Host serine protease ACOT2 assists DENV proliferation by hydrolyzing viral polyproteins.

Dengue fever is a mosquito-borne tropical disease caused by the dengue virus (DENV). The replication of DENV relies on the processing of its genome-encoded polyprotein by both viral protease NS3 (NS3pro) and host proteases. However, the impact of host proteases on DENV proliferation is not well understood. In this study, we utilized fluorophosphonate-based probes (FPs) to investigate the up-regulation of host serine proteases during DENV infection in detail. Among the identified proteases, acyl-CoA thioesterase 2 (ACOT2), an enzyme that hydrolyzes acyl-CoA molecules to generate fatty acids and free CoA, exhibited cleavage activity against DENV polypeptide substrates. Enzymatic assays and virological experiments confirmed that ACOT2 contributes to DENV propagation during the replication stage by cleaving the viral polyprotein. Docking models provided insights into the binding pocket of viral polypeptides and the catalytic mechanism of ACOT2. Notably, this study is the first to demonstrate that ACOT2 functions as a serine protease to hydrolyze protein substrates. These findings offer novel insights into DENV infection, host response, as well as the potential development of innovative antiviral strategies.IMPORTANCEDENV, one of the major pathogens of Dengue fever, remains a significant public health concern in tropical and subtropical regions worldwide. How DENV efficiently hijacks the host and accesses its life cycle with delicate interaction remains to be elucidated. Here, we deconvoluted that the host protease ACOT2 assists the DENV replication and characterized the ACOT2 as a serine protease involved in the hydrolysis of the DENV polypeptide substrate. Our results not only further the understanding of the DENV life cycle but also provide a possibility for the usage of activity-based proteomics to reveal host-virus interactions.

Dauricine interferes with SARS-CoV-2 variants infection by blocking the interface between RBD and ACE2.

The continued viral evolution results in the emergence of various SARS-CoV-2 variants, such as delta or omicron, that are partially resistant to current vaccines and antiviral medicines, posing an increased risk to global public health and raising the importance of continuous development of antiviral medicines. Inhibitor screening targeting the interactions between the viral spike proteins and their human receptor ACE2 represents a promising approach for drug discovery. Here, we demonstrate that the evolutionary trend of the SARS-CoV-2 variants is associated with increased electrostatic interactions between S proteins and ACE2. Virtual screening based on the ACE2-RBD binding interface identified nine monomers of Traditional Chinese medicine (TCM). Furthermore, live-virus neutralization assays revealed that Dauricine, one of the identified monomers, exhibited an antiviral activity with an IC50 range of 18.2 to 33.3 µM for original strain, Delta, and Omicron strains, respectively. The computational study showed that the polycyclic and methoxy groups of Dauricine adhere to the RBD surface through π-π and electrostatic interactions. The discovery of Dauricine is a successful attempt to target viral entry, which will not only help society to respond quickly to viral variants, but also accelerate variant drug development thereby reducing the pressure on health authorities to respond to outbreaks.

Computational and experimental studies of salvianolic acid A targets 3C protease to inhibit enterovirus 71 infection.

Hand, foot, and mouth disease (HFMD) is a common childhood infectious disease caused by enterovirus (EV) infection. EV71 is one of the major pathogens causing hand, foot, and mouth disease and is more likely to cause exacerbation and death than other enteroviruses. Although a monovalent vaccine for EV71 has been developed, there are no clinically available anti-EV71 specific drugs. Here, we performed virtual screening and biological experiments based on the traditional Chinese medicine monomer library. We identified a traditional Chinese medicine monomer, Salvianolic acid A (SA), a polyphenolic compound isolated from Salvia miltiorrhiza. Salvianolic acid A inhibits EV71 virus infection in a concentration-dependent manner, and its antiviral activity is higher than that of other reported natural polyphenols and has a high biosafety. Furthermore, molecular dynamics simulations showed that salvianolic acid A can anchor to E71, a member of the enzyme catalytic triad, and cause H40 to move away from the catalytic center. Meanwhile, molecular mechanics generalized born surface area (MMGBSA) and steered molecular dynamics (SMD) results showed that the P1 group of SA was most easily unbound to the S1 pocket of 3Cpro, which provided theoretical support to further improve the affinity of salvianolic acid A with 3Cpro. These findings suggest that salvianolic acid A is a novel EV71 3Cpro inhibitor with excellent antiviral activity and is a promising candidate for clinical studies.

