International Journal of Molecular Sciences

12 pages, 869 KiB

Open AccessArticle

The Identification of Metabolites and Effects of Albendazole in Alfalfa (Medicago sativa)

by Lucie Raisová Stuchlíková, Martina Navrátilová, Lenka Langhansová, Kateřina Moťková, Radka Podlipná, Barbora Szotáková and Lenka Skálová

Int. J. Mol. Sci. 2020, 21(16), 5943; https://doi.org/10.3390/ijms21165943 - 18 Aug 2020

Cited by 5 | Viewed by 3394

Abstract

Albendazole (ABZ), a widely used anthelmintic drug, enters the environment mainly via livestock excrements. To evaluate the environmental impact of ABZ, the knowledge of its uptake, effects and metabolism in all non-target organisms, including plants, is essential. The present study was designed to [...] Read more.

Albendazole (ABZ), a widely used anthelmintic drug, enters the environment mainly via livestock excrements. To evaluate the environmental impact of ABZ, the knowledge of its uptake, effects and metabolism in all non-target organisms, including plants, is essential. The present study was designed to identify the metabolic pathway of ABZ and to test potential ABZ phytotoxicity in fodder plant alfalfa, with seeds and in vitro regenerants used for these purposes. Alfalfa was chosen, as it may meet manure from ABZ-treated animals in pastures and fields. Alfalfa is often used as a feed of livestock, which might already be infected with helminths. The obtained results showed that ABZ did not inhibit alfalfa seed germination and germ growth, but evoked stress and a toxic effect in alfalfa regenerants. Alfalfa regenerants were able to uptake ABZ and transform it into 21 metabolites. UHPLC-MS/MS analysis revealed three new ABZ metabolites that have not been described yet. The discovery of the parent compound ABZ together with the anthelmintically active and instable metabolites in alfalfa leaves shows that the contact of fodder plants with ABZ-containing manure might represent not only a danger for herbivorous invertebrates, but also may cause the development of ABZ resistance in helminths. Full article

(This article belongs to the Special Issue Identification of Metabolites of Xenobiotics)

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15 pages, 2434 KiB

Open AccessArticle

PPARδ and FOXO1 Mediate Palmitate-Induced Inhibition of Muscle Pyruvate Dehydrogenase Complex and CHO Oxidation, Events Reversed by Electrical Pulse Stimulation

by Hung-Che Chien, Paul L. Greenhaff and Dumitru Constantin-Teodosiu

Int. J. Mol. Sci. 2020, 21(16), 5942; https://doi.org/10.3390/ijms21165942 - 18 Aug 2020

Cited by 7 | Viewed by 4229

Abstract

The mechanisms behind the reduction in muscle pyruvate dehydrogenase complex (PDC)-controlled carbohydrate (CHO) oxidation during chronic high-fat dietary intake are poorly understood, as is the basis of CHO oxidation restoration during muscle contraction. C2C12 myotubes were treated with (300 μM) palmitate or without [...] Read more.

The mechanisms behind the reduction in muscle pyruvate dehydrogenase complex (PDC)-controlled carbohydrate (CHO) oxidation during chronic high-fat dietary intake are poorly understood, as is the basis of CHO oxidation restoration during muscle contraction. C2C12 myotubes were treated with (300 μM) palmitate or without (control) for 16 h in the presence and absence of electrical pulse stimulation (EPS, 11.5 V, 1 Hz, 2 ms). Compared to control, palmitate reduced cell glucose uptake (p < 0.05), PDC activity (p < 0.01), acetylcarnitine accumulation (p < 0.05) and glucose-derived mitochondrial ATP production (p < 0.01) and increased pyruvate dehydrogenase kinase isoform 4 (PDK4) (p < 0.01), peroxisome proliferator-activated receptor alpha (PPARα) (p < 0.01) and peroxisome proliferator-activated receptor delta (PPARδ) (p < 0.01) proteins, and reduced the whole-cell p-FOXO1/t-FOXO1 (Forkhead Box O1) ratio (p < 0.01). EPS rescued palmitate-induced inhibition of CHO oxidation, reflected by increased glucose uptake (p < 0.01), PDC activity (p < 0.01) and glucose-derived mitochondrial ATP production (p < 0.01) compared to palmitate alone. EPS was also associated with less PDK4 (p < 0.01) and PPARδ (p < 0.01) proteins, and lower nuclear p-FOXO1/t-FOXO1 ratio normalised to the cytoplasmic ratio, but with no changes in PPARα protein. Collectively, these data suggest PPARδ, and FOXO1 transcription factors increased PDK4 protein in the presence of palmitate, which limited PDC activity and flux, and blunted CHO oxidation and glucose uptake. Conversely, EPS rescued these metabolic events by modulating the same transcription factors. Full article

(This article belongs to the Special Issue Central and Peripheral Molecular Mechanisms of Metabolism Regulation)

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22 pages, 4513 KiB

Open AccessReview

Histone H1 Post-Translational Modifications: Update and Future Perspectives

by Marta Andrés, Daniel García-Gomis, Inma Ponte, Pedro Suau and Alicia Roque

Int. J. Mol. Sci. 2020, 21(16), 5941; https://doi.org/10.3390/ijms21165941 - 18 Aug 2020

Cited by 55 | Viewed by 10992

Abstract

Histone H1 is the most variable histone and its role at the epigenetic level is less characterized than that of core histones. In vertebrates, H1 is a multigene family, which can encode up to 11 subtypes. The H1 subtype composition is different among [...] Read more.

Histone H1 is the most variable histone and its role at the epigenetic level is less characterized than that of core histones. In vertebrates, H1 is a multigene family, which can encode up to 11 subtypes. The H1 subtype composition is different among cell types during the cell cycle and differentiation. Mass spectrometry-based proteomics has added a new layer of complexity with the identification of a large number of post-translational modifications (PTMs) in H1. In this review, we summarize histone H1 PTMs from lower eukaryotes to humans, with a particular focus on mammalian PTMs. Special emphasis is made on PTMs, whose molecular function has been described. Post-translational modifications in H1 have been associated with the regulation of chromatin structure during the cell cycle as well as transcriptional activation, DNA damage response, and cellular differentiation. Additionally, PTMs in histone H1 that have been linked to diseases such as cancer, autoimmune disorders, and viral infection are examined. Future perspectives and challenges in the profiling of histone H1 PTMs are also discussed. Full article

(This article belongs to the Special Issue Protein Post-translational Modifications in Signal Transduction and Diseases)

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16 pages, 1474 KiB

Open AccessReview

Casein Kinase 1α as a Regulator of Wnt-Driven Cancer

by Chen Shen, Anmada Nayak, Ricardo A. Melendez, Daniel T. Wynn, Joshua Jackson, Ethan Lee, Yashi Ahmed and David J. Robbins

Int. J. Mol. Sci. 2020, 21(16), 5940; https://doi.org/10.3390/ijms21165940 - 18 Aug 2020

Cited by 18 | Viewed by 5750

Abstract

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that [...] Read more.

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators. Full article

(This article belongs to the Special Issue The Wnt Signaling Pathway in Cancer)

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Figure 1
The canonical Wnt signaling pathway. In the Wnt off-state (left), β-catenin, the pivotal transcription coactivator of the Wnt pathway, is degraded by the destruction complex in the cytoplasm. Other Wnt effectors, such as frizzled (Fzd) at the membrane and T cell factor (TCF)/lymphoid enhancer-binding factor 1 (LEF1) transcription factors in the nucleus, are also inhibited to maintain low Wnt activity. In the Wnt on-state (right), Wnt ligands trigger the formation of the signalosome to promote Wnt signal transduction. The function of the destruction complex is inhibited, leading to the stabilization of β-catenin. β-catenin then translocates into the nucleus and binds to TCF/LEF1 to form a transcription complex along with other cofactors to initiate Wnt target transcription. LRP5/6: low-density lipoprotein receptor-related protein 5/6; ZNRF3: E3 ubiquitin ligase zinc- and ring-finger protein 3; RNF43: ring-finger protein 43; Dvl: disheveled; AP2: adapter protein 2; APC: adenomatous polyposis coli; CK1α: casein kinase 1α; GSK3: glycogen synthase kinase 3; Gro/TLE: groucho/transducin-like enhancer of split proteins; Pygo: pygopus; BCL9: B cell lymphoma 9 protein; Ebd1: earthbound 1; RSPO: R-spondin family of secreted ligands; LGR: leucine-rich repeat-containing G-protein coupled receptors; HSPG: heparan sulfate proteoglycans; SIAH1: siah E3 ubiquitin ligase 1; PP2A: protein phosphatase 2A. Full article ">Figure 2
CK1α splice variants. Human CK1α undergoes alternative splicing to produce four splice variants, as shown. These CK1α splice variants are characterized by the insertion of two polypeptide sequences: a long insertion (L) that contains a nuclear localization signal (NLS) into the protein kinase domain, and a short insertion (S) close to the C-terminus. LS: CK1α with both L and S inserts; S: CK1α with only an S insert; NI: CK1α with no insert; SN: CK1α LS with an N-terminal truncation. Full article ">Figure 3
CK1α regulatory subunits. Proteins that have been reported to regulate CK1α are shown. These proteins bind to CK1α and lead to indicated regulatory outcomes of CK1α. GLIPR1: glioma pathogenesis-related protein 1; FAM83G: family with sequence similarity 83G protein; DDX3: DEAD-box RNA helicase 3; MDMX: murine double minute X. Full article ">Figure 4
CK1α activators. (A) The structures of two chemically distinct CK1α activators—pyrvinium and SSTC3—and their inactive analogs—VU-WS211 and SSTC111—respectively, are shown. The red boxes highlight key structures needed for maximal efficacy. (B) A model, highlighting how CK1α activators function to increase the catalytic efficiency of CK1α. (C) A model of the mechanism underlying the differential therapeutic index of CK1α activators in normal tissue and Wnt-driven cancer. Full article ">

19 pages, 3249 KiB

Open AccessArticle

CA9 Silencing Promotes Mitochondrial Biogenesis, Increases Putrescine Toxicity and Decreases Cell Motility to Suppress ccRCC Progression

by Jiatong Xu, Songbiao Zhu, Lina Xu, Xiaohui Liu, Wenxi Ding, Qingtao Wang, Yuling Chen and Haiteng Deng

Int. J. Mol. Sci. 2020, 21(16), 5939; https://doi.org/10.3390/ijms21165939 - 18 Aug 2020

Cited by 9 | Viewed by 3920

Abstract

Carbonic anhydrase IX (CA9), a pH-regulating transmembrane protein, is highly expressed in solid tumors, and particularly in clear cell renal cell carcinoma (ccRCC). The catalytic mechanisms of CA9 are well defined, but its roles in mediating cell migration/invasion and survival in ccRCC remain [...] Read more.

Carbonic anhydrase IX (CA9), a pH-regulating transmembrane protein, is highly expressed in solid tumors, and particularly in clear cell renal cell carcinoma (ccRCC). The catalytic mechanisms of CA9 are well defined, but its roles in mediating cell migration/invasion and survival in ccRCC remain to be determined. Here, we confirmed that the mRNA expression of CA9 in ccRCC was significantly higher than that in para-carcinoma tissues from analysis of the datasets in The Cancer Genome Atlas. CA9 knockdown upregulated oxidative phosphorylation-associated proteins and increased mitochondrial biogenesis, resulting in the reversal of the Warburg phenotype and the inhibition of cell growth. Our study revealed that CA9 knockdown upregulated mitochondrial arginase 2 (ARG2), leading to the accumulation of putrescine, which suppressed ccRCC proliferation. Surfaceomics analysis revealed that CA9 knockdown downregulated proteins associated with extracellular matrix (ECM)—receptor interaction and cell adhesion, resulting in decreased cell migration. CA9 silencing also downregulated amino acid transporters, leading to reduced cellular amino acids. Collectively, our data show that CA9 knockdown suppresses proliferation via metabolic reprogramming and reduced cell migration, reaffirming that CA9 is a potential therapeutic target for ccRCC treatment. Full article

(This article belongs to the Special Issue Targeting Mitochondria in Aging and Disease)

