Environmental magnesium ion affects global gene expression, motility, biofilm formation and virulence of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is widely distributed in marine ecosystems. Magnesium ion (Mg2+) is the second most abundant metal cation in seawater, and plays important roles in the growth and gene expression of V. parahaemolyticus, but lacks the detailed mechanisms. In this study, the RNA sequencing data demonstrated that a total of 1494 genes was significantly regulated by Mg2+. The majority of the genes associated with lateral flagella, exopolysaccharide, type III secretion system 2, type VI secretion system (T6SS) 1, T6SS2, and thermostable direct hemolysin were downregulated. A total of 18 genes that may be involved in c-di-GMP metabolism and more than 80 genes encoding putative regulators were also significantly and differentially expressed in response to Mg2+, indicating that the adaptation process to Mg2+ stress may be strictly regulated by complex regulatory networks. In addition, Mg2+ promoted the proliferative speed, swimming motility and cell adhesion of V. parahaemolyticus, but inhibited the swarming motility, biofilm formation, and c-di-GMP production. However, Mg2+ had no effect on the production of capsular polysaccharide and cytoxicity against HeLa cells. Therefore, Mg2+ had a comprehensive impact on the physiology and gene expression of V. parahaemolyticus.

The mechanisms of exosomes in diabetic foot ulcers healing: a detailed review.

As time goes by, the morbidity of diabetes mellitus continues to rise, and the economic burden of diabetic foot ulcers as a common and serious complication of diabetes is increasing. However, currently there is no unified clinical treatment strategy for this complication, and the therapeutic efficacy is unsatisfactory. Recent studies have revealed that biological effects of exosomes involved in multiple stages of the process of wound closure are similar to source cells. Compared with source cells, exosomes possess lowly immunogenicity, highly stability and easily stored, etc. Accumulating evidence confirmed that exosomes promote diabetic wound healing through various pathways such as promoting angiogenesis, collagen fiber deposition, and inhibiting inflammation. The superior therapeutic efficacy of exosomes in accelerating diabetic cutaneous wound healing has attracted an increasing attention. Notably, the molecular mechanisms of exosomes vary among different sources in the chronic wound closure of diabetes. This review focuses on the specific roles and mechanisms of different cell- or tissue-derived exosomes relevant to wound healing. Additionally, the paper provides an overview of the current pre-clinical and clinical applications of exosomes, illustrates their special advantages in wound repair. Furthermore, we discuss the potential obstacles and various solutions for future research on exosomes in the management of diabetic foot ulcer. The aim is to offer novel insights and approaches for the treatment of diabetic foot ulcer.

Bioorthogonal Peptide Macrocyclization Using Oxime Ligation.

The biocompatible synthesis of constrained peptides is challenging. Oxime ligation is a bioorthogonal technique frequently used for protein bioconjugation. We report a straightforward method to install N-terminal ketones and aminooxy side chains during standard solid-phase peptide synthesis. Cyclization occurs spontaneously after acidic cleavage or in aqueous buffer. We demonstrate the facile synthesis of protease inhibitors with varying conformational constraint. The most constrained peptide displayed an activity 2 orders of magnitude higher than its linear analog.

Pyroptosis inhibition improves the symptom of acute myocardial infarction.

Acute myocardial infarction (AMI), the leading cause of mortality worldwide, is a rapidly developing and irreversible disease. Therefore, proper prompt intervention at the early stage of AMI is crucial for its treatment. However, the molecular features in the early stage have not been clarified. Here, we constructed mouse AMI model and profiled transcriptomes and proteomes at the early stages of AMI progress. Immune system was extensively activated at 6-h AMI. Then, pyroptosis was activated at 24-h AMI. VX-765 treatment, a pyroptosis inhibitor, significantly reduced the infarct size and improved the function of cardiomyocytes. Besides, we identified that WIPI1, specifically expressed in heart, was significantly upregulated at 1 h after AMI. Moreover, WIPI1 expression is significantly higher in the peripheral blood of patients with AMI than healthy control. WIPI1 can serve as a potential early diagnostic biomarker for AMI. It likely decelerates AMI progress by activating autophagy pathways. These findings shed new light on gene expression dynamics in AMI progress, and present a potential early diagnostic marker and a candidate drug for clinical pre-treatment to prolong the optimal cure time.

Challenges of short substrate analogues as SARS-CoV-2 main protease inhibitors.

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.