Embigin facilitates monocarboxylate transporter 1 localization to the plasma membrane and transition to a decoupling state.

Cell-surface ancillary glycoproteins basigin or embigin form heterodimeric complexes with proton-coupled monocarboxylate transporters (MCTs), facilitating the membrane trafficking of MCTs and regulating their transport activities. Here, we determine the cryoelectron microscopy (cryo-EM) structure of the human MCT1-embigin complex and observe that embigin forms extensive interactions with MCT1 to facilitate its localization to the plasma membrane. In addition, the formation of the heterodimer effectively blocks MCT1 from forming a homodimer through a steric hindrance effect, releasing the coupling between two signature motifs and driving a significant conformation change in transmembrane helix 5 (TM5) of MCTs. Consequently, the substrate-binding pocket alternates between states of homodimeric coupling and heterodimeric decoupling states and exhibits differences in substrate-binding affinity, supporting the hypothesis that the substrate-induced motion originating in one subunit of the MCT dimer could be transmitted to the adjacent subunit to alter its substrate-binding affinity.

Discovery of SARS-CoV-2 3CLPro Peptidomimetic Inhibitors through the Catalytic Dyad Histidine-Specific Protein-Ligand Interactions.

As the etiological agent for the coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenges the ongoing efforts of vaccine development and drug design. Due to the accumulating cases of breakthrough infections, there are urgent needs for broad-spectrum antiviral medicines. Here, we designed and examined five new tetrapeptidomimetic anti-SARS-CoV-2 inhibitors targeting the 3C-Like protease (3CLPro), which is highly conserved among coronaviruses and essential for viral replications. We significantly improved the efficacy of a ketoamide lead compound based on high-resolution co-crystal structures, all-atom simulations, and binding energy calculations. The inhibitors successfully engaged the catalytic dyad histidine residue (H41) of 3CLPro as designed, and they exhibited nanomolar inhibitory capacity as well as mitigated the viral loads of SARS-CoV-2 in cellular assays. As a widely applicable design principle, our results revealed that the potencies of 3CLPro-specific drug candidates were determined by the interplay between 3CLPro H41 residue and the peptidomimetic inhibitors.

Nonalcoholic fatty liver disease is associated with worse intestinal complications in patients hospitalized for Clostridioides difficile infection.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease with increasing prevalence worldwide. Clostridioides difficile infection (CDI) remains the most common cause of nosocomial diarrhea in developed countries. AIM: To assess the impact of NAFLD on the outcomes of hospitalized patients with CDI. METHODS: This study was a retrospective cohort study. The Nationwide Inpatient Sample database was used to identify a total of 7239 adults admitted as inpatients with a primary diagnosis of CDI and coexisting NAFLD diagnosis from 2010 to 2014 using ICD-9 codes. Patients with CDI and coexisting NAFLD were compared to those with CDI and coexisting alcoholic liver disease (ALD) and viral liver disease (VLD), individually. Primary outcomes included mortality, length of stay, and total hospitalization charges. Secondary outcomes were in-hospital complications. Multivariate regression was used for outcome analysis after adjusting for possible confounders. RESULTS: CDI with NAFLD was independently associated with lower rates of acute respiratory failure (2.7% vs 4.2%, P < 0.01; 2.7% vs 4.2%, P < 0.05), shorter length of stay (days) (5.75 ± 0.16 vs 6.77 ± 0.15, P < 0.001; 5.75 ± 0.16 vs 6.84 ± 0.23, P <0.001), and lower hospitalization charges (dollars) (38150.34 ± 1757.01 vs 46326.72 ± 1809.82, P < 0.001; 38150.34 ± 1757.01 vs 44641.74 ± 1660.66, P < 0.001) when compared to CDI with VLD and CDI with ALD, respectively. CDI with NAFLD was associated with a lower rate of acute kidney injury (13.0% vs 17.2%, P < 0.01), but a higher rate of intestinal perforation (P < 0.01) when compared to VLD. A lower rate of mortality (0.8% vs 2.7%, P < 0.05) but a higher rate of intestinal obstruction (4.6% vs 2.2%, P = 0.001) was also observed when comparing CDI with NAFLD to ALD. CONCLUSION: Hospitalized CDI patients with NAFLD had more intestinal complications compared to CDI patients with VLD and ALD. Gut microbiota dysbiosis may contribute to the pathogenesis of intestinal complications.

