 33 °C. Data are expressed as mean ± SD (n = 3). Full article ">Figure 4
Semi-continuous culture of CHO/aTF_scFv-Fc2 cells. Cells (10 mL) were seeded in a 50 mL bioreactor tube at a cell density of 0.5 × 107, 1.0 × 107 or 2.0 × 107 cells/mL, and cultured for 12 days. The culture medium was replaced with fresh medium every other day. (a,d) Cell proliferation. (b,e) scFv-Fc concentration. (c,f) scFv-Fc specific production rate. (a–c) 37 °C. (d–f) 33 °C. Cell seeding conditions are as follows: 0.5 × 107 cells/mL (red, 37 °C; blue, 33 °C); 1.0 × 107 cells/mL (pink, 37 °C; light blue, 33 °C); 2.0 × 107 cells/mL (yellow, 37 °C; green, 33 °C). Data are expressed as mean ± SD (n = 3). Full article ">Figure 5
Glucose and lactate concentrations in culture supernatants of semi-continuous cultures using CHO/aTF_scFv-Fc2 cells. Glucose and lactate concentrations in the culture supernatant were measured. (a–c) Glucose concentration. (d–f) Lactate concentration. (a,d) 0.5 × 107 cells/mL (red, 37 °C; blue, 33 °C), (b,e) 1.0 × 107 cells/mL (pink, 37 °C; light blue, 33 °C), (c,f) 2.0 × 107 cells/mL (yellow, 37 °C; green, 33 °C). Data are expressed as mean ± SD (n = 3). Full article ">Figure 6
SDS-PAGE analysis of culture supernatant samples in semi-continuous culture. (a,b) 37 °C, (c,d) 33 °C. (a,c) Reducing conditions. (b,d) Non-reducing conditions. Lane M, molecular weight marker; lane W, water; lanes 1–6, culture supernatant collected on day 2 (lane 1), day 4 (lane 2), day 6 (lane 3), day 8 (lane 4), day 10 (lane 5), and day 12 (lane 6); lane N, fresh medium; lane P, purified scFv-Fc (2 µg). Full article ">Figure 7
Structure analysis of N-linked glycans modified in scFv-Fc produced by CHO/aTF_scFv-Fc2 cells in semi-continuous culture. (a) HILIC-UPLC elusion profiles. Culture temperature: 37 °C (upper), 33 °C (lower). (b) Percentage of N-linked glycans detected via HILIC-UPLC analysis. Pink column, 37 °C; blue column, 33 °C. Each symbol in the schematic diagram is as follows: red squares, N-acetylglucosamine; blue circles, mannose; green circles, galactose; yellow triangles, fucose. Data are expressed as mean ± SD (n = 3). Full article ">Figure 8
Transcriptome analysis using DNA microarray of CHO/aTF_scFv-Fc2 cells in semi-continuous culture. (a) Venn diagrams of the number of changes in gene expression at low temperature (33 °C) versus normal temperature (37 °C) cultured at seeding cell densities of 0.5 × 107, 1.0 × 107 and 2.0 × 107 cells/mL. (Left), 2-fold up-regulation; (right), 2-fold down-regulation. (b) Genes that commonly fluctuated upward under each seeding cell density condition at low temperature. In order to narrow down the number of genes, variable genes were extracted under the condition of Z-score > 5. The rate of change in expression under the seeding condition of 1.0 × 107 cells/mL that resulted in the highest antibody production is listed in descending order. The gene symbols and names are shown in <a href="#app1-cells-12-02638" class="html-app">Table S2</a>. (c) Functional clustering analysis. We grouped each seeding cell density condition with normal-temperature and low-temperature cultures, and extracted genes with fold change >2 or <0.5 and Z-score > 2 or <−2 via DAVID (<a href="https://david.ncifcrf.gov/" target="_blank">https://david.ncifcrf.gov/</a> (accessed on 15 November 2023)), which was used for clustering analysis. (Left) Up-regulation. (Right) Down-regulation. Biological process (blue bars), cellular component (orange bars), molecular function (green bars). Full article ">

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