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CA9 knockdown inhibits cell growth in clear cell renal cell carcinoma (ccRCC) cells. (A) CA9 expression profile across all tumor samples and paired normal tissues. The transcriptome datasets were obtained from The Cancer Genome Atlas (TCGA). Tumor abbreviations are listed in <a href="#app1-ijms-21-05939" class="html-app">Table S1</a>. (B) The mean mRNA level of CA9 in ccRCC tissues (num(T) = 523) and normal tissues (num(N) = 72) from TCGA data. (C) mRNA expression of CA9 decreased in 786-O-CA9-KD cells compared with control cells, measured by quantitative real-time PCR (qPCR). ACTB was used as a control. (D) Western blotting of CA9 revealed that the expression of CA9 was reduced in 786-O-CA9-KD cells. β-actin was used as a control. (E) Knockdown of CA9 in 786-O cells inhibited cell growth compared with control cells. (F) Knockdown of CA9 in 769-P cells inhibited cell growth. (G) Overexpression of CA9 in 786-O cells promoted cell proliferation. Cell proliferation curves measured by the Cell Counting Kit-8 (CCK-8) assays. Significance was calculated by Student’s t-test. **** p < 0.0001, ** p < 0.01, * p < 0.05; n = 3, mean ± SEM. Full article ">Figure 2
Functional classification of differentially expressed proteins (DEPs) in proteomics analysis of 786-O CA9 knockdown cells compared with control cells. (A) Experimental variations of proteomics analysis between 786-O-CA9-KD cells and control cells. (B) A volcano plot of proteins based on p-values and ratios of protein expressions in 786-O-CA9-KD cells compared with control cells. Blue and red dots indicate the downregulated and upregulated proteins with significant difference (p-values < 0.05, ratios ≤ 0.75 or ≥1.33), respectively. (C) The upregulated and downregulated proteins after CA9 knockdown in 786-O cells were classified by Kyoto Encyclopedia of Genes and Genomes (KEGG) BlastKOALA. (D) Numbers of upregulated and downregulated proteins participating in genetic information processing. Most mitochondrial biogenesis proteins were upregulated in CA9 knockdown cells. (E) Representative canonical pathways enriched in DEPs after CA9 knockdown in 786-O cells, analyzed by IPA (Ingenuity Pathway Analysis). Full article ">Figure 3
CA9 knockdown increases mitochondrial biogenesis in 786-O cells. (A) Sixteen oxidative phosphorylation (OXPHOS)-related proteins and (B) 28 mitochondrial ribosomal proteins were upregulated in CA9 knockdown cells (n = 3, mean ± SEM). (C) CA9 knockdown increased MitoTracker staining intensity (**** p < 0.0001, n = 4, mean ± SEM). (D) Western blot images of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear factor erythroid 2-related factor 2 (NRF2), mitochondrial transcription factor 1 (TFAM), ATP synthase subunit delta (ATP5D) and ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1) in control cells and CA9-KD cells. β-actin was used as a control. CA9 knockdown enhanced protein expressions of key factors in mitochondrial biogenesis in 786-O cells. Full article ">Figure 4
CA9 knockdown increases putrescine production from arginine and inhibits cell growth. (A) A volcano plot of metabolites in 786-O-CA9-KD cells compared with control cells. The plot displays the fold changes of metabolite contents between CA9-KD cells and control cells against the p-values (−log10). Blue and red dots indicate the decreased and increased metabolites with significant difference (p-values < 0.05, fold changes ≤ 0.75 or ≥1.33), respectively. (B) Boxplots of metabolite intensity in putrescine synthesis pathway in CA9-KD cells and control cells (n = 5, mean ± SEM). (C) Schematic representation of putrescine biosynthesis from 13C6-arginine; red dots represent heavy carbon with 13C labeling while black dots represent light carbon (12C). The blue rectangles indicate metabolites and orange ovals indicate related enzymes. ODC, ornithine decarboxylase; ARG2, arginase 2; ASS1, argininosuccinate synthase 1; ASL, argininosuccinate lyase; SMS, spermine synthase; OTC, ornithine carbamoyltransferase. (D) Isotope relative abundance of M+5 ornithine, M+5 citrulline, and M+4 putrescine in 786-O-CA9-KD cells and control cells. Cells were supplemented with 13C6-arginine for 12 h prior to quantify metabolite levels via mass spectroscopy analysis (n = 3, mean ± SEM). (E) Expression ratios of enzymes involved in the putrescine synthesis between 786-O-CA9-KD cells and control cells (n = 3, mean ± SEM). (F) ARG2 and ODC were upregulated after CA9 knockdown, while ASS1 and ASL were downregulated, confirmed by Western blotting. β-actin was used as a control. (G) Putrescine inhibited the growth of 786-O cells. The viability was assessed by the CCK-8 assay (n = 3, mean ± SEM). **** p < 0.0001, *** p < 0.001, * p < 0.05. Full article ">Figure 5
CA9 silencing downregulated expressions of amino acid transporters and proteins associated with cell motility in ccRCC cells. (A) Abundance of amino acids in 786-O-CA9-KD cells and control cells. Most amino acids were reduced in CA9 knockdown cells (**** p < 0.0001, *** p < 0.001, ns: no significance; n = 5, mean ± SEM). (B) Experimental design for the surfaceomics analysis. (C) Six amino acid transporters were downregulated in CA9 knockdown cells (n = 3, mean ± SEM). (D) mammalian target of rapamycin (mTOR) pathway was inactivated in CA9 knockdown cells, assessed by Western blotting of phosphor-mTOR (Ser2448), phospho-p70S6K (Thr389), and phospho-4EBP1 (Thr37/46). β-actin was used as a control. (E) Numbers of upregulated and downregulated surface proteins involved in cell motility. (F) The downregulated surface proteins in CA9 knockdown cells analyzed by Gene Ontology with DAVID 6.8. (G) Cell migration was inhibited in 786-O-CA9-KD cells compared with that in control cells, assessed by the wound healing assay. Cells were imaged at 0, 12, and 24 h after scratching. Scale bar: 200 μm. Full article ">

22 pages, 1979 KiB

Open AccessReview

Role of Adipose Tissue-Derived Autotaxin, Lysophosphatidate Signaling, and Inflammation in the Progression and Treatment of Breast Cancer

by David N. Brindley, Xiaoyun Tang, Guanmin Meng and Matthew G. K. Benesch

Int. J. Mol. Sci. 2020, 21(16), 5938; https://doi.org/10.3390/ijms21165938 - 18 Aug 2020

Cited by 33 | Viewed by 5090

Abstract

Autotaxin (ATX) is a secreted enzyme that produces lysophosphatidate (LPA), which signals through six G-protein coupled receptors, promoting tumor growth, metastasis, and survival from chemotherapy and radiotherapy. Many cancer cells produce ATX, but breast cancer cells express little ATX. In breast tumors, ATX [...] Read more.

Autotaxin (ATX) is a secreted enzyme that produces lysophosphatidate (LPA), which signals through six G-protein coupled receptors, promoting tumor growth, metastasis, and survival from chemotherapy and radiotherapy. Many cancer cells produce ATX, but breast cancer cells express little ATX. In breast tumors, ATX is produced by tumor-associated stroma. Breast tumors are also surrounded by adipose tissue, which is a major bodily source of ATX. In mice, a high-fat diet increases adipocyte ATX production. ATX production in obesity is also increased because of low-level inflammation in the expanded adipose tissue. This increased ATX secretion and consequent LPA signaling is associated with decreased adiponectin production, which results in adverse metabolic profiles and glucose homeostasis. Increased ATX production by inflamed adipose tissue may explain the obesity-breast cancer association. Breast tumors produce inflammatory mediators that stimulate ATX transcription in tumor-adjacent adipose tissue. This drives a feedforward inflammatory cycle since increased LPA signaling increases production of more inflammatory mediators and cyclooxygenase-2. Inhibiting ATX activity, which has implications in breast cancer adjuvant treatments, attenuates this cycle. Targeting ATX activity and LPA signaling may potentially increase chemotherapy and radiotherapy efficacy, and decrease radiation-induced fibrosis morbidity independently of breast cancer type because most ATX is not derived from breast cancer cells. Full article

(This article belongs to the Special Issue Unraveling the Involvement of the Adipose Tissue in Breast Cancer Progression)

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22 pages, 1091 KiB

Open AccessReview

Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives

by Dong Li, Hong Yang, Feng Xiong, Xiangmin Xu, Wen-Bo Zeng, Fei Zhao and Min-Hua Luo

Int. J. Mol. Sci. 2020, 21(16), 5937; https://doi.org/10.3390/ijms21165937 - 18 Aug 2020

Cited by 9 | Viewed by 5837

Abstract

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded [...] Read more.

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool. Full article

(This article belongs to the Special Issue Herpes Simplex Virus: From Reactivation to Assembly)

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Figure 1
Genomes of current H129-derived tracers, and anterograde tracing schematics. (A–E). Genome schematics of current H129-derived polysynaptic tracers. (A) Wildtype H129 (H129-wt). The genome of HSV-1 strain H129 (H129) is composed of 2 regions, unique long (UL) and unique short (US), which are flanked by terminal repeat (TR) and internal repeat (IR), respectively. (B) H129dTK-TT. The coding gene of tdTomato and codon-modified TK (mTK) is linked by the sequence encoding 2A self-cleaving peptide (2A). A LoxP-Stop-LoxP element is placed between CAG promoter (PCAG) and tdTomato-2A-TK cassette. WPRE is used as an enhancer, and poly(A) (pA) is used to stop the transcription. The whole expression cassette is inserted into the middle of the TK gene (UL23) in the H129 genome. The original TK is knocked out by being split into 3′- and 5′- parts. (C) H129-EGFP. The EGFP expression cassette with CMV promoter (PCMV) and poly(A) is inserted into the H129 genome between UL26/26.5 and UL27. (D) H129-G4. The BAC sequence is inserted into the H129 genome between UL22 and UL23 to generate BAC-H129. The PCMV controlled binary-GFP expression cassette is composed of a membrane-bound EGFP (mEGFP) and an EGFP with a 2A linker. Two copies of the binary-GFP expression cassette are inserted into the H129 genome. One is placed between the BAC sequence and UL23, and the other between US7 and US8. (E) H129-H8. The GFP expression cassette is composed of EGFP controlled by human ubiquitin C gene promoter (PhUbc) and WPRE-pA, which are all flanked by AAV2-ITR. The AAV replicase expression cassette is composed of the AAV2 Rep gene (AAV-Rep) and poly(A) (pA) controlled by PCMV. Both cassettes are inserted into the H129 genome between UL37 and UL38. (F,G). Genome schematic of current H129-derived monosynaptic tracers. (F) H129-dTK-tdT. The tdTomato expression cassette driven by PCMV is inserted into BAC-H129 to replace the TK gene (UL23), resulting in TK deletion. (G) H129-dTK-T2. Another identical tdTomato expression cassette (PCMV-tdT) is inserted into the genome of H129-dTK-tdT, between US7 and US8. (H,I). Schematic diagram of polysynaptic and monosynaptic tracing. (H) Polysynaptic tracing. The H129 polysynaptic tracers (indicated by the enveloped virion with green genome) are replication-competent. After being injected into the brain region (indicated by syringe and white circle), they infect the local neurons, replicate, and express fluorescent proteins to label the neurons (indicated as green). The produced progeny virions transmit anterogradely to the next order downstream neurons, repeat the replication/transmission processes, and label further downstream neurons. The ideal polysynaptic tracers should not label upstream neurons (indicated as gray) via terminal pickup or retrograde transmission. (I) Monosynaptic tracing. The H129 monosynaptic tracers (indicated by the enveloped virion with red genome) are replication-incompetent due to certain gene deletion. The helper virus, mostly AAV (indicated by the unenveloped virion with green genome), expresses the deficient gene and the fluorescent protein of a different color (indicated as green). After being injected into the same brain region (indicated by syringes and gray circle), the helper virus expresses the gene to complimentarily support the replication of deficient H129 monosynaptic tracer in trans. The produced progeny virions transmit anterogradely to the next order neurons, where there is no helper virus. Then the monosynaptic tracers stop transmitting to the further downstream regions. During the monosynaptic transmission, the monosynaptic tracers label the neurons by expressing the fluorescent protein (indicated as red). Therefore, the initial coinfected neurons (starter neurons, indicated as yellow) are labeled by both H129 tracer (red) and helper (green), and the second-order neurons are labeled only by H129 tracers (indicated as red). No further downstream neurons or upstream neurons were labeled (indicated as gray). Full article ">Figure 2
The limitations of current H129-derived tracers. (A–C). Limitations of labeling intensity. (A) Some tracers label the neurons with relatively low intensity. (B) Current monosynaptic tracers do not replicate after monosynaptically transmitting to postsynaptic neurons. Labeling intensity in second-order neurons is very low, making the neurites invisible even after immunostaining. (C) The labeling intensity of the entire neuron is not evenly distributed. The soma is labeled much more brightly than the neurites, resulting in axon and axonal terminals that are difficult to observe or even invisible. (D–G). Limitations of the tracer transmission. (D) H129-derived tracers mainly invade the neuron from the soma and transmit further (solid arrow). However, they can also be potentially picked up by the axonal terminal of the upstream neurons (dashed arrows), and label them with the fluorescent protein. (E) Besides the predominant anterograde transmission (solid arrows), H129-derived tracers were reported to potentially transmit retrogradely and label upstream neurons (dashed arrows). (F) H129 may potentially transmit to adjacent neurons from varicosity (indicated as the enlarged region of the axon) (dashed arrows). (G) H129 may potentially infect astrocytes. In astrocytes, H129-dTK may replicate in the absence of helper virus, and the progeny virions may transmit to adjacent neurons (dashed arrows). (H,I). Limitations of cytotoxicity. (H) The polysynaptic tracers (indicated by the enveloped virion with green genome) are replication-competent. Their anterograde transmission requires tracer replication and progeny production. The viral replication causes severe damage or even neuron death (indicated by the cracks). (I) Monosynaptic tracers (indicated by the enveloped virion with red genome) are replication-deficient in the absence of a helper virus. Due to replication deficiency, only a few viral proteins are synthesized, leading to less damage to the cells. Therefore, they show attenuated toxicity to the postsynaptic neurons after monosynaptic transmission (the red neuron). However, under the assistance of the helper virus (indicated by the unenveloped virion with green genome), H129-derived monosynaptic tracers replicate in the starter neurons (the yellow neuron) and cause severe damage to the starter neurons (indicated by the cracks). Full article ">

18 pages, 1506 KiB

Open AccessReview

Molecular and Cellular Factors Associated with Racial Disparity in Breast Cancer

by Manish Charan, Ajeet K. Verma, Shahid Hussain, Swati Misri, Sanjay Mishra, Sarmila Majumder, Bhuvaneswari Ramaswamy, Dinesh Ahirwar and Ramesh K. Ganju

Int. J. Mol. Sci. 2020, 21(16), 5936; https://doi.org/10.3390/ijms21165936 - 18 Aug 2020

Cited by 17 | Viewed by 5422

Abstract

Recent studies have demonstrated that racial differences can influence breast cancer incidence and survival rate. African American (AA) women are at two to three fold higher risk for breast cancer than other ethnic groups. AA women with aggressive breast cancers show worse prognoses [...] Read more.

Recent studies have demonstrated that racial differences can influence breast cancer incidence and survival rate. African American (AA) women are at two to three fold higher risk for breast cancer than other ethnic groups. AA women with aggressive breast cancers show worse prognoses and higher mortality rates relative to Caucasian (CA) women. Over the last few years, effective treatment strategies have reduced mortality from breast cancer. Unfortunately, the breast cancer mortality rate among AA women remains higher compared to their CA counterparts. The focus of this review is to underscore the racial differences and differential regulation/expression of genetic signatures in CA and AA women with breast cancer. Moreover, immune cell infiltration significantly affects the clinical outcome of breast cancer. Here, we have reviewed recent findings on immune cell recruitment in the tumor microenvironment (TME) and documented its association with breast cancer racial disparity. In addition, we have extensively discussed the role of cytokines, chemokines, and other cell signaling molecules among AA and CA breast cancer patients. Furthermore, we have also reviewed the distinct genetic and epigenetic changes in AA and CA patients. Overall, this review article encompasses various molecular and cellular factors associated with breast cancer disparity that affects mortality and clinical outcome. Full article

(This article belongs to the Special Issue Cytokines/Chemokines in Cancer Metastasis)

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15 pages, 1782 KiB

Open AccessArticle

Amorphization of Thiamine Chloride Hydrochloride: Effects of Physical State and Polymer Type on the Chemical Stability of Thiamine in Solid Dispersions

by Seda Arioglu-Tuncil, Adrienne L. Voelker, Lynne S. Taylor and Lisa J. Mauer

Int. J. Mol. Sci. 2020, 21(16), 5935; https://doi.org/10.3390/ijms21165935 - 18 Aug 2020

Cited by 9 | Viewed by 3258

Abstract

Thiamine is an essential micronutrient, but delivery of the vitamin in supplements or foods is challenging because it is unstable under heat, alkaline pH, and processing/storage conditions. Although distributed as a crystalline ingredient, thiamine chloride hydrochloride (TClHCl) likely exists in the amorphous state, [...] Read more.

Thiamine is an essential micronutrient, but delivery of the vitamin in supplements or foods is challenging because it is unstable under heat, alkaline pH, and processing/storage conditions. Although distributed as a crystalline ingredient, thiamine chloride hydrochloride (TClHCl) likely exists in the amorphous state, specifically in supplements. Amorphous solids are generally less chemically stable than their crystalline counterparts, which is an unexplored area related to thiamine delivery. The objective of this study was to document thiamine degradation in the amorphous state. TClHCl:polymer dispersions were prepared by lyophilizing solutions containing TClHCl and amorphous polymers (pectin and PVP (poly[vinylpyrrolidone])). Samples were stored in controlled temperature (30–60 °C) and relative humidity (11%) environments for 8 weeks and monitored periodically by X-ray diffraction (to document physical state) and HPLC (to quantify degradation). Moisture sorption, glass transition temperature (T_g), intermolecular interactions, and pH were also determined. Thiamine was more labile in the amorphous state than the crystalline state and when present in lower proportions in amorphous polymer dispersions, despite increasing T_g values. Thiamine was more stable in pectin dispersions than PVP dispersions, attributed to differences in presence and extent of intermolecular interactions between TClHCl and pectin. The results of this study can be used to control thiamine degradation in food products and supplements to improve thiamine delivery and decrease rate of deficiency. Full article

(This article belongs to the Special Issue Functional Mechanism of B-Vitamins and Their Metabolites 2.0)

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Figure 1
X-ray powder diffraction patterns of TClHCl:PVP solid dispersions (SDs) prepared from (A) 50TClHCl:50PVP to 90TClHCl:10PVP, in which all samples exhibited partial crystallinity, and (B) 1TClHCl:99PVP to 40TClHCl:60PVP immediately following lyophilization, in which all samples were amorphous. Full article ">Figure 2
X-ray powder diffraction patterns of (A) 5TClHCl:95PVP and (B) 5TClHCl:95PEC solid dispersions (SDs) stored at 11% RH and 30–60 °C on day 56. Full article ">Figure 3
(A) Chemical stability of thiamine in various ratios of TClHCl:PVP solid dispersions (SDs) stored at 11% RH and 60 °C for 56 days. (B) First-order degradation regression lines of thiamine in various ratios of TClHCl:PVP dispersions stored at 11% RH and 60 °C for 56 days. Full article ">Figure 4
Chemical stability of thiamine in (A) 5TClHCl:95PEC and 5TClHCl:95PVP physical mixtures (PMs) compared to solid dispersions (SDs) stored at 11% RH and 60 °C for 56 days, (B) 5TClHCl:95PEC solid dispersions (SDs) stored at 11% RH and 30–60 °C for 56 days, and (C) 5TClHCl:95PVP solid dispersions (SDs) stored at 11% RH and 30–60 °C for 56 days. Full article ">Figure 5
Moisture sorption profiles of (A) PVP, PEC, and TClHCl at 25 °C; (B) TClHCl, 40TClHCl:60PVP, 5TClHCl:95PVP, 50TClHCl:50PEC, and 5TClHCl:95PEC solid dispersions (SDs) at 25 °C, with insert graph highlighting initial moisture sorption differences between SDs from 5–50% RH; and (C) 40TClHCl:60PVP, 5TClHCl:95PVP, 50TClHCl:50PEC, and 5TClHCl:95PEC physical mixtures (PMs) at 25 °C. The arrows in the profiles identify 11% RH, the desiccator storage RH, indicating that the moisture contents of all samples were similar at the storage RH. Full article ">Figure 6
Chemical structures of (A) thiamine, (B) pectin, and (C) PVP. Full article ">

32 pages, 1886 KiB

Open AccessReview

Matrix Metalloproteinases in Age-Related Macular Degeneration (AMD)

by Luis García-Onrubia, Fco. Javier Valentín-Bravo, Rosa M. Coco-Martin, Rogelio González-Sarmiento, J. Carlos Pastor, Ricardo Usategui-Martín and Salvador Pastor-Idoate

Int. J. Mol. Sci. 2020, 21(16), 5934; https://doi.org/10.3390/ijms21165934 - 18 Aug 2020

Cited by 36 | Viewed by 6207

Abstract

Age-related macular degeneration (AMD) is a complex, multifactorial and progressive retinal disease affecting millions of people worldwide. In developed countries, it is the leading cause of vision loss and legal blindness among the elderly. Although the pathogenesis of AMD is still barely understood, [...] Read more.

Age-related macular degeneration (AMD) is a complex, multifactorial and progressive retinal disease affecting millions of people worldwide. In developed countries, it is the leading cause of vision loss and legal blindness among the elderly. Although the pathogenesis of AMD is still barely understood, recent studies have reported that disorders in the regulation of the extracellular matrix (ECM) play an important role in its etiopathogenesis. The dynamic metabolism of the ECM is closely regulated by matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). The present review focuses on the crucial processes that occur at the level of the Bruch’s membrane, with special emphasis on MMPs, TIMPs, and the polymorphisms associated with increased susceptibility to AMD development. A systematic literature search was performed, covering the years 1990–2020, using the following keywords: AMD, extracellular matrix, Bruch’s membrane, MMPs, TIMPs, and MMPs polymorphisms in AMD. In both early and advanced AMD, the pathological dynamic changes of ECM structural components are caused by the dysfunction of specific regulators and by the influence of other regulatory systems connected with both genetic and environmental factors. Better insight into the pathological role of MMP/TIMP complexes may lead to the development of new strategies for AMD treatment and prevention. Full article

(This article belongs to the Special Issue Molecular Biology of Age-Related Macular Degeneration (AMD) 2.0)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Age-related macular degeneration (AMD) is an eye disease affecting the macula, a central region in the retina. Individuals affected by AMD in its advanced stage may experience a profound loss of central vision. (A,B) Color pictures of retina with changes typical for early stages of AMD, typified by the presence of numerous large drusen, more or less confluent, and associated (or not) with retinal pigment epithelium (RPE) abnormalities (arrow). (C,D) Color and autofluorescence (AF) pictures of fundus for retina with changes typical for dry AMD. (C) The advanced form of dry AMD is typified by the presence of central geographic atrophy (GA) showing a sharply demarcated atrophic lesion of the outer retina, resulting from the loss of photoreceptors, RPE, and choriocapillaris (asterisk). (D) GA areas typically appear as dark patches in fundus AF images, and can be clearly delineated (asterisk). Full article ">Figure 2
(A–F) Color, optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and AF pictures of fundus for retina with changes typical for wet AMD. (A) Wet AMD is characterized by abnormal angiogenesis (choroidal neovascularization (CNV)), causing recurrent retinal exudation, subretinal hemorrhage, retinal or pigment detachment and, in the final stages of the disease, subretinal fibrosis (disciform scar). (B) AF showing confluent atrophic patches (asterisk) with a banded pattern of increased AF in the junction. The CNV can be seen in the OCT-angiography (black star); (C) the structural OCT enables the identification of the abnormal vascular tree (white star) and the presence of subretinal fluid (arrows) (D). By doing an FFA, we can also confirm the presence of the CNV: (E) Early phase: stippled hyperfluorescence with adjacent masking areas by blood or subretinal fibrosis; (F) Late phase: The hyperfluorescence increases irregularly due to the presence of progressive leakage (black arrow-head). Full article ">Figure 3
Mechanisms for Pro Matrix Metalloproteinase Activation. ProMMP-2 is the only MMP activated on the cell surface by MT-1MMP (MMP-14); this activation requires the trimolecular complex MT1-MMP/TIMP-2/proMMP-2 and the dimerization of the MT1-MMP. Extracellular activation is applicable to many secreted MMPs, such as proMMP-1,3,7,8,9,10,12, and 13, which are activated by a wide type of proteinases. Furin-activated, secreted proMMPs, such as proMMP-11, 14, 23, and 28 are activated intracellularly due to the removal of propeptides by the action of proprotein convertases such as furin. MMP: matrix metalloproteinases; TIMP: tissue metalloproteinase inhibitor; Cl: C-terminal domain of TIMP-2; F: furin recognition site; Zn: zinc of the active site. Full article ">

26 pages, 2750 KiB

Open AccessReview

How Do Molecular Dynamics Data Complement Static Structural Data of GPCRs

by Mariona Torrens-Fontanals, Tomasz Maciej Stepniewski, David Aranda-García, Adrián Morales-Pastor, Brian Medel-Lacruz and Jana Selent

Int. J. Mol. Sci. 2020, 21(16), 5933; https://doi.org/10.3390/ijms21165933 - 18 Aug 2020

Cited by 37 | Viewed by 6533

Abstract

G protein-coupled receptors (GPCRs) are implicated in nearly every physiological process in the human body and therefore represent an important drug targeting class. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided multiple static structures of GPCRs in complex with various signaling [...] Read more.

G protein-coupled receptors (GPCRs) are implicated in nearly every physiological process in the human body and therefore represent an important drug targeting class. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) have provided multiple static structures of GPCRs in complex with various signaling partners. However, GPCR functionality is largely determined by their flexibility and ability to transition between distinct structural conformations. Due to this dynamic nature, a static snapshot does not fully explain the complexity of GPCR signal transduction. Molecular dynamics (MD) simulations offer the opportunity to simulate the structural motions of biological processes at atomic resolution. Thus, this technique can incorporate the missing information on protein flexibility into experimentally solved structures. Here, we review the contribution of MD simulations to complement static structural data and to improve our understanding of GPCR physiology and pharmacology, as well as the challenges that still need to be overcome to reach the full potential of this technique. Full article

(This article belongs to the Special Issue Computer Simulation on Membrane Receptors and Lipid Bilayers)

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(a) Number of G protein-coupled receptors (GPCRs) structures available in GPCRdb [<a href="#B4-ijms-21-05933" class="html-bibr">4</a>,<a href="#B5-ijms-21-05933" class="html-bibr">5</a>] over time. (b) Number of publications per year indexed at Thomson Reuters’ Web of Science that contain the topics “molecular dynamics” and (“GPCR” or “GPCRs”). The exponential growth of successful GPCR research based on molecular dynamics (MD) simulations is evidenced by the rapid upsurge in the number of publications per year related to this subject. Full article ">Figure 2
Schematic view of the ligand-protein interaction results that can be obtained with the GPCRmd server [<a href="#B53-ijms-21-05933" class="html-bibr">53</a>]. Specifically, the GPCRmd Workbench module of the server enables interactive visualization (GPCRmd Viewer) and analysis (GPCRmd Toolkit) for individual simulations, including ligand-protein interactions among others. Figure obtained from the GPCRmd server [<a href="#B53-ijms-21-05933" class="html-bibr">53</a>]. Full article ">Figure 3
(a) Flowchart summarizing the stages of a MD simulation. (b) Example of a GPCR molecular system, including the β-2 adrenergic receptor (β2AR, blue) with a full agonist in the binding site (orange) in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane (tails in light brown, heads colored by heteroatom). The system is solvated with water (red) and ionized with sodium (green) and chloride (purple) ions. Full article ">Figure 4
Example of different parameters analyzed in a 500 ns-long MD simulation of the A2A receptor (A2AR). (a) Root mean square deviation (RMSD) profile taking as reference the first frame of the simulation, which is superimposed to the rest of the frames. RMSD values (i.e., structural differences with respect to the reference frame) increase over the simulation time until the system reaches a stable conformation after 100 ns. (b) Root mean square fluctuation (RMSF) profile displaying the values of all the alpha carbons in the protein. Higher RMSF values correspond to flexible loops, while lower ones belong to transmembrane helices, where residues are stabilized by the secondary structure. (c) Radius of gyration (RG) profile where the RG fluctuates around the same value during the simulation, indicating that the system does not suffer any big change in compactness. (d) Superimposition of 25 representative frames of the simulated receptor. The relative mobility of loop regions contrasts with the rigidness of the transmembrane helices. Full article ">Figure 5
Pattern of total interaction frequency of several MD simulations of GPCRs, extracted from the GPCRmd Receptor Meta-analysis tool (<a href="https://submission.gpcrmd.org/contmaps/" target="_blank">https://submission.gpcrmd.org/contmaps/</a>) of the GPCRmd server [<a href="#B53-ijms-21-05933" class="html-bibr">53</a>]. Columns represent interacting residue pairs according to Ballesteros-Weinstein residue numbering [<a href="#B142-ijms-21-05933" class="html-bibr">142</a>], whereas rows represent different simulations. The color of each cell shows the frequency in which any type of non-covalent interaction occurs during the simulation. Results are clustered based on the interaction frequencies of the simulations. This clustering is able to separate simulations according to the receptor subtype, showing that different receptor subtypes present differentiated interaction patterns. Full article ">

40 pages, 1923 KiB

Open AccessReview

Deciphering SARS-CoV-2 Virologic and Immunologic Features

by Grégorie Lebeau, Damien Vagner, Étienne Frumence, Franck Ah-Pine, Xavier Guillot, Estelle Nobécourt, Loïc Raffray and Philippe Gasque

Int. J. Mol. Sci. 2020, 21(16), 5932; https://doi.org/10.3390/ijms21165932 - 18 Aug 2020

Cited by 30 | Viewed by 25882

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and its associated pathology, COVID-19, have been of particular concerns these last months due to the worldwide burden they represent. The number of cases requiring intensive care being the critical point in this epidemic, a better understanding [...] Read more.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and its associated pathology, COVID-19, have been of particular concerns these last months due to the worldwide burden they represent. The number of cases requiring intensive care being the critical point in this epidemic, a better understanding of the pathophysiology leading to these severe cases is urgently needed. Tissue lesions can be caused by the pathogen or can be driven by an overwhelmed immune response. Focusing on SARS-CoV-2, we and others have observed that this virus can trigger indeed an immune response that can be dysregulated in severe patients and leading to further injury to multiple organs. The purpose of the review is to bring to light the current knowledge about SARS-CoV-2 virologic and immunologic features. Thus, we address virus biology, life cycle, tropism for many organs and how ultimately it will affect several host biological and physiological functions, notably the immune response. Given that therapeutic avenues are now highly warranted, we also discuss the immunotherapies available to manage the infection and the clinical outcomes. Full article

(This article belongs to the Section Molecular Immunology)

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Figure 1
SARS-CoV-2 supposed life cycle. (A) Entry of SARS-CoV-2 in target cell expressing ACE2 (or another receptor, CD147 have been evoked but need to be confirmed). (B) Uncoating and releasing SARS-CoV-2 single stranded positive RNA genome. (C) Translation of replicase–transcriptase complex directly from RNA genome. (D) RNA genome replication due to a negative template. (E) Nested production of subgenomic RNA encoding for structural proteins. (F) Translation of viral S, E and M inserted in endoplasmic reticulum. (G) Nucleocapsid coupled to the genome, forming nucleoprotein, combine to S, E and M to form a mature virion (H). (I) Exocytosis of SARS-CoV-2. Full article ">Figure 2
The two evoked routes of entry for SARS-CoV-2 to date. Angiotensin-converting-enzyme 2 (ACE2), which has been described as an interferon-stimulated gene (ISG), is a route of entry for SARS-CoV-2. Additionally, CD147 is evoked as a potential second route of entry. Based on a previous study with SARS-CoV, an interaction with Cyclophilin A is possible. The blue background corresponds to cells expressing ACE2, whereas the red background is representing cells expressing CD147. Solid arrows correspond to a direct activity involving ACE2, dotted arrows correspond to an indirect promoting activity. Full article ">Figure 3
Tropism and multiple organ injuries in SARS-CoV-2 infection. SARS-CoV-2 infection has been associated with multiple organ injuries due to viral tropism. Among injured organs (and targeted cell) we can find: lung (type II pneumocyte), heart (cardiomyocyte), liver (cholangiocyte), spleen and lymph nodes (macrophage), kidney and brain. Full article ">Figure 4
Mild versus severe immune response during SARS-CoV-2 infection. In regards to cytokine signature during SARS-CoV-2, mild and moderate cases showed a controlled response with higher expression of IL-1β, IL-1RA, IL-2RA, IL-6, IL-7, IL-8, IL-9, IL-10, basic FGF, G-CSF, GM-CSF, HGF, IFNγ, IP-10, MCP-1, MIP-1a, MIP-1b, PDGF, TNF-α and VEGF. While, a cytokine-induced immunopathological mechanism has been observed with an increase of IL-2, IL-7, IL-17, IL-10, MCP-1, MIP-1a and TNF-α in severe cases, leading to a bystander effect. Full article ">Figure 5
Viral sensing, innate antiviral response and immune evasion. Potential mechanisms of SARS-CoV-2 immune evasion based on previous studies on MERS-CoV (brown) and SARS-CoV (blue). Some mechanisms are inhibiting viral sensing, whereas others are directed against the innate antiviral response. Solid arrows correspond to a direct promoting activity, dotted arrows correspond to an indirect promoting activity and T-bars correspond to a direct inhibitory activity. Full article ">

29 pages, 1197 KiB

Open AccessReview

Necroptosis in Hepatosteatotic Ischaemia-Reperfusion Injury

by Raji Baidya, Darrell H. G. Crawford, Jérémie Gautheron, Haolu Wang and Kim R. Bridle

Int. J. Mol. Sci. 2020, 21(16), 5931; https://doi.org/10.3390/ijms21165931 - 18 Aug 2020

Cited by 23 | Viewed by 5010

Abstract

While liver transplantation remains the sole treatment option for patients with end-stage liver disease, there are numerous limitations to liver transplantation including the scarcity of donor livers and a rise in livers that are unsuitable to transplant such as those with excess steatosis. [...] Read more.

While liver transplantation remains the sole treatment option for patients with end-stage liver disease, there are numerous limitations to liver transplantation including the scarcity of donor livers and a rise in livers that are unsuitable to transplant such as those with excess steatosis. Fatty livers are susceptible to ischaemia-reperfusion (IR) injury during transplantation and IR injury results in primary graft non-function, graft failure and mortality. Recent studies have described new cell death pathways which differ from the traditional apoptotic pathway. Necroptosis, a regulated form of cell death, has been associated with hepatic IR injury. Receptor-interacting protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL) are thought to be instrumental in the execution of necroptosis. The study of hepatic necroptosis and potential therapeutic approaches to attenuate IR injury will be a key factor in improving our knowledge regarding liver transplantation with fatty donor livers. In this review, we focus on the effect of hepatic steatosis during liver transplantation as well as molecular mechanisms of necroptosis and its involvement during liver IR injury. We also discuss the immune responses triggered during necroptosis and examine the utility of necroptosis inhibitors as potential therapeutic approaches to alleviate IR injury. Full article

(This article belongs to the Special Issue New Frontiers in Organ Preservation and Hepatoprotection)

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Figure 1
Overview of the process of ischemia-reperfusion (IR) injury. Upon depletion of oxygen during the ischaemic stage, mitochondria initiate anaerobic metabolism and ATP production decreases. Further, ion-exchange pump channel dysfunction and pH level decreases leading to cell swelling. During the reperfusion stage, mitochondrial swelling and accumulation of H+, Na+ and K+ result in oxidative stress leading to the excessive production of ROS. This induces cell injury, leading to cell death. The figure is modified from Reference [<a href="#B48-ijms-21-05931" class="html-bibr">48</a>]. Full article ">Figure 2
TNFα-induced cell death pathway. TNFα stimulates TNFR1 to generate complex I by recruiting TRADD, TRAF2 and 5, RIPK1 and cIAP1/2. Polyubiquitination of RIPK1 in complex I will activate the NF-κB pathway, whereas polyubiquitination of RIPK1 by CLYD shifts complex I to cytoplasm to form complex II. Activation of CASPASE8 will result in activation of CASPASE3 and cells undergo apoptosis. Upon inhibition of CASPASE8, activation and phosphorylation of RIPK1 leads to recruitment of RIPK3 and further recruits MLKL to form the necrosome. Further activation of PGAM5 and DRP1 results in ROS production in mitochondria and induces necroptosis. Activation of TLR3/ TLR4 by PAMPs or LPS, activates Toll–IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF) and RIPK3 binding and triggers necroptosis. Abbreviations: TNF, tumour necrosis factor; TRADD, TNFRSF1A-associated via death domain; TRAF, TNF receptor-associated factors; cIAP, cellular inhibitor of apoptosis protein; CYLD, deubiquitinase cylindromatosis; FADD, FAS-associated death domain; MLKL, mediator mixed-lineage kinase domain like; RIPK, receptor-interacting protein kinase; PGAM5, phosphoglycerate mutase 5; Drp1, dynamin-related protein 1; ROS, reactive oxygen species; TLR3/4, TNF-like death receptors 3/4; PAMPs, pathogen-associated molecular patterns; LPS, lipopolysaccharide. Figure is modified from References [<a href="#B84-ijms-21-05931" class="html-bibr">84</a>,<a href="#B91-ijms-21-05931" class="html-bibr">91</a>,<a href="#B92-ijms-21-05931" class="html-bibr">92</a>]. Full article ">

11 pages, 2615 KiB

Open AccessArticle

Interactions between the Intrinsically Disordered Regions of hnRNP-A2 and TDP-43 Accelerate TDP-43′s Conformational Transition

by Wan-Chin Chiang, Ming-Hsuan Lee, Tsai-Chen Chen and Jie-rong Huang

Int. J. Mol. Sci. 2020, 21(16), 5930; https://doi.org/10.3390/ijms21165930 - 18 Aug 2020

Cited by 8 | Viewed by 3766

Abstract

Most biological functions involve protein–protein interactions. Our understanding of these interactions is based mainly on those of structured proteins, because encounters between intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are much less studied, regardless of the fact that more [...] Read more.

Most biological functions involve protein–protein interactions. Our understanding of these interactions is based mainly on those of structured proteins, because encounters between intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are much less studied, regardless of the fact that more than half eukaryotic proteins contain IDRs. RNA-binding proteins (RBPs) are a large family whose members almost all have IDRs in addition to RNA binding domains. These IDRs, having low sequence similarity, interact, but structural details on these interactions are still lacking. Here, using the IDRs of two RBPs (hnRNA-A2 and TDP-43) as a model, we demonstrate that the rate at which TDP-43′s IDR undergoes the neurodegenerative disease related α-helix-to-β-sheet transition increases in relation to the amount of hnRNP-A2′s IDR that is present. There are more than 1500 RBPs in human cells and most of them have IDRs. RBPs often join the same complexes to regulate genes. In addition to the structured RNA-recognition motifs, our study demonstrates a general mechanism through which RBPs may regulate each other’s functions through their IDRs. Full article

(This article belongs to the Special Issue Structural Biology of Proteins and Peptides)

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Figure 1
Schematic illustration of the intrinsically disordered regions (IDRs) of TDP-43 and hnRNP-A2 in this study. (A,B) The domains of (A) TDP-43 and (B) hnRNP-A2. The level of protein disorder was predicted using the PONDR VSL2 algorithm [<a href="#B36-ijms-21-05930" class="html-bibr">36</a>]. The fragments used in this study are indicated with red-dashed lines. (C) Schematic representation of the α-helical, self-association, and liquid-liquid phase separation propensities of TDP-43 reported in previous publications [<a href="#B37-ijms-21-05930" class="html-bibr">37</a>,<a href="#B38-ijms-21-05930" class="html-bibr">38</a>,<a href="#B39-ijms-21-05930" class="html-bibr">39</a>]. The molecular details of the effects of hnRNP-A2 remain elusive. Full article ">Figure 2
The interaction between the intrinsically disordered regions (IDRs) of TDP-43 and hnRNP-A2. (A) Overlaid NMR HSQC spectra of 20 µM 15N TDP-43266–414 alone (red) and in the presence of 20 µM 14N-hnRNP-A2288–341 (green). (B) Overlaid NMR HSQC spectra of 20 µM 15N- hnRNP-A2288–341 alone (red) and in the presence of 20 µM 14N-TDP-43266–414 (green). (C) Circular dichroism spectra of 20 µM TDP-43266–414 (blue), hnRNP-A2288–341 (red), and a one-to-one mixture of the two (purple) overlaid on the summed spectra of the two proteins alone (broken purple line). (D) Fits using BeStSel [<a href="#B41-ijms-21-05930" class="html-bibr">41</a>,<a href="#B42-ijms-21-05930" class="html-bibr">42</a>] of the mixture (left panel) and numerically summed TDP-43/hnRNP-A2 spectra (right) and the proportion of secondary structure components obtained. The residuals of the fits are shown as black bars. Full article ">Figure 3
The time evolution of NMR HSQC and circular dichroism spectra indicates that conformational change in TDP-43266–414 is accelerated by the presence of hnRNP-A2288–341. (A–C) Time sequences of NMR HSQC spectra of 15N-TDP-43 (A) in the presence and (C) in the absence of hnRNP-A2288–341, and (B) of 15N hnRNP-A2288–341 in the presence of TDP-43266–414. Assignments are shown for the cross-peaks whose position or intensity clearly change over time. (D–F) Time sequences of NMR peak intensity profiles as a function of residue number for (D) TDP-43266–414 mixed with hnRNP-A2288–341 and (E,F) for TDP-43266–414 alone. (G) Primary sequence of TDP-43266–414 with residues highlighted in black if the corresponding NMR intensity decreases over time. The α-helical and Q/N rich regions are also indicated. (H–J) Circular dichroism spectra at different incubation times of (H) the mixture sample, (I) TDP-43266–414 alone and (J) hnRNP-A2288–341 only. Full article ">Figure 4
Increasing the amount of hnRNP-A2288–341 accelerates transitions from α-helix to β-sheet in TDP-43266–414. (A–D) Comparisons of the NMR HSQC spectrum of TDP-43266–414 in the absence of hnRNP-A2288–341 after 22 h of incubation (red) with the spectra of (A) TDP-43266–414 alone after ~1 h incubation (black), (B) a 1:1 mixture of TDP-43266–414 and hnRNP-A2288–341 after ~9 h of incubation, (C) a 1:3 mixture of TDP-43266–414 and hnRNP-A2288–341 after ~3 h of incubation, and (D) a 1:5 mixture of TDP-43266–414 and hnRNP-A2288–341 after less than 1 h of incubation. (E–H) Proportions of secondary structure elements derived from CD measurements (upper panel: α-helix; lower panel: antiparallel β-sheet) for the same mixtures at different incubation times. Full article ">Figure 5
A schematic illustration of the proposed model. In the absence of hnRNP-A2, the IDR of TDP-43 is in equilibrium with both α-helix and coil conformations but tends to aggregate in its coil form (left panel). When the α-helical region of TDP-43′s IDR weakly contacts the IDR of hnRNP-A2 (pink), the coil population of TDP-43 is increased, which enhances its tendency to aggregate (right panel). Full article ">

14 pages, 621 KiB

Open AccessReview

Taste the Pain: The Role of TRP Channels in Pain and Taste Perception

by Edwin N. Aroke, Keesha L. Powell-Roach, Rosario B. Jaime-Lara, Markos Tesfaye, Abhrarup Roy, Pamela Jackson and Paule V. Joseph

Int. J. Mol. Sci. 2020, 21(16), 5929; https://doi.org/10.3390/ijms21165929 - 18 Aug 2020

Cited by 39 | Viewed by 8490

Abstract

Transient receptor potential (TRP) channels are a superfamily of cation transmembrane proteins that are expressed in many tissues and respond to many sensory stimuli. TRP channels play a role in sensory signaling for taste, thermosensation, mechanosensation, and nociception. Activation of TRP channels (e.g., [...] Read more.

Transient receptor potential (TRP) channels are a superfamily of cation transmembrane proteins that are expressed in many tissues and respond to many sensory stimuli. TRP channels play a role in sensory signaling for taste, thermosensation, mechanosensation, and nociception. Activation of TRP channels (e.g., TRPM5) in taste receptors by food/chemicals (e.g., capsaicin) is essential in the acquisition of nutrients, which fuel metabolism, growth, and development. Pain signals from these nociceptors are essential for harm avoidance. Dysfunctional TRP channels have been associated with neuropathic pain, inflammation, and reduced ability to detect taste stimuli. Humans have long recognized the relationship between taste and pain. However, the mechanisms and relationship among these taste–pain sensorial experiences are not fully understood. This article provides a narrative review of literature examining the role of TRP channels on taste and pain perception. Genomic variability in the TRPV1 gene has been associated with alterations in various pain conditions. Moreover, polymorphisms of the TRPV1 gene have been associated with alterations in salty taste sensitivity and salt preference. Studies of genetic variations in TRP genes or modulation of TRP pathways may increase our understanding of the shared biological mediators of pain and taste, leading to therapeutic interventions to treat many diseases. Full article

(This article belongs to the Section Biochemistry)

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23 pages, 1561 KiB

Open AccessArticle

Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study

by Chris R. Taitt, Tomasz A. Leski, Michael G. Prouty, Gavin W. Ford, Vireak Heang, Brent L. House, Samuel Y. Levin, Jennifer A. Curry, Adel Mansour, Hanan El Mohammady, Momtaz Wasfy, Drake Hamilton Tilley, Michael J. Gregory, Matthew R. Kasper, James Regeimbal, Paul Rios, Guillermo Pimentel, Brook A. Danboise, Christine E. Hulseberg, Elizabeth A. Odundo, Abigael N. Ombogo, Erick K. Cheruiyot, Cliff O. Philip and Gary J. Vora Show full author list Hide full author list

Int. J. Mol. Sci. 2020, 21(16), 5928; https://doi.org/10.3390/ijms21165928 - 18 Aug 2020

Cited by 7 | Viewed by 4327

Abstract

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due [...] Read more.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included bla_TEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, bla_OXA-48 or bla_NDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum β-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care. Full article

(This article belongs to the Special Issue Drug Resistance Mechanisms in Bacteria)

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(A) Number of antimicrobial resistance determinants (ARDs) detected in tested population, by category; numbers in parentheses indicate total ARDs represented on microarray for that category. BLA = β-lactams (53); AG = aminoglycosides (44); MAC = macrolide (40); TET = tetracyclines (38); GLY = glycopeptides (13); ANS = ansamycins (1); MUP = mupirocin (1); PHE = phenicols (20); LIN = lincosamides (6); MLS = macrolides/lincosamides/streptogramins (13); FQ = fluoroquinolones (4); QUA = quaternary amines (2); STR = streptothricin; PT = platensimycin + platencin (1); SUL = sulfonamides (3); AMP = antimicrobial peptides (1); TMP = diaminopyrimidine. (B) Number of ARDs detected per isolate. (C) Prevalence of unique ARDs detected in >10 isolates. Full article ">Figure 2
Numbers of unique ARDs observed for Campylobacter spp. (yellow), E. coli (orange), Shigella spp. (green), Salmonella (blue), and all species (black) detected in isolates from each collection site. Full article ">Figure 3
Box and whisker charts showing number of unique ARDs per isolate for (A) E. coli; (B) Shigella spp.; (C) Salmonella; (D) each population as a whole (including Campylobacter spp.). Black squares in each chart indicate mean values. Full article ">Figure 4
Pie charts showing carriage rates of ARDs conferring resistance to β-lactams, aminoglycosides, tetracyclines, and trimethoprim among E. coli, Shigella spp., and Salmonella. Black pie slices indicate the percentage of isolates that were negative for all tested ARDs in that category. Note that many strains carried multiple β-lactamase and aminoglycoside ARDs. Full article ">

29 pages, 578 KiB

Open AccessReview

Hypothyroidism-Induced Nonalcoholic Fatty Liver Disease (HIN): Mechanisms and Emerging Therapeutic Options

by Daniela Maria Tanase, Evelina Maria Gosav, Ecaterina Neculae, Claudia Florida Costea, Manuela Ciocoiu, Loredana Liliana Hurjui, Claudia Cristina Tarniceriu and Mariana Floria

Int. J. Mol. Sci. 2020, 21(16), 5927; https://doi.org/10.3390/ijms21165927 - 18 Aug 2020

Cited by 37 | Viewed by 11347

Abstract

Nonalcoholic fatty liver disease (NAFLD) is an emerging worldwide problem and its association with other metabolic pathologies has been one of the main research topics in the last decade. The aim of this review article is to provide an up-to-date correlation between hypothyroidism [...] Read more.

Nonalcoholic fatty liver disease (NAFLD) is an emerging worldwide problem and its association with other metabolic pathologies has been one of the main research topics in the last decade. The aim of this review article is to provide an up-to-date correlation between hypothyroidism and NAFLD. We followed evidence regarding epidemiological impact, immunopathogenesis, thyroid hormone-liver axis, lipid and cholesterol metabolism, insulin resistance, oxidative stress, and inflammation. After evaluating the influence of thyroid hormone imbalance on liver structure and function, the latest studies have focused on developing new therapeutic strategies. Thyroid hormones (THs) along with their metabolites and thyroid hormone receptor β (THR-β) agonist are the main therapeutic targets. Other liver specific analogs and alternative treatments have been tested in the last few years as potential NAFLD therapy. Finally, we concluded that further research is necessary as well as the need for an extensive evaluation of thyroid function in NAFLD/NASH patients, aiming for better management and outcome. Full article

(This article belongs to the Special Issue Thyroid Hormones and NAFLD: New Insights)

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18 pages, 3801 KiB

Open AccessReview

The Significance of Flavonoids in the Process of Biological Nitrogen Fixation

by Wei Dong and Yuguang Song

Int. J. Mol. Sci. 2020, 21(16), 5926; https://doi.org/10.3390/ijms21165926 - 18 Aug 2020

Cited by 49 | Viewed by 6316 | Correction

Abstract

Nitrogen is essential for the growth of plants. The ability of some plant species to obtain all or part of their requirement for nitrogen by interacting with microbial symbionts has conferred a major competitive advantage over those plants unable to do so. The [...] Read more.

Nitrogen is essential for the growth of plants. The ability of some plant species to obtain all or part of their requirement for nitrogen by interacting with microbial symbionts has conferred a major competitive advantage over those plants unable to do so. The function of certain flavonoids (a group of secondary metabolites produced by the plant phenylpropanoid pathway) within the process of biological nitrogen fixation carried out by Rhizobium spp. has been thoroughly researched. However, their significance to biological nitrogen fixation carried out during the actinorhizal and arbuscular mycorrhiza–Rhizobium–legume interaction remains unclear. This review catalogs and contextualizes the role of flavonoids in the three major types of root endosymbiosis responsible for biological nitrogen fixation. The importance of gaining an understanding of the molecular basis of endosymbiosis signaling, as well as the potential of and challenges facing modifying flavonoids either quantitatively and/or qualitatively are discussed, along with proposed strategies for both optimizing the process of nodulation and widening the plant species base, which can support nodulation. Full article

(This article belongs to the Section Biochemistry)

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20 pages, 8064 KiB

Open AccessArticle

CD163 as a Biomarker in Colorectal Cancer: The Expression on Circulating Monocytes and Tumor-Associated Macrophages, and the Soluble Form in the Blood

by Daniëlle Krijgsman, Natasja L. De Vries, Morten N. Andersen, Anni Skovbo, Rob A.E.M. Tollenaar, Holger J. Møller, Marianne Hokland and Peter J.K. Kuppen

Int. J. Mol. Sci. 2020, 21(16), 5925; https://doi.org/10.3390/ijms21165925 - 18 Aug 2020

Cited by 29 | Viewed by 4757

Abstract

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An [...] Read more.

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression. Full article

(This article belongs to the Collection Feature Papers in Molecular Oncology)

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Figure 1
Sample availability for the measurement of monocytes, sCD163 and tumor-associated macrophages (TAMs) in colorectal cancer (CRC) patients and healthy donors. Monocytes, sCD163 and TAMs were studied in 78 CRC patients. TAMs and monocytes were studied in 72 and 47 CRC patients, respectively. Additionally, sCD163 levels were studied in 64 pre-operative and 44 post-operative patients. Finally, monocytes were studied in 10 healthy donors, whereas sCD163 levels were studied in 40 healthy donors. The numbers in the figure indicate the number of patients in each subgroup with overlapping samples. Abbreviations: CRC (colorectal cancer), ELISA (enzyme-linked immunosorbent assay), PBMC (peripheral blood mononuclear cells), sCD163 (soluble CD163), TAM (tumor-associated macrophages). Full article ">Figure 2
sCD163 levels in the serum of CRC patients and healthy donors as measured by the enzyme-linked immunosorbent assay (ELISA) in relation to clinicopathological parameters. (A) Comparison of sCD163 serum levels in healthy donors (N = 40) and pre-operative CRC patients (N = 64), and the change in sCD163 levels in CRC patients after surgery (N = 39). (B) Association between the sCD163 levels in CRC patients and TNM stage (stage 0/I, N = 15; stage II/III, N = 43; stage IV, N = 6). (C) Association between the sCD163 levels and clinical outcome in CRC patients. Kaplan–Meier curves for the overall survival (OS) are shown for TNM stage 0–IV CRC patients (N = 64) and Kaplan–Meier curves for disease-free survival (DFS) are shown for the TNM stage 0–III CRC patients (N = 58). Stratifications were based on the median sCD163 level (2.0 mg/L). The bars ((A), left figure; (B)) show the median sCD163 level with a 95% confidence interval (CI) whereas the dotted lines show the reference sCD163 levels (0.7–3.9 mg/L). Statistically significant p-values (≤0.05) are indicated in bold. Abbreviations: CI (confidence interval), CRC (colorectal cancer), DFS (disease-free survival), HD (healthy donor), OS (overall survival), sCD163 (soluble CD163), TNM (tumor, node, metastasis). Full article ">Figure 3
Distribution of monocyte subsets and their level of CD163 expression in the peripheral blood of CRC patients and healthy donors as measured by flow cytometry in relation to clinicopathological parameters (A) Comparison of the total monocyte percentage, monocyte subset distribution (CD14++CD16− classical, CD14++CD16+ intermediate and CD14+CD16++ nonclassical monocytes), and CD163 expression level on these monocyte subsets between healthy donors (N = 10) and CRC patients (N = 47). (B) Association between the total monocyte percentage and TNM stage (stage 0/I, N = 14; stage II/III, N = 25; stage IV, N = 8), differentiation grade (well/moderate, N = 34; poor, N = 11) and tumor-positive lymph nodes (no, N = 30; yes, N = 17) in CRC patients. (C) Association between the percentage of classical monocytes and differentiation grade (well/moderate, N = 34; poor, N = 11) in CRC patients. (D) Association between the total monocyte percentage and clinical outcome in CRC patients. Kaplan–Meier curves for the OS are shown for TNM stage 0–IV CRC patients (N = 47) and Kaplan–Meier curves for the DFS are shown for stage 0–III CRC patients (N = 39). Stratifications were based on the median total monocyte percentage (24.9%). The bars in figure (A–C) show the median with a 95% CI. Statistically significant p-values (≤0.05) are indicated in bold. Abbreviations: CI (confidence interval), CRC (colorectal cancer), HD (healthy donor), MFI (median fluorescence intensity), PBMCs (peripheral blood mononuclear cells), TNM (tumor, node, metastasis). Full article ">Figure 4
TAM subsets in primary colorectal tumors as visualized by multiplex immunofluorescence. (A) Image of M0 (CD68+iNOS−CD163−), M1 (CD68+iNOS+CD163−), M2 (CD68+iNOS−CD163+) and M3 (CD68+iNOS+CD163+) TAMs. (B) Representative images of colorectal tumors with high numbers of stromal TAMs (sTAMs) and intraepithelial TAMs (ieTAMs) (white: DAPI; red: cytokeratin+ tumor epithelium; blue: CD68+ TAMs). The white arrows indicate examples of TAMs with indicated phenotypes (A) or localizations (B). Abbreviations: CRC (colorectal cancer), iNOS (inducible nitric oxide synthase), ieTAM (intraepithelial TAM), sTAM (stromal TAM), TAM (tumor-associated macrophage). Full article ">Figure 5
Distribution of the TAM subsets in the stromal and intraepithelial compartments of primary colorectal tumors as measured by multiplex immunofluorescence in relation to clinicopathological parameters. (A) Distribution of the sTAM and ieTAM subsets (CD68+iNOS−CD163− M0, CD68+iNOS+CD163− M1, CD68+iNOS−CD163+ M2 and CD68+iNOS+CD163+ M3 TAMs) in primary colorectal tumors (N = 72 and N = 68, respectively). (B) Associations between the percentage of M2 ieTAMs and TNM stage (stage 0/I, N = 14; stage II/III, N = 46; stage IV, N = 8), and between the percentage of M2 ieTAMs and M2 ieTAM density and tumor differentiation grade (well/moderate, N = 55; poor, N = 12) in primary colorectal tumors. The bars show the median with a 95% CI. Statistically significant p-values (≤0.05) are indicated in bold. Abbreviations: CI (confidence interval), CRC (colorectal cancer), iNOS (inducible nitric oxide synthase), ieTAM (intraepithelial TAM), sTAM (stromal TAM), TAM (tumor-associated macrophage), TNM (Tumor, Node, Metastasis). Full article ">

18 pages, 3409 KiB

Open AccessArticle

Osteoclasts’ Ability to Generate Trenches Rather Than Pits Depends on High Levels of Active Cathepsin K and Efficient Clearance of Resorption Products

by Xenia G. Borggaard, Dinisha C. Pirapaharan, Jean-Marie Delaissé and Kent Søe

Int. J. Mol. Sci. 2020, 21(16), 5924; https://doi.org/10.3390/ijms21165924 - 18 Aug 2020

Cited by 23 | Viewed by 4087

Abstract

Until recently, it was well-accepted that osteoclasts resorb bone according to the resorption cycle model. This model is based on the assumption that osteoclasts are immobile during bone erosion, allowing the actin ring to be firmly attached and thereby provide an effective seal [...] Read more.

Until recently, it was well-accepted that osteoclasts resorb bone according to the resorption cycle model. This model is based on the assumption that osteoclasts are immobile during bone erosion, allowing the actin ring to be firmly attached and thereby provide an effective seal encircling the resorptive compartment. However, through time-lapse, it was recently documented that osteoclasts making elongated resorption cavities and trenches move across the bone surface while efficiently resorbing bone. However, it was also shown that osteoclasts making rounded cavities and pits indeed resorb bone while they are immobile. Only little is known about what distinguishes these two different resorption modes. This is of both basic and clinical interest because these resorption modes are differently sensitive to drugs and are affected by the gender as well as age of the donor. In the present manuscript we show that: 1. levels of active cathepsin K determine the switch from pit to trench mode; 2. pit and trench mode depend on clathrin-mediated endocytosis; and 3. a mechanism integrating release of resorption products and membrane/integrin recycling is required for prolongation of trench mode. Our study therefore contributes to an improved understanding of the molecular and cellular determinants for the two osteoclastic bone resorption modes. Full article

(This article belongs to the Special Issue New Insights in Osteoclasts’ Biology)

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25 pages, 9171 KiB

Open AccessArticle

Oxidant-Induced Alterations in the Adipocyte Transcriptome: Role of the Na,K-ATPase Oxidant Amplification Loop

by Komal Sodhi, James Denvir, Jiang Liu, Juan R. Sanabria, Yiliang Chen, Roy Silverstein, Zijian Xie, Nader G. Abraham and Joseph I. Shapiro

Int. J. Mol. Sci. 2020, 21(16), 5923; https://doi.org/10.3390/ijms21165923 - 18 Aug 2020

Cited by 7 | Viewed by 2783

Abstract

(1) Background: Recently we have noted that adipocyte specific expression of the peptide, NaKtide, which was developed to attenuate the Na,K-ATPase oxidant amplification loop, could ameliorate the phenotypical features of uremic cardiomyopathy. We performed this study to better characterize the cellular transcriptomes that [...] Read more.

(1) Background: Recently we have noted that adipocyte specific expression of the peptide, NaKtide, which was developed to attenuate the Na,K-ATPase oxidant amplification loop, could ameliorate the phenotypical features of uremic cardiomyopathy. We performed this study to better characterize the cellular transcriptomes that are involved in various biological pathways associated with adipocyte function occurring with renal failure. (2) Methods: RNAseq was performed on the visceral adipose tissue of animals subjected to partial nephrectomy. Specific expression of NaKtide in adipocytes was achieved using an adiponectin promoter. To better understand the cause of gene expression changes in vivo, 3T3L1 adipocytes were exposed to indoxyl sulfate (IS) or oxidized low density lipoprotein (oxLDL), with and without pNaKtide (the cell permeant form of NaKtide). RNAseq was also performed on these samples. (3) Results: We noted a large number of adipocyte genes were altered in experimental renal failure. Adipocyte specific NaKtide expression reversed most of these abnormalities. High correlation with some cardiac specific phenotypical features was noted amongst groups of these genes. In the murine adipocytes, both IS and oxLDL induced similar pathway changes as were noted in vivo, and pNaKtide appeared to reverse these changes. Network analysis demonstrated tremendous similarities between the network revealed by gene expression analysis with IS compared with oxLDL, and the combined in vitro dataset was noted to also have considerable similarity to that seen in vivo with experimental renal failure. (4) Conclusions: This study suggests that the myriad of phenotypical features seen with experimental renal failure may be fundamentally linked to oxidant stress within adipocytes. Full article

(This article belongs to the Special Issue Cardiotonic Steroids: From Toxins to Hormones)

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18 pages, 3375 KiB

Open AccessArticle

The Role of Atypical Cannabinoid Ligands O-1602 and O-1918 on Skeletal Muscle Homeostasis with a Focus on Obesity

by Anna C. Simcocks, Lannie O’Keefe, Kayte A. Jenkin, Lauren M. Cornall, Esther Grinfeld, Michael L. Mathai, Deanne H. Hryciw and Andrew J. McAinch

Int. J. Mol. Sci. 2020, 21(16), 5922; https://doi.org/10.3390/ijms21165922 - 18 Aug 2020

Cited by 11 | Viewed by 3313

Abstract

O-1602 and O-1918 are atypical cannabinoid ligands for GPR55 and GPR18, which may be novel pharmaceuticals for the treatment of obesity by targeting energy homeostasis regulation in skeletal muscle. This study aimed to determine the effect of O-1602 or O-1918 on markers of [...] Read more.

O-1602 and O-1918 are atypical cannabinoid ligands for GPR55 and GPR18, which may be novel pharmaceuticals for the treatment of obesity by targeting energy homeostasis regulation in skeletal muscle. This study aimed to determine the effect of O-1602 or O-1918 on markers of oxidative capacity and fatty acid metabolism in the skeletal muscle. Diet-induced obese (DIO) male Sprague Dawley rats were administered a daily intraperitoneal injection of O-1602, O-1918 or vehicle for 6 weeks. C₂C₁₂ myotubes were treated with O-1602 or O-1918 and human primary myotubes were treated with O-1918. GPR18 mRNA was expressed in the skeletal muscle of DIO rats and was up-regulated in red gastrocnemius when compared with white gastrocnemius. O-1602 had no effect on mRNA expression on selected markers for oxidative capacity, fatty acid metabolism or adiponectin signalling in gastrocnemius from DIO rats or in C₂C₁₂ myotubes, while APPL2 mRNA was up-regulated in white gastrocnemius in DIO rats treated with O-1918. In C₂C₁₂ myotubes treated with O-1918, PGC1α, NFATc1 and PDK4 mRNA were up-regulated. There were no effects of O-1918 on mRNA expression in human primary myotubes derived from obese and obese T2DM individuals. In conclusion, O-1602 does not alter mRNA expression of key pathways important for skeletal muscle energy homeostasis in obesity. In contrast, O-1918 appears to alter markers of oxidative capacity and fatty acid metabolism in C₂C₁₂ myotubes only. GPR18 is expressed in DIO rat skeletal muscle and future work could focus on selectively modulating GPR18 in a tissue-specific manner, which may be beneficial for obesity-targeted therapies. Full article

(This article belongs to the Special Issue Peripheral Targets in Obesity: Pathologies and Therapeutics)

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16 pages, 2400 KiB

Open AccessArticle

P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation

by Jia-hua Hu, Cole Malloy and Dax A. Hoffman

Int. J. Mol. Sci. 2020, 21(16), 5921; https://doi.org/10.3390/ijms21165921 - 18 Aug 2020

Cited by 8 | Viewed by 3582

Abstract

The subthreshold, transient A-type K⁺ current is a vital regulator of the excitability of neurons throughout the brain. In mammalian hippocampal pyramidal neurons, this current is carried primarily by ion channels comprising Kv4.2 α-subunits. These channels occupy the somatodendritic domains of these [...] Read more.