4-Iminooxazolidin-2-One as a Bioisostere of Cyanohydrin Suppresses EV71 Proliferation by Targeting 3Cpro.

The fatal pathogen enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease (HFMD), which leads to serious neurological syndromes. While there are no effective clinical agents available for EV71 treatment thus far, EV71 3C protease (3Cpro), a cysteine protease encoded by the virus, has become a promising drug target for discovery of antiviral drugs, given that it plays a crucial role in virus proliferation and interferes with host cell function. Here, we report two inhibitors of EV71 3Cpro, FOPMC and FIOMC, that were developed from previously reported cyanohydrin derivative (R)-1 by replacing the acyl cyanohydrin group with 4-iminooxazolidin-2-one. FOPMC and FIOMC have potent antiviral activity and dramatically improved metabolic stability. These two inhibitors demonstrated broad anti-EV effects on various cell lines and five epidemic viral strains. We further illuminated the binding models between 3Cpro and FOPMC/FIOMC through molecular docking and molecular dynamics simulations. The substitution of an acyl cyanohydrin group with 4-iminooxazolidin-2-one does make FOPMC and FIOMC potent anti-EV71 drug candidates as universal nonclassical bioisosteres with a cyanohydrin moiety. IMPORTANCE EV71 is one of the most epidemic agents of HFMD. Thus far, there are no antiviral drugs available for clinical usage. The conserved EV71 3Cpro plays pivotal roles in virus proliferation and defense host immunity, as well as having no homology in host cells, making it a most promising antiviral target. In this work, we identified that propyl- and isopropyl-substituted 4-iminooxazolidin-2-one moieties (FOPMC and FIOMC) effectively inhibited five epidemic viral strains in rhabdomyosarcoma (RD), HEK-293T, and VeroE6 cell lines. The inhibition mechanism was also illustrated with molecular docking and molecular dynamics (MD) simulations. The successful replacement of the labile cyanohydrin greatly improved the stability and pharmacokinetic properties of (R)-1, making 4-iminooxazolidin-2-one a nonclassical bioisosteric moiety of cyanohydrin. This discovery addressed a critical issue of the primitive structural scaffold of these promising anti-EV71 inhibitors and could lead to their development as broad-spectrum anti-EV agents.

Characteristics and Inpatient Outcomes of Primary Biliary Cholangitis and Autoimmune Hepatitis Overlap Syndrome.

