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.

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.

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.

Pwp1 is required for the differentiation potential of mouse embryonic stem cells through regulating Stat3 signaling.

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self-renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-40 repeat-containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self-renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation-seq results revealed that the PWP1-occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs.

Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

BACKGROUND: Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. RESULTS: We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. CONCLUSIONS: The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

Phylogenetic affinity of tree shrews to Glires is attributed to fast evolution rate.

Previous phylogenetic analyses have led to incongruent evolutionary relationships between tree shrews and other suborders of Euarchontoglires. What caused the incongruence remains elusive. In this study, we identified 6845 orthologous genes between seventeen placental mammals. Tree shrews and Primates were monophyletic in the phylogenetic trees derived from the first or/and second codon positions whereas tree shrews and Glires formed a monophyly in the trees derived from the third or all codon positions. The same topology was obtained in the phylogeny inference using the slowly and fast evolving genes, respectively. This incongruence was likely attributed to the fast substitution rate in tree shrews and Glires. Notably, sequence GC content only was not informative to resolve the controversial phylogenetic relationships between tree shrews, Glires, and Primates. Finally, estimation in the confidence of the tree selection strongly supported the phylogenetic affiliation of tree shrews to Primates as a monophyly.

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.

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.

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.

The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development.

It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with ∼90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.

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.

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.

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.

MIClique: An algorithm to identify differentially coexpressed disease gene subset from microarray data.

Computational analysis of microarray data has provided an effective way to identify disease-related genes. Traditional disease gene selection methods from microarray data such as statistical test always focus on differentially expressed genes in different samples by individual gene prioritization. These traditional methods might miss differentially coexpressed (DCE) gene subsets because they ignore the interaction between genes. In this paper, MIClique algorithm is proposed to identify DEC gene subsets based on mutual information and clique analysis. Mutual information is used to measure the coexpression relationship between each pair of genes in two different kinds of samples. Clique analysis is a commonly used method in biological network, which generally represents biological module of similar function. By applying the MIClique algorithm to real gene expression data, some DEC gene subsets which correlated under one experimental condition but uncorrelated under another condition are detected from the graph of colon dataset and leukemia dataset.

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.

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.

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.

Sequential local least squares imputation estimating missing value of microarray data.

Missing values in microarray data can significantly affect subsequent analysis, thus it is important to estimate these missing values accurately. In this paper, a sequential local least squares imputation (SLLSimpute) method is proposed to solve this problem. It estimates missing values sequentially from the gene containing the fewest missing values and partially utilizes these estimated values. In addition, an automatic parameter selection algorithm, which can generate an appropriate number of neighboring genes for each target gene, is presented for parameter estimation. Experimental results confirmed that SLLSimpute method exhibited better estimation ability compared with other currently used imputation methods.

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.