Structure-Based Discovery of the SARS-CoV-2 Main Protease Noncovalent Inhibitors from Traditional Chinese Medicine.

Traditional Chinese medicine (TCM) has been extensively employed for the treatment of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is demand for discovering more SARS-CoV-2 Mpro inhibitors with diverse scaffolds to optimize anti-SARS-CoV-2 lead compounds. In this study, comprehensive in silico and in vitro assays were utilized to determine the potential inhibitors from TCM compounds against SARS-CoV-2 Mpro, which is an important therapeutic target for SARS-CoV-2. The ensemble docking analysis of 18263 TCM compounds against 15 SARS-CoV-2 Mpro conformations identified 19 TCM compounds as promising candidates. Further in vitro testing validated three compounds as inhibitors of SARS-CoV-2 Mpro and showed IC50 values of 4.64 ± 0.11, 7.56 ± 0.78, and 11.16 ± 0.26 µM, with EC50 values of 12.25 ± 1.68, 15.58 ± 0.77, and 29.32 ± 1.25 µM, respectively. Molecular dynamics (MD) simulations indicated that the three complexes remained stable over the last 100 ns of production run. An analysis of the binding mode revealed that the active compounds occupy different subsites (S1, S2, S3, and S4) of the active site of SARS-CoV-2 Mpro via specific poses through noncovalent interactions with key amino acids (e.g., HIS 41, ASN 142, GLY 143, MET 165, GLU 166, or GLN 189). Overall, this study provides evidence indicating that the three natural products obtained from TCM could be further used for anti-COVID-19 research, justifying the investigation of Chinese herbal medicinal ingredients as bioactive constituents for therapeutic targets.

Liquid-Liquid phase separation in bacteria.

Cells are the essential building blocks of living organisms, responsible for carrying out various biochemical reactions and performing specific functions. In eukaryotic cells, numerous membrane organelles have evolved to facilitate these processes by providing specific spatial locations. In recent years, it has also been discovered that membraneless organelles play a crucial role in the subcellular organization of bacteria, which are single-celled prokaryotic microorganisms characterized by their simple structure and small size. These membraneless organelles in bacteria have been found to undergo Liquid-Liquid phase separation (LLPS), a molecular mechanism that allows for their assembly. Through extensive research, the occurrence of LLPS and its role in the spatial organization of bacteria have been better understood. Various biomacromolecules have been identified to exhibit LLPS properties in different bacterial species. LLPS which is introduced into synthetic biology applies to bacteria has important implications, and three recent research reports have shed light on its potential applications in this field. Overall, this review investigates the molecular mechanisms of LLPS occurrence and its significance in bacteria while also considering the future prospects of implementing LLPS in synthetic biology.

A multidimensional atlas of human glioblastoma-like organoids reveals highly coordinated molecular networks and effective drugs.

Recent advances in the genomics of glioblastoma (GBM) led to the introduction of molecular neuropathology but failed to translate into treatment improvement. This is largely attributed to the genetic and phenotypic heterogeneity of GBM, which are considered the major obstacle to GBM therapy. Here, we use advanced human GBM-like organoid (LEGO: Laboratory Engineered Glioblastoma-like Organoid) models and provide an unprecedented comprehensive characterization of LEGO models using single-cell transcriptome, DNA methylome, metabolome, lipidome, proteome, and phospho-proteome analysis. We discovered that genetic heterogeneity dictates functional heterogeneity across molecular layers and demonstrates that NF1 mutation drives mesenchymal signature. Most importantly, we found that glycerol lipid reprogramming is a hallmark of GBM, and several targets and drugs were discovered along this line. We also provide a genotype-based drug reference map using LEGO-based drug screen. This study provides new human GBM models and a research path toward effective GBM therapy.

The relationship between Listeria infections and host immune responses: Listeriolysin O as a potential target.

Listeria monocytogenes (Lm), a foodborne bacterium, can infect people and has a high fatality rate in immunocompromised individuals. Listeriolysin O (LLO), the primary virulence factor of Lm, is critical in regulating the pathogenicity of Lm. This review concludes that LLO may either directly or indirectly activate a number of host cell viral pathophysiology processes, such as apoptosis, pyroptosis, autophagy, necrosis and necroptosis. We describe the invasion of host cells by Lm and the subsequent removal of Lm by CD8 T cells and CD4 T cells upon receipt of the LLO epitopes from major histocompatibility complex class I (MHC-I) and major histocompatibility complex class II (MHC-II). The development of several LLO-based vaccines that make use of the pore-forming capabilities of LLO and the immune response of the host cells is then described. Finally, we conclude by outlining the several natural substances that have been shown to alter the three-dimensional conformation of LLO by binding to particular amino acid residues of LLO, which reduces LLO pathogenicity and may be a possible pharmacological treatment for Lm.

SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway.

Interferon-gamma (IFN-γ) signaling is necessary for the proinflammatory activation of macrophages but IFN-γ-independent pathways, for which the initiating stimuli and downstream mechanisms are lesser known, also contribute. Here we identify, by high-content screening, SEPTIN2 (SEPT2) as a negative regulation of IFN-γ-independent macrophage autoactivation. Mechanistically, endoplasmic reticulum (ER) stress induces the expression of SEPT2, which balances the competition between acetylation and ubiquitination of heat shock protein 5 at position Lysine 327, thereby alleviating ER stress and constraining M1-like polarization and proinflammatory cytokine release. Disruption of this negative feedback regulation leads to the accumulation of unfolded proteins, resulting in accelerated M1-like polarization, excessive inflammation and tissue damage. Our study thus uncovers an IFN-γ-independent macrophage proinflammatory autoactivation pathway and suggests that SEPT2 may play a role in the prevention or resolution of inflammation during infection.

Development of probiotic E. coli Nissle 1917 for ß-alanine production by using protein and metabolic engineering.

ß-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing ß-alanine from glucose. First, an aspartate-α-decarboxylase variant ADCK43Y with improved activity was identified and over-expressed in EcN, promoting the titer of ß-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of ß-alanine to 0.92 g/L. Third, the yield of ß-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L ß-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of ß-alanine via fermentation. KEY POINTS: • Escherichia coli Nissle 1917 (EcN) was engineered as a ß-alanine producing cell factory • Identification of a decarboxylase variant ADCK43Y with improved activity • Directing the metabolic flux to L-ASP and expressing ADCK43Y elevated the titer of ß-alanine to 11.9 g/L.

Bacteria and macrophages in the tumor microenvironment.

Cancer and microbial infections are significant worldwide health challenges. Numerous studies have demonstrated that bacteria may contribute to the emergence of cancer. In this review, we assemble bacterial species discovered in various cancers to describe their variety and specificity. The relationship between bacteria and macrophages in cancer is also highlighted, and we look for ample proof to establish a biological basis for bacterial-induced macrophage polarization. Finally, we quickly go over the potential roles of metabolites, cytokines, and microRNAs in the regulation of the tumor microenvironment by bacterially activated macrophages. The complexity of bacteria and macrophages in cancer will be revealed as we gain a better understanding of their pathogenic mechanisms, which will lead to new therapeutic approaches for both inflammatory illnesses and cancer.

Functional studies of HLA and its role in SARS-CoV-2: Stimulating T cell response and vaccine development.

In the biological immune process, the major histocompatibility complex (MHC) plays an indispensable role in the expression of HLA molecules in the human body when viral infection activates the T-cell response to remove the virus. Since the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2019, how to address and prevent SARS-CoV-2 has become a common problem facing all mankind. The T-cell immune response activated by MHC peptides is a way to construct a defense line and reduce the transmission and harm of the virus. Presentation of SARS-CoV-2 antigen is associated with different types of HLA phenotypes, and different HLA phenotypes induce different immune responses. The prediction of SARS-CoV-2 mutation information and the design of vaccines based on HLAs can effectively activate autoimmunity and cope with virus mutations, which can provide some references for the prevention and treatment of SARS-CoV-2.

Small molecule Z363 co-regulates TAF10 and MYC via the E3 ligase TRIP12 to suppress tumour growth.

BACKGROUND: The MYC oncoprotein, also known as the master regulator of genes, is a transcription factor that regulates numerous physiological processes, including cell cycle control, apoptosis, protein synthesis and cell adhesion, among others. MYC is overexpressed in approximately 70% of human cancers. Given its pervasive role in cancer biology, MYC down-regulation has become an attractive cancer treatment strategy. METHODS: The CRISPR/Cas9 method was used to produce KO cell models. Western blot was used to analyzed the expressions of MYC and TATA-binding proteinassociated factors 10 (TAF10) in cancer cells (MCF7, A549, HepG2 cells) Cell culture studies were performed to determine the mechanisms by which small molecules (Z363119456, Z363) affects MYC and TAF10 expressions and functions. Mouse studies were carried out to investigate the impact of Z363 regulation on tumor growth. RESULTS: Z363 activate Thyroid hormone Receptor-interacting Protein 12 (TRIP12), which phosphorylates MYC at Thr58, resulting in MYC ubiquitination and degradation and thereby regulating MYC target genes. Importantly, TRIP12 also induces TAF10 degradation, which reduces MYC protein levels. TRIP12, an E3 ligase, controls MYC levels both directly and indirectly by inhibiting MYC or TAF10 activity. CONCLUSIONS: In summary,these results demonstrate the anti-cancer properties of Z363, a small molecule that is co-regulated by TAF10 and MYC.

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection.

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.

MicroRNAs as immune regulators and biomarkers in tuberculosis.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is one of the most lethal infectious disease worldwide, and it greatly affects human health. Some diagnostic and therapeutic methods are available to effectively prevent and treat TB; however, only a few systematic studies have described the roles of microRNAs (miRNAs) in TB. Combining multiple clinical datasets and previous studies on Mtb and miRNAs, we state that pathogens can exploit interactions between miRNAs and other biomolecules to avoid host mechanisms of immune-mediated clearance and survive in host cells for a long time. During the interaction between Mtb and host cells, miRNA expression levels are altered, resulting in the changes in the miRNA-mediated regulation of host cell metabolism, inflammatory responses, apoptosis, and autophagy. In addition, differential miRNA expression can be used to distinguish healthy individuals, patients with TB, and patients with latent TB. This review summarizes the roles of miRNAs in immune regulation and their application as biomarkers in TB. These findings could provide new opportunities for the diagnosis and treatment of TB.

Targeted pharmacokinetics and bioinformatics screening strategy reveals JAK2 as the main target for Xin-Ji-Er-Kang in treatment of MIR injury.

BACKGROUND AND PURPOSE: Xin-Ji-Er-Kang (XJEK) is traditional Chinese formula presented excellent protective effects on several heart diseases, but the potential components and targets are still unclear. The aim of this study is to elucidate the effective components of XJEK and reveal its potential mechanism of cardioprotective effect in myocardial ischemia-reperfusion (MIR) injury. EXPERIMENTAL APPROACH: Firstly, the key compounds in XJEK, plasma and heart tissue were analyzed by high resolution mass spectrometry. Bioinformatics studies were also involved to disclose the potential targets and the binding sites for the key compounds. Secondly, to study the protective effect of XJEK on MIR injury and related mechanism, mice subjected to MIR surgery and gavage administered with XJEK for 6 weeks. Cardiac function parameters and apoptosis level of cardiac tissue were assessed. The potential mechanism was further verified by knock down of target protein in vitro. RESULTS: Pharmacokinetics studies showed that Sophora flavescens alkaloids, primarily composed with matrine, are the key component of XJEK. And, through bioinformatic analysis, we speculated JAK2 could be the potential target for XJEK, and could form stable hydrogen bonds with matrine. Administration of XJEK and matrine significantly improved heart function and reduced apoptosis of cardiomyocytes by increasing the phosphorylation of JAK2 and STAT3. The anti-apoptosis effect of XJEK and matrine was also observed on AC16 cells, and could be reversed by co-treatment with JAK2 inhibitor AG490 or knock-down of JAK2. CONCLUSION: XJEK exerts cardioprotective effect on MIR injury, which may be associated with the activation of JAK2/STAT3 signaling pathway.

Folic acid Ameliorates the Declining Quality of Sodium Fluoride-Exposed Mouse Oocytes through the Sirt1/Sod2 Pathway.

Excessive sodium fluoride (NaF) intake interferes with reproductive function in humans and animals; however, strategies to prevent these effects are still underexplored. Here, we showed that in vivo and in vitro supplementation of folic acid (FA) efficaciously improved the quality of NaF-exposed oocytes. FA supplementation not only increased ovulation of oocytes from NaF-treated mice but also enhanced oocyte meiotic competency and fertilization ability by restoring the spindle/chromosome structure. Moreover, FA supplementation could exert a beneficial effect on NaF- exposed oocytes by restoring mitochondrial function, eliminating reactive oxygen species accumulation to suppress apoptosis. We also found that FA supplementation restored the defective phenotypes in oocytes through a Sirt1/Sod2-dependent mechanism. Inhibition of Sirt1 with EX527 abolished the FA-mediated improvement in NaF-exposed oocyte quality. Collectively, our data indicated that FA supplementation is a feasible approach to protect oocytes from NaF-related deterioration.

ISOC1 Modulates Inflammatory Responses in Macrophages through the AKT1/PEX11B/Peroxisome Pathway.

Inflammation underlies a variety of physiological and pathological processes and plays an essential role in shaping the ensuing adaptive immune responses and in the control of pathogens. However, its physiological functions are not completely clear. Using a LPS-treated RAW264.7 macrophage inflammation model, we found that the production of inflammatory cytokines in ISOC1-deficient cells was significantly higher than that in the control group. It was further proved that ISOC1 deficiency could activate AKT1, and the overactivation of AKT1 could reduce the stability of PEX11B through protein modification, thereby reducing the peroxisome biogenesis and thus affecting inflammation. In this study, we reported for the first time the role of ISOC1 in innate immunity and elucidated the mechanism by which ISOC1 regulates inflammation through AKT1/PEX11B/peroxisome. Our results defined a new role of ISOC1 in the regulatory mechanism underlying the LPS-induced inflammatory response.

Challenging Bendable Organic Single Crystal and Transistor Arrays with High Mobility and Durability toward Flexible Electronics.

Bendable organic single crystals are promising candidates for flexible electronics owing to their superior charge-transport properties. However, large-area high-quality organic single crystals are rarely available on the polymer substrates generally used in flexible electronics. Here, a surface-assisted assembly strategy based on a polymer modification, poly(amic acid) (PAA), is developed to grow large-area organic singe crystals on polymer substrates using a simple drop-casting method. The unique surface properties of PAA that enable molecular solution superwetting and promote molecular ordered assembly produce an extraordinary self-driven "meniscus-guided coating" behavior, enabling the fabrication of millimeter-sized, highly aligned organic single crystals for a variety of organic semiconductors. Organic field-effect transistors based on a mode molecule of 2,7-dioctyl[1]benzothieno[3,2-b][1]benzothiophene demonstrate the highest (average) mobility of 18.6 cm2 V-1 s-1 (15.9 cm2 V-1 s-1 ), attractively low operating voltage of -3 V, and high flexible durability. The results shed light on the large-area fabrication of organic single crystals on polymer dielectrics toward high-performance and integrated plastic electronics.

Vitamin C-induced competitive binding of HIF-1α and p53 to ubiquitin E3 ligase CBL contributes to anti-breast cancer progression through p53 deacetylation.

Vitamin C (VC), in regard to its effectiveness against tumors, has had a controversial history in cancer treatment. However, the anticancer mechanisms of VC are not fully understood. Here, we reported that VC exerted an anticancer effect on cancer cell and xenograft models via inhibiting HIF-1α-dependent cell proliferation and promoting p53-dependent cell apoptosis. To be specific, VC modulated the competitive binding of HIF-1α and p53 to their common E3 ubiquitin ligase CBL, thereby inhibiting tumorigenesis. Moreover, VC treatment activated SIRT1, resulting in p53 deacetylation and CBL-p53 complex dissociation, which in turn facilitated CBL recruitment of HIF-1α for ubiquitination in a proteasome-dependent manner. Altogether, our results provided a mechanistic rationale for exploring the therapeutic use of VC in cancer therapy.

A Novel ERK2 Degrader Z734 Induces Apoptosis of MCF-7 Cells via the HERC3/p53 Signaling Pathway.

Breast cancer is one of the leading causes of death worldwide, and synthetic chemicals targeting specific proteins or various molecular pathways for tumor suppression, such as ERK inhibitors and degraders, have been intensively investigated. The targets of ERK participate in the regulation of critical cellular mechanisms and underpin the progression of anticancer therapy. In this study, we identified a novel small molecule, which we named Z734, as a new mitogen-activated protein kinase 1 (ERK2) degrader and demonstrated that Z734 inhibits cell growth by inducing p53-mediated apoptotic pathways in human breast cancer cells. Treatment with Z734 resulted in the inhibition of cancer cell proliferation, colony formation and migration invasion, as well as cancer cell death via apoptosis. In addition, the Co-IP and GST pulldown assays indicated that the HECT and RLD domains containing E3 ubiquitin protein ligase 3 (HERC3) could directly interact with ERK2 through the HECT domain, promoting ERK2 ubiquitination. We also observed a strong link between HERC3 and p53 for the modulation of apoptosis. HERC3 can increase the protein and phosphorylation levels of p53, which further promotes apoptotic activity. In a xenograft mouse model, the effect was obtained in a treatment group that combined Z734 with lapatinib compared with that of the single-treatment groups. In summary, our results indicated that Z734 actively controls the development of breast cancer through apoptosis, and HERC3 may mediate ERK2 and p53 signaling, which offers new potential targets for clinical therapy.

A combination of tetramethylpyrazine hydrochloride and butylphthalide on serum S100B, CRP, Hcy levels and NIHSS score in patients with acute cerebral infarction: A retrospective study.

To investigate the effect of tetramethylpyrazine hydrochloride combined with butylphthalide on serum S100B, CRP, Hcy and NIHSS score in patients with acute cerebral infarction. 80 patients with acute cerebral infarction treated in our hospital from February 2019 to February 2021 were selected for retrospective analysis, and according to different treatment methods, the patients were equally divided into control group (conventional treatment) and experimental group (tetramethylpyrazine hydrochloride and butylphthalide). After treatment, the total effective rate of patients in the experimental group was significantly higher than that in the control group (P<0.05); the levels of serum S100B, CRP and Hcy, and NHISS scores in the two groups decreased, and the experimental group was significantly lower than the control group (P<0.05); the ADL scores of the two groups increased and the experimental group witnessed higher score (P<0.05); the number of patients in the experimental group with scores of 0-2 and 5 were significantly larger than that in the control group (P<0.05). The combination of tetramethylpyrazine hydrochloride and butylphthalide emanates a promising result in the treatment of patients with ACI. It reduces serum S100B, CRP and Hcy levels, protects nerve tissue, and improves nerve function, and thus merits clinical application.

Self-Calibration 3D Hybrid SERS Substrate and Its Application in Quantitative Analysis.

Surface-enhanced Raman spectroscopy (SERS) has been widely applied in many fields as a sensitive vibrational fingerprint technique. However, SERS faces challenges in quantitative analysis due to the heterogeneity of hot spots. An internal standard (IS) strategy has been employed for correcting the variation of hot spots. However, the method suffers from limitations due to the competitive adsorption between the IS and the target analyte. In this work, we combined the IS strategy with the 3D hybrid nanostructures to develop a bifunctional SERS substrate. The substrate had two functional units. The bottom self-assembly layer consisted of Au@IS@SiO2 nanoparticles, which provided a stable reference signal and functioned as the calibration unit. The top one consisted of appropriate-sized Au octahedrons for the detection of target analytes, which was the detection unit. Within the 3D hybrid nanostructure, the calibration unit improved the SERS performance of the detection unit, which was demonstrated by the 6-fold increase of SERS intensity when compared with the 2D substrate. Meanwhile, the reproducibility of the detection was greatly improved by correcting the hot spot changes through the calibration unit. Two biomedical molecules of cotinine and creatinine in ultrapure water and artificial urine, respectively, were sensitively determined by the 3D hybrid substrate. We expect that the developed bifunctional 3D substrate will open up new ways to advance the applications of SERS.

Long-term exposure to copper induces mitochondria-mediated apoptosis in mouse hearts.

Copper is a trace element necessary for the normal functioning of organisms, but excessive copper contents may be toxic to the heart. The goal of this study was to investigate the role of excessive copper accumulation in mitochondrial damage and cell apoptosis inhibition. In vivo, the heart copper concentration and cardiac troponin I (c-TnI) and N-terminal forebrain natriuretic peptide (NT-pro-BNP) levels increased in the copper-laden model group compared to those of the control group. Histopathological and ultrastructural observations revealed that the myocardial collagen volume fraction (CVF), perivascular collagen area (PVCA) and cardiomyocyte cross-sectional area (CSA) were markedly elevated in the copper-laden model group compared with the control group. Furthermore, transmission electron microscopy (TEM) showed that the mitochondrial double-layer membrane was incomplete in the copper-laden model groups. Furthermore, cytochrome C (Cyt-C) expression was downregulated in mitochondria but upregulated in the cytoplasm in response to copper accumulation. In addition, Bcl-2 expression decreased, while Bax and cleaved caspase-3 levels increased. These results indicate that copper accumulation in cardiomyocyte mitochondria induces mitochondrial injury, and Cyt-C exposure and induces apoptosis, further resulting in heart damage.