BACKGROUND AND AIMS: Primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH) are hepatobiliary diseases of presumed immune-mediated origin that have been shown to overlap. The aim of this retrospective trial was to use national data to examine the characteristics and outcomes of patients hospitalized with overlapping PBC and AIH (PBC/AIH). METHODS: The National Inpatient Sample was used to identify hospitalized adult patients with PBC, AIH, and PBC/AIH from 2010 to 2014 by International Classification of Diseases-Ninth Edition Revision codes; patients with hepatitis B virus and hepatitis C virus infection were excluded. Primary outcomes measures were in-hospital outcomes that included mortality, respiratory failure, septic shock, length of stay, and total hospital charges. Secondary outcomes were the clinical characteristics of PBC/AIH, including the comorbid extrahepatic autoimmune disease pattern and complications of cirrhosis. RESULTS: A total of 3,478 patients with PBC/AIH were included in the study. PBC/AIH was associated with higher rates of Sjögren's syndrome (p<0.001; p<0.001), lower rates of Crohn's disease (p<0.05; p<0.05), and higher rates of cirrhosis-related complications when compared to PBC or AIH alone. There were similar rates of mortality between the PBC/AIH, PBC, and AIH groups. The PBC/AIH group had higher rates of septic shock when compared to the PBC group (p<0.05) and AIH group (p<0.05) after adjusting for possible confounders. CONCLUSIONS: PBC/AIH is associated with a lower rate of Crohn's disease, a higher rate of Sjögren's syndrome, higher rates of cirrhosis-related complications, and significantly increased risk of septic shock compared to PBC and AIH individually.

Reversible covalent inhibitors suppress enterovirus 71 infection by targeting the 3C protease.

As one of the principal etiological agents of hand, foot, and mouth disease (HFMD), enterovirus 71 (EV71) is associated with severe neurological complications or fatal diseases, while without effective medications thus far. Here we applied dually activated Michael acceptor to develop a series of reversible covalent compounds for EV71 3C protease (3Cpro), a promising antiviral drug target that plays an essential role during viral replication by cleaving the precursor polyprotein, inhibiting host protein synthesis, and evading innate immunity. Among them, cyanoacrylate and Boc-protected cyanoarylamide derivatives (SLQ-4 and SLQ-5) showed effective antiviral activity against EV71. The two inhibitors exhibited broad antiviral effects, acting on RD, 293T, and Vero cell lines, as well as on EV71 A, B, C, CVA16, and CVB3 viral strains. We further determined the binding pockets between the two inhibitors and 3Cpro based on docking studies. These results, together with our previous studies, provide evidence to elucidate the mechanism of action of these two reversible covalent inhibitors and contribute to the development of clinically effective medicines to treat EV71 infections.

Health Related Social Needs Among Chinese American Primary Care Patients During the COVID-19 Pandemic: Implications for Cancer Screening and Primary Care.

Research Objective: Initiatives to address social determinants of health (SDOH) and measure health-related social needs (HRSN) within clinic settings are increasing. However, few have focused on the specific needs of Asian Americans (AA). We examine the prevalence of HRSN during a period spanning the COVID-19 pandemic to inform strategies to improve cancer screening and primary care among AA patients. Methods: We implemented a self-administered HRSN screening tool in English and Chinese, traditional (T) or simplified (S) text, within a hospital-affiliated, outpatient primary care practice predominantly serving AA in New Jersey. HRSN items included food insecurity, transportation barriers, utility needs, interpersonal violence, housing instability, immigration history, and neighborhood perceptions on cohesion and trust. We conducted medical chart reviews for a subset of participants to explore the relationship between HRSN and history of cancer screening. Results: Among 236 participants, most were Asian (74%), non-US born (79%), and privately insured (57%). One-third responded in Chinese (37%). Half reported having ≥1 HRSN. Interpersonal violence was high across all participants. Transportation needs were highest among Chinese-T participants, while food insecurity and housing instability were higher among Chinese-S participants. Lower-income patients had higher odds of having ≥2 HRSN (OR:2.53, 95% CI: 1.12, 5.98). Older age and public insurance/uninsured were significantly associated with low neighborhood perceptions. Conclusions: We observed higher than anticipated reports of HRSN among primary care patients in a suburban, hospital-affiliated practice serving AA. Low neighborhood perceptions, particularly among Chinese-S participants, highlight the importance of addressing broader SDOH among insured, suburban AA patients. These study findings inform the need to augment HRSN identification to adequately address social needs that impact health outcomes and life course experiences for Asian patients. As HRSN measuring efforts continue, and COVID-19's impact on the health of minority communities emerge, it will be critical to develop community-specific referral pathways to connect AA to resources for HRSN and continue to address more upstream social determinants of health for those who are disproportionately impacted.