Epidemiological and clinical characteristics of 671 COVID-19 patients in Henan Province, China.

BACKGROUND: Despite many reports on the characteristics of coronavirus disease 2019 (COVID-19) in Wuhan, China, relatively little is known about the transmission features of COVID-19 outside Wuhan, especially at the provincial level. METHODS: We collected epidemiological, demographic, clinical, laboratory, radiological and occupation information, along with contact history, of 671 patients with laboratory-confirmed COVID-19 reported from January 23 to February 5, 2020, in Henan province, China. We described characteristics of these cases, compared the diagnostic accuracy and features of blood testing, computed tomography (CT) scans and X-rays, and analysed SARS-CoV-2 transmission sources and patients' occupations in Henan province. RESULTS: The mean age of patients in this case series was 43 years, 56.2% were male and 22.4% had coexisting medical disorders. The death rate was 0.3%. Fourteen patients did not show any symptoms. Lymphocyte percentage was associated with disease severity (χ2 = 6.71, P = 0.035) but had a large variation in each sample group. The mean time from illness onset to diagnosis was 5.6 days. A total of 330 patients had ever lived in or visited Wuhan, 150 had contact with confirmed cases, 323 had been to a hospital and 119 had been to a wet market. There were 33 patients who did not have a traceable transmission source, with 21.2% of these being farmers and 15.2% being workmen. CONCLUSIONS: Lymphocyte percentage was a sign of severe COVID-19 in general but was not a good diagnostic index. Longer time from illness onset to diagnosis was associated with higher COVID-19 severity, older age, higher likelihood of having coexisting cardiovascular diseases including hypertension, and being male. Farming was found to be a high-risk occupation in Henan province, China.

Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum.

Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.

Increase in DNA Damage by MYCN Knockdown Through Regulating Nucleosome Organization and Chromatin State in Neuroblastoma.

As a transcription factor, MYCN regulates myriad target genes including the histone chaperone FACT. Moreover, FACT and MYCN expression form a forward feedback loop in neuroblastoma. It is unclear whether MYCN is involved in chromatin remodeling in neuroblastoma through regulation of its target genes. We showed here that MYCN knockdown resulted in loss of the nucleosome-free regions through nucleosome assembly in the promoters of genes functionally enriched for DNA repair. The active mark H3K9ac was removed or replaced by the repressive mark H3K27me3 in the promoters of double-strand break repair-related genes upon MYCN knockdown. Such chromatin state alterations occurred only in MYCN-bound promoters. Consistently, MYCN knockdown resulted in a marked increase in DNA damage in the treatment with hydroxyurea. In contrast, nucleosome reorganization and histone modification changes in the enhancers largely included target genes with tumorigenesis-related functions such as cell proliferation, cell migration, and cell-cell adhesion. The chromatin state significantly changed in both MYCN-bound and MYCN-unbound enhancers upon MYCN knockdown. Furthermore, MYCN knockdown independently regulated chromatin remodeling in the promoters and the enhancers. These findings reveal the novel epigenetic regulatory role of MYCN in chromatin remodeling and provide an alternative potential epigenetic strategy for MYCN-driven neuroblastoma treatment.

H3K27me3 Signal in the Cis Regulatory Elements Reveals the Differentiation Potential of Progenitors During Drosophila Neuroglial Development.

Drosophila neural development undergoes extensive chromatin remodeling and precise epigenetic regulation. However, the roles of chromatin remodeling in establishment and maintenance of cell identity during cell fate transition remain enigmatic. Here, we compared the changes in gene expression, as well as the dynamics of nucleosome positioning and key histone modifications between the four major neural cell types during Drosophila neural development. We find that the neural progenitors can be separated from the terminally differentiated cells based on their gene expression profiles, whereas nucleosome distribution in the flanking regions of transcription start sites fails to identify the relationships between the progenitors and the differentiated cells. H3K27me3 signal in promoters and enhancers can not only distinguish the progenitors from the differentiated cells but also identify the differentiation path of the neural stem cells (NSCs) to the intermediate progenitor cells to the glial cells. In contrast, H3K9ac signal fails to identify the differentiation path, although it activates distinct sets of genes with neuron-specific and glia-related functions during the differentiation of the NSCs into neurons and glia, respectively. Together, our study provides novel insights into the crucial roles of chromatin remodeling in determining cell type during Drosophila neural development.

Ferroptosis is governed by differential regulation of transcription in liver cancer.