The subthreshold, transient A-type K⁺ current is a vital regulator of the excitability of neurons throughout the brain. In mammalian hippocampal pyramidal neurons, this current is carried primarily by ion channels comprising Kv4.2 α-subunits. These channels occupy the somatodendritic domains of these principle excitatory neurons and thus regulate membrane voltage relevant to the input–output efficacy of these cells. Owing to their robust control of membrane excitability and ubiquitous expression in the hippocampus, their dysfunction can alter network stability in a manner that manifests in recurrent seizures. Indeed, growing evidence implicates these channels in intractable epilepsies of the temporal lobe, which underscores the importance of determining the molecular mechanisms underlying their regulation and contribution to pathologies. Here, we describe the role of p38 kinase phosphorylation of a C-terminal motif in Kv4.2 in modulating hippocampal neuronal excitability and behavioral seizure strength. Using a combination of biochemical, single-cell electrophysiology, and in vivo seizure techniques, we show that kainic acid-induced seizure induces p38-mediated phosphorylation of Thr607 in Kv4.2 in a time-dependent manner. The pharmacological and genetic disruption of this process reduces neuronal excitability and dampens seizure intensity, illuminating a cellular cascade that may be targeted for therapeutic intervention to mitigate seizure intensity and progression. Full article

(This article belongs to the Special Issue P38 Signaling Pathway)

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p38 mitogen-activated protein kinase (MAPK) contributed to kainic acid-induced seizure in WT mice but not Kv4.2TA mice. (A) Time course of mean behavioral seizure score following kainic acid injection. The mean behavioral seizure score was significantly reduced in Kv4.2TA mice compared to WT mice. Furthermore, p38 inhibitor SB 203580 significantly reduced behavioral seizure score following kainic acid injection in WT mice but not in Kv4.2TA mice, n = 13–15 for each group, two-way ANOVA, * p < 0.05. (B) Total behavioral seizure score for each group, n = 13–15 for each group, t-test, * p < 0.05. (C) Latency to stage 3 seizure for each group. n = 13–15 for each group, t-test, ** p < 0.01. Full article ">Figure 2
Seizure induced by kainic acid triggers Kv4.2 T607 phosphorylation in a time-dependent manner in mouse hippocampus. (A) Time course of Kv4.2 phosphorylation at Thr602 and Thr607 by kainic acid administration (25 mg/kg, i.p.) in mouse hippocampus. (B) Statistical analysis of kainic acid-induced phosphorylation of Kv4.2 at Thr607 in mouse hippocampus, n = 3–8 in each group, t-test, ** p < 0.01. (C) Statistical analysis of kainic acid-induced phosphorylation of Kv4.2 at Thr602 in mouse hippocampus, n = 3–8 in each group, t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 3
p38 MAPK contributes to kainic acid-induced Kv4.2 phosphorylation at T607. (A) SB 203580, a potent p38 inhibitor (20 mg/kg, i.p., 15 min), blocked kainic acid-induced phosphorylation of Kv4.2 T607 in mouse hippocampus. (B) Statistical analysis of the effect of SB 203580 on kainic acid-induced phosphorylation of Kv4.2 at Thr607 in mouse hippocampus, n = 4–6 in each group, t-test, ** p < 0.01. Full article ">Figure 4
p38 MAPK colocalizes with Kv4.2. (A) HEK293T cells were transfected with GFP-Kv4.2 and Flag-p38. Cells were fixed and stained with GFP and Flag to show co-localization. Scale bar: 20 μm. (B) High magnification images and line scan analysis of colocalization. Scale bar: 5 μm. (C) Mouse brains were co-stained with Kv4.2 and pp38 antibody. Phosphorylated p38 is localized in the cell body and dendrites as well. Scale bar: 20 μm. (D) High magnification images showing Kv4.2 and pp38 colocalized in dendrites, as indicated with arrow heads. Scale bar: 5 μm. Full article ">Figure 5
Kainic acid activates p38 MAPK in both WT and Kv4.2TA mice. (A) Immunostaining analysis showed p38 phosphorylation increased with kainic acid administration (25 mg/kg, i.p., 30 min) in mouse hippocampus, n = 26 cells in each group, t-test, *** p < 0.001. (B) Western blot analysis showed p38 phosphorylation increased with kainic acid administration (25 mg/kg, i.p., 30 min) in hippocampus in both WT and Kv4.2TA mice, n = 4–6 cells in each group, t-test, *** p < 0.001. Full article ">Figure 6
p38 impacts hippocampal pyramidal neuron excitability through Kv4.2. (A) Current step of +300 pA induces repetitive firing in pyramidal neurons recorded from WT and Kv4.2TA mice with or without SB 203580 treatment. Scale 40 mV/250 ms. Square current inset 300 pA. (B) Sequential somatic current injections increasing in magnitude reveal p38 kinase inhibition reduces AP firing frequency in WT hippocampal neurons at +300 pA relative to vehicle (n = 15 in vehicle, n = 19 in treatment; two-way ANOVA, * p < 0.05). Kv4.2TA neurons display reduced firing frequency at +300 pA relative to WT in vehicle, which is augmented in the presence of SB 203580 such that current magnitudes of +200 and +250 pA also exhibit significant differences (n = 18 in vehicle, n = 14 in SB 203580; two-way ANOVA, * p < 0.05; *** p < 0.001). (C) Inter-spike intervals measured between the first two spikes in a train evoked by 150 pA injection display no significant difference among groups. Kruskal–Wallis test, p > 0.05. (D) Ramp current injections evoke repetitive firing in all pyramidal neurons recorded in each condition. Arrow indicates point at which action potential (AP) threshold, rheobase, and latency to fire were measured. Ramp current inset 400 pA/s. (E) Minimum current to elicit AP firing at threshold (rheobase) is not significantly different among the populations. One-way ANOVA, p > 0.05. (F) Latency to fire in response to ramp injection is not significantly different among populations. Kruskal–Wallis test, p > 0.05. Full article ">

27 pages, 10738 KiB

Open AccessReview

Ursolic Acid-Based Derivatives as Potential Anti-Cancer Agents: An Update

by Vuyolwethu Khwaza, Opeoluwa O. Oyedeji and Blessing A. Aderibigbe

Int. J. Mol. Sci. 2020, 21(16), 5920; https://doi.org/10.3390/ijms21165920 - 18 Aug 2020

Cited by 125 | Viewed by 8675

Abstract

Ursolic acid is a pharmacologically active pentacyclic triterpenoid derived from medicinal plants, fruit, and vegetables. The pharmacological activities of ursolic acid have been extensively studied over the past few years and various reports have revealed that ursolic acid has multiple biological activities, which [...] Read more.

Ursolic acid is a pharmacologically active pentacyclic triterpenoid derived from medicinal plants, fruit, and vegetables. The pharmacological activities of ursolic acid have been extensively studied over the past few years and various reports have revealed that ursolic acid has multiple biological activities, which include anti-inflammatory, antioxidant, anti-cancer, etc. In terms of cancer treatment, ursolic acid interacts with a number of molecular targets that play an essential role in many cell signaling pathways. It suppresses transformation, inhibits proliferation, and induces apoptosis of tumor cells. Although ursolic acid has many benefits, its therapeutic applications in clinical medicine are limited by its poor bioavailability and absorption. To overcome such disadvantages, researchers around the globe have designed and developed synthetic ursolic acid derivatives with enhanced therapeutic effects by structurally modifying the parent skeleton of ursolic acid. These structurally modified compounds display enhanced therapeutic effects when compared to ursolic acid. This present review summarizes various synthesized derivatives of ursolic acid with anti-cancer activity which were reported from 2015 to date. Full article

(This article belongs to the Collection Bioactive Natural Compounds for Therapeutics and Nutraceutical Applications)

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4 pages, 183 KiB

Open AccessEditorial

Basic and Translational Models of Cooperative Oncogenesis

by Helena E. Richardson, Julia B. Cordero and Daniela Grifoni

Int. J. Mol. Sci. 2020, 21(16), 5919; https://doi.org/10.3390/ijms21165919 - 18 Aug 2020

Cited by 5 | Viewed by 2704

Abstract

n/a Full article

(This article belongs to the Special Issue Basic and Translational Models of Cooperative Oncogenesis)

17 pages, 499 KiB

Open AccessReview

PD-L1 in Systemic Immunity: Unraveling Its Contribution to PD-1/PD-L1 Blockade Immunotherapy

by Ana Bocanegra, Ester Blanco, Gonzalo Fernandez-Hinojal, Hugo Arasanz, Luisa Chocarro, Miren Zuazo, Pilar Morente, Ruth Vera, David Escors and Grazyna Kochan

Int. J. Mol. Sci. 2020, 21(16), 5918; https://doi.org/10.3390/ijms21165918 - 18 Aug 2020

Cited by 19 | Viewed by 6425

Abstract

The use of monoclonal antibodies targeting PD-1/PD-L1 axis completely changed anticancer treatment strategies. However, despite the significant improvement in overall survival and progression-free survival of patients undergoing these immunotherapy treatments, the only clinically accepted biomarker with some prediction capabilities for the outcome of [...] Read more.

The use of monoclonal antibodies targeting PD-1/PD-L1 axis completely changed anticancer treatment strategies. However, despite the significant improvement in overall survival and progression-free survival of patients undergoing these immunotherapy treatments, the only clinically accepted biomarker with some prediction capabilities for the outcome of the treatment is PD-L1 expression in tumor biopsies. Nevertheless, even when having PD-L1-positive tumors, numerous patients do not respond to these treatments. Considering the high cost of these therapies and the risk of immune-related adverse events during therapy, it is necessary to identify additional biomarkers that would facilitate stratifying patients in potential responders and non-responders before the start of immunotherapies. Here, we review the utility of PD-L1 expression not only in tumor cells but in immune system cells and their influence on the antitumor activity of immune cell subsets. Full article

(This article belongs to the Special Issue PD-L1, a Master Regulator of Immunity 2.0)

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13 pages, 2754 KiB

Open AccessArticle

Evaluation of the Individual and Combined Toxicity of Fumonisin Mycotoxins in Human Gastric Epithelial Cells

by Song Yu, Bingxuan Jia, Na Liu, Dianzhen Yu and Aibo Wu

Int. J. Mol. Sci. 2020, 21(16), 5917; https://doi.org/10.3390/ijms21165917 - 18 Aug 2020

Cited by 33 | Viewed by 3926

Abstract

Fumonisin contaminates food and feed extensively throughout the world, causing chronic and acute toxicity in human and animals. Currently, studies on the toxicology of fumonisins mainly focus on fumonisin B1 (FB1). Considering that FB1, fumonisin B2 (FB2) and fumonisin B3 (FB3) could coexist [...] Read more.

Fumonisin contaminates food and feed extensively throughout the world, causing chronic and acute toxicity in human and animals. Currently, studies on the toxicology of fumonisins mainly focus on fumonisin B1 (FB1). Considering that FB1, fumonisin B2 (FB2) and fumonisin B3 (FB3) could coexist in food and feed, a study regarding a single toxin, FB1, may not completely reflect the toxicity of fumonisin. The gastrointestinal tract is usually exposed to these dietary toxins. In our study, the human gastric epithelial cell line (GES-1) was used as in vitro model to evaluate the toxicity of fumonisin. Firstly, we found that they could cause a decrease in cell viability, and increase in membrane leakage, cell death and the induction of expression of markers for endoplasmic reticulum (ER) stress. Their toxicity potency rank is FB1 > FB2 >> FB3. The results also showed that the synergistic effect appeared in the combinations of FB1 + FB2 and FB1 + FB3. Nevertheless, the combinations of FB2 + FB3 and FB1 + FB2 + FB3 showed a synergistic effect at low concentration and an antagonistic effect at high concentration. We also found that myriocin (ISP-1) could alleviate the cytotoxicity induced by fumonisin in GES-1 cells. Finally, this study may help to determine or optimize the legal limits and risk assessment method of mycotoxins in food and feed and provide a potential method to block the fumonisin toxicity. Full article

(This article belongs to the Special Issue Natural and Synthetic Toxins: Molecular Aspects and Development Treatment Strategy)

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Figure 1
The chemical structure of major fumonisin B: (A) fumonisin B1; (B) fumonisin B2; and (C) fumonisin B3. Full article ">Figure 2
The cytotoxicity of fumonisins B (FBs) on the human gastric epithelial cell line (GES-1) was shown to decrease the cell viability rate and increase the LDH leakage rate. (A) Cell viability was assessed by the cell count kit-8 cell proliferation assay. (B) Cell membrane integrity was determined by detecting the LDH leakage from the cell media using a cytotoxicity LDH detection kit. These data represented the mean ± SEM of the three individual experiments (** p < 0.01, *** p < 0.001, analysis of variance (ANOVA) test). Full article ">Figure 3
FB1-induced cell death in GES-1 cells. (A) Dead cells were marked with Annexin V-FITC/PI dye. The GES-1 cell death rate was analyzed by flow cytometry. (B) These data represented the mean ± SEM of the three individual experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, analysis of variance (ANOVA) test). Full article ">Figure 4
FBs induced ER stress in the GES-1 cells. The levels of ER stress markers were determined by immunoblotting (A) and real-time PCR (B) after treatment with 20 μM FBs for 48 h. The data represented the mean ± SEM of the three individual experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, analysis of variance (ANOVA) test). Full article ">Figure 5
Interactive effects of FB1, FB2 and FB3 on the GES-1 cell viability. (A–D) GES-1 cells were treated with fumonisin alone or their mixtures for 48 h. The data represented the mean ± SEM of the three individual experiments (** p < 0.01, *** p < 0.001, analysis of variance (ANOVA) test). Full article ">Figure 6
Myriocin (ISP-1) alleviated FB-induced GES-1 cytotoxicity. (A) Effect of ISP-1 on FB1-induced cytotoxicity. (B) Effect of ISP-1 on FB2-induced cytotoxicity. (C) Effect of ISP-1 on FB3 induced cytotoxicity. The data represented the mean ± SEM of the three individual experiments (** p < 0.01, *** p < 0.001, analysis of variance (ANOVA) test). Full article ">

19 pages, 12775 KiB

Open AccessArticle

Toll-Like Receptor 5 of Golden Pompano Trachinotus ovatus (Linnaeus 1758): Characterization, Promoter Activity and Functional Analysis

by Ke-Cheng Zhu, Meng Wu, Dian-Chang Zhang, Hua-Yang Guo, Nan Zhang, Liang Guo, Bao-Suo Liu and Shi-Gui Jiang

Int. J. Mol. Sci. 2020, 21(16), 5916; https://doi.org/10.3390/ijms21165916 - 18 Aug 2020

Cited by 27 | Viewed by 3251

Abstract

Toll-like receptors (TLRs), as important pattern recognition receptors, represent a significant component of fish immune systems and play an important role in resisting the invasion of pathogenic microorganisms. The TLR5 subfamily contains two types of TLR5, the membrane form of TLR5 (TLR5M) and [...] Read more.