Reflux esophagitis is associated with higher risks of acute stroke and transient ischemic attacks in patients hospitalized with atrial fibrillation: A nationwide inpatient sample analysis.

ABSTRACT: Reflux esophagitis (RE) is a subset of gastroesophageal reflux disease (GERD) with endoscopic evidence of esophageal inflammation, which has been linked to an increased incidence of atrial fibrillation (AF). However, data on the effect of RE on patient outcomes is limited. We sought to examine the potential association of RE with outcomes of patients with AF in a nationwide study.The National Inpatient Sample (NIS) database was queried to identify hospitalized adult patients with AF and RE between 2010 and 2014. Primary outcomes included inpatient mortality, length of stay (LOS), and total hospital charges. AF related complications such as acute stroke, transient ischemic attack (TIA) and acute heart failure were assessed as secondary outcomes. Propensity score matching and multivariate regression analysis were used.Six lakh sixty seven thousands five hundred twenty patients were admitted for primary diagnosis of AF out of which 5396 had a secondary diagnosis of RE. In the AF with RE cohort, the average age was 73.6 years, 41.5% were male, and 79.9% were Caucasian. There was a greater prevalence of concomitant dyslipidemia, chronic liver disease and chronic pulmonary disease (P < .01) when compared to the AF without RE cohort. Patients with AF and RE also had higher incidence of acute strokes and TIAs (P < .05), longer LOS (P < .001), and higher hospital charges (P < .05) with no difference in acute heart failure (P = .08), hospital mortality (P = .12), or CHA2DS2-VASc score (P = .67).In hospitalized patients with AF, RE was associated with a higher rate of acute stroke and TIAs, longer LOS, and greater hospital charges.

Characterisation and application of Halomonas shantousis SWA25, a halotolerant bacterium with multiple biogenic amine degradation activity.

Biogenic amines are identified as toxicological substances in foods and may have detrimental effects on consumers' health. In recent years, the application of microorganisms that can degrade biogenic amines has become an emerging method for their reduction. The degradation characteristics and application potential of a salt-tolerant bacterium Halomonas shantousis SWA25 were investigated in this study. H. shantousis SWA25 exhibited degradation activity against eight biogenic amines at 10­40°C (optimum, 30­40°C) and pH 3.0­9.0 (optimum, 6.0­7.0) in the presence of 0­20% (w/v) NaCl (optimum, 0%). Specifically, H. shantousis SWA25 degraded all tryptamine (TRY) and tyramine (TYR) in 6 h, all phenethylamine (PHE) in 9 h, 66.7% of histamine (HIM), 52.4% of cadaverine (CAD), 48.0% of spermidine (SPD), 42.9% of putrescine (PUT) and 42.0% of spermine (SPM) in 20 h at 30°C and pH 7.0 with shaking at 120 r min−1. The enzymes from H. shantousis SWA25 responsible for degradation of biogenic amines were mainly amine oxidases located on the cell membrane. Further studies showed that H. shantousis SWA25 effectively degraded TRY, PHE, PUT, CAD, HIM and TYR in commercial fish sauce and soy sauce samples. Nevertheless, significant SPD and SPM degradation were not observed due to low initial concentrations. Therefore, H. shantousis SWA25 can be applied as a potential biogenic amines degradation bacterium in foods.

Halomonas shantousis sp. nov., a novel biogenic amines degrading bacterium isolated from Chinese fermented fish sauce.