Ferroptosis is an outcome of metabolic disorders and closely linked to liver cancer. However, the mechanism underlying the fine regulation of ferroptosis in liver cancer remains unclear. Here, we have identified two categories of genes: ferroptosis up-regulated factors (FUF) and ferroptosis down-regulated factors (FDF), which stimulate and suppress ferroptosis by affecting the synthesis of GSH. Furthermore, FUF are controlled by one transcription factor HIC1, while FDF controlled by another transcription factor HNF4A. Occurrence of ferroptosis might depend on the histone acetyltransferase KAT2B. Upon stimulation of ferroptosis, dissociation of KAT2B prevents HNF4A from binding to the FDF promoter. This effect happens prior to the recruitment of KAT2B to the FUF promoter, which facilitates HIC1 binding to transcribe FUF. Clinically, HIC1 and HNF4A conversely correlate with tumor stage in liver cancer. Patients with lower HIC1 and higher HNF4A exhibit poorer prognostic outcomes. Disrupting the balance between HIC1 and HNF4A might be helpful in treating liver cancer.

Dynamic placement of the linker histone H1 associated with nucleosome arrangement and gene transcription in early Drosophila embryonic development.

The linker histone H1 is critical to maintenance of higher-order chromatin structures and to gene expression regulation. However, H1 dynamics and its functions in embryonic development remain unresolved. Here, we profiled gene expression, nucleosome positions, and H1 locations in early Drosophila embryos. The results show that H1 binding is positively correlated with the stability of beads-on-a-string nucleosome organization likely through stabilizing nucleosome positioning and maintaining nucleosome spacing. Strikingly, nucleosomes with H1 placement deviating to the left or the right relative to the dyad shift to the left or the right, respectively, during early Drosophila embryonic development. H1 occupancy on genic nucleosomes is inversely correlated with nucleosome distance to the transcription start sites. This inverse correlation reduces as gene transcription levels decrease. Additionally, H1 occupancy is lower at the 5' border of genic nucleosomes than that at the 3' border. This asymmetrical pattern of H1 occupancy on genic nucleosomes diminishes as gene transcription levels decrease. These findings shed new lights into how H1 placement dynamics correlates with nucleosome positioning and gene transcription during early Drosophila embryonic development.

Periplaneta Americana Extract May Attenuate Renal Fibrosis through Inhibiting Janus Tyrosine Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway.

BACKGROUND: Periplaneta americana is one of the ancient insect groups with the strongest vitality. Periplaneta americana extract (PAE) has been explored as an alternative remedy for many diseases. Although much progress has been made in the study about PAE, the role of the drug in renal disease is rarely reported, especially in renal fibrosis. This study was designed to evaluate the renoprotective effect of PAE treatment to renal fibrosis. METHOD: An in vivo, unilateral ureteral obstruction (UUO) mouse model was built. Then the mice were treated with PAE (100 mg/kg body weight) once daily by oral gavage, again starting on the day of UUO and continued for 1 week. At the end of 1 week, the mice were sacrificed; kidney samples were collected for further analysis. In vitro, Boston University mouse proximal tubular cells were plated in 35-mm dishes at a density of 0.3 * 106 cells/dish. Then the cells were treated with 5-ng/mL TGF-ß1 in serum-free DMEM medium for an indicated length of time. The experimental groups were pretreated with the indicated concentrations of PAE (0.3125 mg/mL). The cells were further cultured for 24 h, and then cells were monitored morphologically or collected for biochemical analyses. RESULTS: Both in vivo and vitro PAE inhibits the expression of FN and alpha-smooth muscle actin and suppresses renal fibrosis. Importantly, PAE protects against renal fibrosis by inhibiting Janus tyrosine kinase 2 (JAK)/signal transducer and activator of transcription 3 (STAT) tyrosine phosphorylation. CONCLUSION: PAE attenuates renal fibrosis through the suppression of the JAK2/STAT3 pathway.

Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b.

Androgenetic haploid embryonic stem cells (AG-haESCs) hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC) mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood.

Role of Stat3 Signaling in Control of EMT of Tubular Epithelial Cells During Renal Fibrosis.