Toll-like receptors (TLRs), as important pattern recognition receptors, represent a significant component of fish immune systems and play an important role in resisting the invasion of pathogenic microorganisms. The TLR5 subfamily contains two types of TLR5, the membrane form of TLR5 (TLR5M) and the soluble form of TLR5 (TLR5S), whose detailed functions have not been completely elucidated. In the present study, we first identified two genes, TLR5M (ToTLR5M) and TLR5S (ToTLR5S), from golden pompano (Trachinotus ovatus). The full-length ToTLR5M and ToTLR5S cDNA are 3644 bp and 2329 bp, respectively, comprising an open reading frame (ORF) of 2673 bp, encoding 890 amino acids, and an ORF of 1935 bp, encoding 644 amino acids. Both the ToTLR5s possess representative TLR domains; however, only ToTLR5M has transmembrane and intracellular TIR domains. Moreover, the transcription of two ToTLR5s was significantly upregulated after stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and flagellin in both immune-related tissues (liver, intestine, blood, kidney, and skin) and nonimmune-related tissue (muscle). Furthermore, the results of bioinformatic and promoter analysis show that the transcription factors GATA-1 (GATA Binding Protein 1), C/EBPalpha (CCAAT Enhancer Binding Protein Alpha), and ICSBP (Interferon (IFN) consensus sequence binding protein) may play a positive role in moderating the expression of two ToTLR5s. Overexpression of ToTLR5M and ToTLR5S notably increases NF-κB (nuclear factor kappa-B) activity. Additionally, the binding assay revealed that two rToTLR5s can bind specifically to bacteria and pathogen-associated molecular patterns (PAMPs) containing Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Escherichia coli, Photobacterium damselae, Staphylococcus aureus, Aeromonas hydrophila, LPS, poly(I:C), flagellin, and peptidoglycan (PGN). In conclusion, the present study may help to elucidate the function of ToTLR5M/S and clarify their possible roles in the fish immune response to bacterial infection. Full article

(This article belongs to the Section Molecular Biology)

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19 pages, 3232 KiB

Open AccessArticle

Biochemical Characterization and Crystal Structure of a Novel NAD⁺-Dependent Isocitrate Dehydrogenase from Phaeodactylum tricornutum

by Shi-Ping Huang, Lu-Chun Zhou, Bin Wen, Peng Wang and Guo-Ping Zhu

Int. J. Mol. Sci. 2020, 21(16), 5915; https://doi.org/10.3390/ijms21165915 - 18 Aug 2020

Cited by 5 | Viewed by 3382

Abstract

The marine diatom Phaeodactylum tricornutum originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from P. tricornutum (PtIDH1) was expressed, purified, and identified in detail [...] Read more.

The marine diatom Phaeodactylum tricornutum originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from P. tricornutum (PtIDH1) was expressed, purified, and identified in detail through enzymatic characterization. Kinetic analysis showed that PtIDH1 is NAD⁺-dependent and has no detectable activity with NADP⁺. The catalytic efficiency of PtIDH1 for NAD⁺ is 0.16 μM⁻¹·s⁻¹ and 0.09 μM⁻¹·s⁻¹ in the presence of Mn²⁺ and Mg²⁺, respectively. Unlike other bacterial homodimeric NAD-IDHs, PtIDH1 activity was allosterically regulated by the isocitrate. Furthermore, the dimeric structure of PtIDH1 was determined at 2.8 Å resolution, and each subunit was resolved into four domains, similar to the eukaryotic homodimeric NADP-IDH in the type II subfamily. Interestingly, a unique and novel C-terminal EF-hand domain was first defined in PtIDH1. Deletion of this domain disrupted the intact dimeric structure and activity. Mutation of the four Ca²⁺-binding sites in the EF-hand significantly reduced the calcium tolerance of PtIDH1. Thus, we suggest that the EF-hand domain could be involved in the dimerization and Ca²⁺-coordination of PtIDH1. The current report, on the first structure of type II eukaryotic NAD-IDH, provides new information for further investigation of the evolution of the IDH family. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Phylogenetic analysis of IDHs from different species. The analysis involved 45 IDH sequences and a neighbor-joining tree with 1000 bootstraps and was created by MEGA 7.0. The UniProt entry numbers are noted in parentheses. PtIDH1 is marked by a purple star. Full article ">Figure 2
Structure-based amino acid sequence alignment of PtIDH1 with other IDHs. (A) Comparison of the N-terminal amino acid sequences of PtIDH1 with other IDHs. The putative mitochondrial targeting peptide sequences are underlined in black, and the possible cleavage sites are indicated by dots. (B) The substrate and metal ion binding conserved amino acid residues of the IDH family are indicated by triangles. (C) The putative conserved residues implicated in PtIDH1 coenzyme binding are compared with other homomeric NAD(P)-IDHs. The residues that directly or indirectly interact with the 2′-phosphate of NADP+ are indicated by stars. (D) Comparison of the C-terminal amino acid sequence of PtIDH1 with other IDHs. The putative EF-hand domain sequence is underlined in black. The Ca2+-binding sites are indicated by red clubs. The secondary structures of human cytosolic NADP-IDH (PDB entry: 1T0L) was placed above the alignment. Full article ">Figure 3
Overexpression, purification, and oligomeric state determination of PtIDH1. (A) Gel filtration chromatography elution profile of PtIDH1 from the Superdex 200 (10/300) column. The lower right insert panel shows the protein purity detection by 12% SDS-PAGE. (B) Elution profile of dimeric PtIDH1 (Peak 2 from A). All the flow rates of gel filtration chromatography were 0.5 mL·min−1. Full article ">Figure 4
Hill plots of PtIDH1 activity. The nH values of PtIDH1 for isocitrate were 1.43 ± 0.14 and 1.36 ± 0.03 in the presence of Mn2+ and Mg2+, respectively. The mean ± SD values were obtained from three independent replicates. Full article ">Figure 5
Effect of pH, temperature, and NADH on the NAD+-linked activity of PtIDH1. (A) Effect of pH on the activity of PtIDH1 in the presence of Mn2+ and Mg2+, respectively. (B) Effect of temperature on the activity of PtIDH1 in the presence of Mn2+ and Mg2+. (C) Heat-inactivation profiles of PtIDH1 in the presence of Mn2+ and Mg2+. (D) Effect of NADH on the activity of PtIDH1 with Mn2+. All values are displayed as the means of at least three independent measurements. Full article ">Figure 5 Cont.
Effect of pH, temperature, and NADH on the NAD+-linked activity of PtIDH1. (A) Effect of pH on the activity of PtIDH1 in the presence of Mn2+ and Mg2+, respectively. (B) Effect of temperature on the activity of PtIDH1 in the presence of Mn2+ and Mg2+. (C) Heat-inactivation profiles of PtIDH1 in the presence of Mn2+ and Mg2+. (D) Effect of NADH on the activity of PtIDH1 with Mn2+. All values are displayed as the means of at least three independent measurements. Full article ">Figure 6
The structural characterizations of PtIDH1. (A) Overall structure of PtIDH1-Apo. PtIDH1 shows a dimeric structure and contains two subunits, which are colored light orange (subunit A) or spring-green (subunit B). (B) View of the monomer of the PtIDH1. Each subunit contains four domains: a large domain, a small domain, a clasp domain, and an EF-hand domain, which are colored green, cyan, yellow, and orange, respectively. (C) Monomer comparison of PtIDH1 (green) and HcIDH (light blue) shows the difference in the C-terminus (in red). (D) Overlay of the monomer of PtIDH (green) and AtIDH (yellow). The arrows, pointing towards the red ribbon of PtIDH1, represent the structural differences between PtIDH1 and AtIDH. Full article ">Figure 7
Characterization of the mutants. (A) Overexpression and purification of PtIDH1-EF. Gel filtration chromatography elution profile of PtIDH1-EF from the Superdex 200 (10/300) column with a flow rate of 0.5 mL·min−1. The inset panel shows the protein purity detection by 12% SDS-PAGE. (B) Modeling studies of Ca2+ co-ordination by the EF-hand motif of PtIDH1. Ca2+ coordinating residues (X, Y, Z, -Y, -Z, and -X) are indicated as sticks and are labeled. The Ca2+ ion and the water molecule are indicated in yellow and red, respectively. Side chain, sc; backbone, bb; water molecule, w. (C) Circular dichroism (CD) spectra of the wild-type PtIDH1 and its mutant (M4A). (D) Effect of Ca2+ on the activity of wild-type PtIDH1 and its mutant (M4A). The assay mixtures were prepared as described for a standard reaction in the presence of 2 mM Mn2+, and the Ca2+ concentration was changed by extra addition. A reaction mixture without added Ca2+ was used as a control. *** p < 0.001 vs. control. Full article ">

23 pages, 4350 KiB

Open AccessArticle

Comparative Transcriptomic Analysis of the Development of Sepal Morphology in Tomato (Solanum Lycopersicum L.)

by Jingyi Liu, Meijing Shi, Jing Wang, Bo Zhang, Yushun Li, Jin Wang, Ahmed. H. El-Sappah and Yan Liang

Int. J. Mol. Sci. 2020, 21(16), 5914; https://doi.org/10.3390/ijms21165914 - 18 Aug 2020

Cited by 20 | Viewed by 4490

Abstract

Sepal is an important component of the tomato flower and fruit that typically protects the flower in bud and functions as a support for petals and fruits. Moreover, sepal appearance influences the commercial property of tomato nowadays. However, the phenotype information and development [...] Read more.

Sepal is an important component of the tomato flower and fruit that typically protects the flower in bud and functions as a support for petals and fruits. Moreover, sepal appearance influences the commercial property of tomato nowadays. However, the phenotype information and development mechanism of the natural variation of sepal morphology in the tomato is still largely unexplored. To study the developmental mechanism and to determine key genes related to downward sepal in the tomato, we compared the transcriptomes of sepals between downward sepal (dsp) mutation and the wild-type by RNA sequencing and found that the differentially expressed genes were dominantly related to cell expansion, auxin, gibberellins and cytokinin. dsp mutation affected cell size and auxin, and gibberellins and cytokinin contents in sepals. The results showed that cell enlargement or abnormal cell expansion in the adaxial part of sepals in dsp. As reported, auxin, gibberellins and cytokinin were important factors for cell expansion. Hence, dsp mutation regulated cell expansion to control sepal morphology, and auxin, gibberellins and cytokinin may mediate this process. One ARF gene and nine SAUR genes were dramatically upregulated in the sepal of the dsp mutant, whereas seven AUX/IAA genes were significantly downregulated in the sepal of dsp mutant. Further bioinformatic analyses implied that seven AUX/IAA genes might function as negative regulators, while one ARF gene and nine SAUR genes might serve as positive regulators of auxin signal transduction, thereby contributing to cell expansion in dsp sepal. Thus, our data suggest that 17 auxin-responsive genes are involved in downward sepal formation in the tomato. This study provides valuable information for dissecting the molecular mechanism of sepal morphology control in the tomato. Full article

(This article belongs to the Collection Genetics and Molecular Breeding in Plants)

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Int. J. Mol. Sci., Volume 21, Issue 16 (August-2 2020) – 353 articles

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