A Gram-negative, aerobic, short rod-shaped and non-motile bacterium, designated SWA25(T), was isolated from Chinese fermented fish sauce in Shantou, Guangdong Province, China. Strain SWA25(T) was moderately halophilic, formed colourless colonies and grew at 10-45 °C (optimum, 37 °C) and pH 4-9 (optimum, 6-7) in the presence of 0.5-22.5 % (w/v) NaCl (optimum, 3 %). The major cellular fatty acids (>10 %) were identified as C18:1 ω7C, C16:0, C16:1 ω7c, and C19:0 cyclo ω8c, and the predominant respiratory ubiquinone was Q-9. The genomic DNA G+C content was 61.3 ± 2.1 mol %. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SWA25(T) belonged to the genus Halomonas in the family Halomonadaceae. The closest relatives were Halomonas xianhensis A-1(T) (96.5 % 16S rRNA gene sequence similarity), H. lutea DSM 23508(T) (96.5 %) and H. muralis LMG 20969(T) (96.1 %). DNA-DNA hybridization assays showed 30.7 ± 2.6 % relatedness between strain SWA25(T) and H. xianhensis A-1(T), and 39.4 ± 4.1 % between strain SWA25(T) and H. lutea DSM 23508(T). On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain SWA25(T) should be placed in the genus Halomonas as a representative of a novel species. The name Halomonas shantousis sp. nov. is proposed, with SWA25(T)(=CCTCC AB 2013151(T) = JCM 19368(T)) as the type strain.

Epigenetic silencing of miR-126 contributes to tumor invasion and angiogenesis in colorectal cancer.

microRNAs (miRNAs) have been reported to play a crucial role in regulating a variety of genes pivotal for tumor metastasis. miR-126 is well known as one of the angiogenesis regulatory miRNAs. Recent studies have reported controversial roles of miR-126 in tumor progression. In this study, we sought to investigate the potential roles of miR-126 in colorectal cancer (CRC). By real-time PCR, miR-126 was shown to be downregulated in primary CRC tissues and cell lines. Restoration of miR-126 in CRC cells inhibited cell growth, migration and invasion. Using both in silico prediction and immunoblotting, we found that vascular endothelial growth factor (VEGF) was a target of miR-126. The interaction of miR-126 on the 3'UTR of VEGF mRNA was validated by luciferase reporter assay. Mechanistically, we found that the silencing of miR-126 was induced by promoter methyl-ation of its host gene, EGFL7. Treatment with 5-aza-CdR restored miR-126 expression and thereby led to a decline in VEGF expression. Functionally, due to suppression of VEGF, enhanced miR-126 expression inhibited tumor neovasculature triggered by CRC cells. In conclusion, our findings suggest that DNA methylation-induced silencing of miR-126 contributes, at least in part, to tumor invasion and angiogenesis in CRC, through upregulation of VEGF expression. miR-126 may be a potential target for the therapeutic strategy against CRC.

[Toll like receptors and malaria parasites invasion: progress and prospective].

To deeply study the molecular mechanism of infection and reproduction of malaria parasites, this review began with co-relationship between toll like receptors (TLRs) and the parasites, further discussed how the parasites were recognized by TLRs and activated downstream inflammation reactions when the mechanical barrier of host was broken through. Some large molecules involved into the process had the potential to be novel drug targets.

[An in vitro test of propolis against Trichomonas vaginalis].

Cultured T. vaginalis was used for the anti-trichomonas test at different times, concentrations of propolis and densities of the parasites. After being cultured for 0, 6, 12, 24 hrs, the survival rate of the parasites was (91.50+/-3.11)%, (43.00+/-6.83)%, (22.25+/-5.32)% and (11.50+/-5.74)% respectively with a significant difference between propolis group and the control. Under the concentrations of 0.24, 0.48, 0.96, 1.92, 3.84 and 7.68 (mg/ml) with 24 hrs culture, the survival rate was (88.00+/-5.29)%, (92.67+/-4.16)%, (90.0+/-6.00)%, (84.00+/-4.00)%, (2.67+/-1.15)%, and 0 respectively. The results showed that propolis possesses clear in vitro anti-trichomonas activity which is relevant to the duration of culture and the concentration of the agent.