BACKGROUND/AIMS: Transforming growth factor ß 1 (TGFß1) plays a critical role in the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs) during renal injury, a major cause of acute renal failure, renal fibrosis and obstructive nephropathy. However, the underlying molecular mechanisms remain ill-defined. Here, we addressed this question. METHODS: Expression of TGFß1, Snail, and phosphorylated Stat3 was examined by immunohistochemistry in the kidney after induction of unilateral ureteral obstruction (UUO) in mice. In vitro, primary TECs were purified by flow cytometry, and then challenged with TGFß1 with/without presence of specific inhibitors for phosphorylation of SMAD3 or Stat3. Protein levels were determined by Western blotting. RESULTS: We detected significant increases in Snail and phosphorylated Stat3, an activated form for Stat3, in the kidney after induction of UUO in mice. In vitro, TGFß1-challenged primary TECs upregulated Snail, in a SMAD3/Stat3 dependent manner. CONCLUSION: Our study sheds light on the mechanism underlying the EMT of TECs after renal injury, and suggests Stat3 signaling as a promising innovative therapeutic target for prevention of renal fibrosis.

Smad5 acts as an intracellular pH messenger and maintains bioenergetic homeostasis.

Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.

Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment.

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling.

Cytotoxic T-lymphocyte-associated protein 4 +49A/G polymorphisms contribute to the risk of type 1 diabetes in children: An updated systematic review and meta-analysis with trial sequential analysis.

Type 1 diabetes (T1D) is a heritable disease associated with multiple genetic variants. This systematic review and meta-analysis assessed the correlation between cytotoxic T-lymphocyte-associated protein 4(CTLA-4) +49A/G polymorphisms and the risk of T1D in children. The random effects model was used to estimate the related odds ratios (ORs) and 95% confidence intervals (CIs). Trial sequential analysis (TSA) was used to determine whether the currently available evidence was sufficient and conclusive. Our results indicated that CTLA-4 gene polymorphisms significantly increased the risk of childhood T1D in an allelic model (G vs. A: OR=1.33, 95%CI=1.19-1.48; I2=44.0% and P=0.001for heterogeneity) and a codominant model (GG vs. AA: OR=1.75, 95%CI=1.37-2.24; I2=57.5% and P=0.001for heterogeneity; GA vs. AA: OR=1.26, 95%CI=1.09-1.46; I2=40.4% and P=0.036for heterogeneity). Subgroup analysis results indicated that the ORs were higher in the Asian population (ORallelic model=1.60, ORGG vs. AA=2.46 and ORGA vs. AA=1.58) than the Caucasian population (ORallelic model==1.24, ORGG vs. AA=1.55 and ORGA vs. AA=1.19). The TSA results indicated that the evidence of the effect was sufficient. In conclusion, CTLA4 +49A/G polymorphisms increased the risk of T1D in children, and CTLA4 +49A/G can be considered to be a genetic marker for T1D in children.

Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos.

Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. We show here that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. Our results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification.

Comparison of HLA-A, -B and -DRB1 Loci Polymorphism between Kidney Transplants of Uremia Patients and Healthy Individuals in Central China.

Chronic kidney disease is becoming a global public health problem, which will usually cause uremia at the end stage of chronic kidney failure. So far, kidney transplant is the most effective and proper therapy for uremia, however, the short supply of matched donor kidney has been a persistent bottleneck for transplantation. HLA matching of HLA-A, -B and -DRB1 loci is very important for the allocation of kidney transplants. In this study, we investigated genotypes of HLA-A, -B and -DRB1 loci based on 1,464 uremia patients and 10,000 unrelated healthy individuals in Henan province of China, and compared the frequency distribution of these HLA alleles and corresponding haplotypes between patient and healthy groups. We detected 23 HLA-A, 49 HLA-B and 17 HLA-DRB1 alleles in total. The predominant alleles of HLA-A, -B and -DRB1 loci in patients are the same as those in healthy group. The seven most frequent alleles account for about 87%, 50%, and 77% at HLA-A, -B and -DRB1 loci, respectively. The haplotypes (combinations of HLA-A, -B, and -DRB1) with significantly different frequency between patients and controls mostly account for less than 1%. Overall, this suggests that HLA matching is not a potential difficulty for kidney transplant of uremia patients. However, three of the top seven frequent HLA-DRB1 alleles have a significantly different distribution in patients and controls, while only one alleles for HLA-B and zero for HLA-A loci. These HLA-DRB1 alleles may be closely associated with uremia. This study sheds new lights on the composition and difference of HLA genotypes in uremia patients and healthy populations in Central China that can serve as a guide to HLA matching for kidney transplants and a resource for HLA typing-related studies.

Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos.

Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. Here, we reveal the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells.