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Introduction: Autism spectrum disorder (ASD) is an increasing concern among the Iraqi Arab population. The genetic alterations that cause ASD are likely to converge at the synapse. This study investigated polymorphisms in the GABA A receptor subunit (GABRG3) and the RELN gene as putative biomarkers of ASD in a pediatric population in Iraq., Methods: The case control study included 60 patients with a clinical diagnosis of ASD (mild, moderate, or severe) according to DSM-IV criteria and matched healthy controls (n = 60). Blood samples were collected for DNA genotyping of SNPs rs736707 and rs208129 for RELN and GABRG3 using allele specific PCR. Assessment of genotype and allele distributions in patient groups used odd ratios (OR) with 95% confidence intervals and the Chi-square test. All statistical analysis was performed used SPSS software., Result: The patient cohort was highly consanguineous, with increased ratio (p > 0.05) of males to females (3:1) in both ASD (mean age, 6.66 ± 3.05) and controls (mean age, 5.76 ± 2.3). Both GABRG3 rs208129 genotypes TT (OR 4.33, p = 0.0015) and TA (OR 0.259, P = 0.008), and the T and A alleles were significantly associated with ASD. The RELN rs736707 TC genotype (OR 2.626, P = 0.034) was the only significant association with ASD., Conclusion: GABRG3 SNP rs208129 is a leading biomarker to predict genetic vulnerability to ASD in Iraqi Arabs. Expanded SNP panels and increased sample sizes are required for future GABRG3 studies, and to reach a consensus on RELN utility. Future ASD screening programs in Iraq should include genetic metrics in addition to clinical phenotype assessments., (© 2022. The Author(s).)

Novel coronavirus disease 2019 (COVID-19) poses a global threat, due to its fluctuating frequency and lethality. Published data revealed associations of COVID-19 susceptibility and severity with host genetic polymorphisms in renin-angiotensin-aldosterone system (RAAS)-related genes including angiotensin-converting enzyme (ACE)1, ACE2, and transmembrane protease (TMPRSS)2. However, the findings remain inconclusive. Accordingly, we aimed to clarify associations of genetic variants in those genes with COVID-19 susceptibility and severity using a systematic review with meta-analysis. From inception through 1 July 2021, a literature search was performed using PubMed, Scopus, Web of Science, and Cochrane Library databases. Allelic distributions for each polymorphism were calculated as pooled odds ratios (OR) with 95% confidence intervals (CI) to assess the strength of association. A total of 3333 COVID-19 patients and 5547 controls from 11 eligible studies were included. From a systematic review, ACE1 rs1799752, ACE1 rs4646994, ACE2 rs2285666, and TMPRSS2 rs12329760 were identified as common polymorphisms of RAAS-related genes. Meta-analysis showed a significant association between TMPRSS2 rs12329760 C-allele and an increased risk of developing severe COVID-19 (OR = 1.32, 95% CI: 1.01, 1.73). Likewise, additional meta-analyses uncovered that both ACE1 rs4646994 DD-genotype and ACE2 rs2285666 GG-genotype carriers had a significantly increased risk of developing severe COVID-19 (OR = 2.06, 95% CI: 1.45, 2.93; OR = 2.14, 95% CI: 1.26, 3.66; respectively). Genetic polymorphisms of ACE1 rs4646994 DD-genotype, ACE2 rs2285666 GG-genotype, and TMPRSS2 rs12329760 CC-genotype and C-allele may serve as predictive models of COVID-19 severity., (© 2022 John Wiley & Sons Ltd.)

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17β-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 μg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19., Competing Interests: The authors have declared that no competing interests exist.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

Staphylococcus aureus biofilms are the pathogenic factor in the spread of infection and are more pronounced in multidrug-resistant strains of S. aureus, where high expression of proteases is observed. Among various proteases, Serine protease (SspA) and cysteine protease Staphopain B (SspB) are known to play a key role in the biofilm formation and removal of biofilms. In earlier studies, we have reported Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) exhibits anti-S. aureus and anti-biofilm properties by elevating the expression of the protease. In this study, the effect of DBTMP on the activities of SspA, and SspB of S. aureus was evaluated. The SspA and SspB genes of S. aureus ATCC12600 were sequenced (Genbank accession numbers: MZ456982 and MW574006). In S. aureus active SspA is formed by proteolytic cleavage of immature SspA, to get this mature SspA (mSspA), we have PCR amplified the mSspA sequence from the SspA gene. The mSspA and SspB genes were cloned, expressed, and characterized. The pure recombinant proteins rSspB and rmSspA exhibited a single band in SDS-PAGE with a molecular weight of 40 and 30 KD, respectively. The activities of rmSspA and rSspB are 32.33 and 35.45 Units/mL correspondingly. DBTMP elevated the activities of rmSspA and rSspB by docking with respective enzymes. This compound disrupted the biofilms formed by the multidrug-resistant strains of S. aureus and further prevented biofilm formation. These findings explain that DBTMP possesses anti-S. aureus and anti-biofilm features., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tissue fibrosis and organ dysfunction are hallmarks of age-related diseases including heart failure, but it remains elusive whether there is a common pathway to induce both events. Through single-cell RNA-seq, spatial transcriptomics, and genetic perturbation, we elucidate that high-temperature requirement A serine peptidase 3 (Htra3) is a critical regulator of cardiac fibrosis and heart failure by maintaining the identity of quiescent cardiac fibroblasts through degrading transforming growth factor-β (TGF-β). Pressure overload downregulates expression of Htra3 in cardiac fibroblasts and activated TGF-β signaling, which induces not only cardiac fibrosis but also heart failure through DNA damage accumulation and secretory phenotype induction in failing cardiomyocytes. Overexpression of Htra3 in the heart inhibits TGF-β signaling and ameliorates cardiac dysfunction after pressure overload. Htra3-regulated induction of spatio-temporal cardiac fibrosis and cardiomyocyte secretory phenotype are observed specifically in infarct regions after myocardial infarction. Integrative analyses of single-cardiomyocyte transcriptome and plasma proteome in human reveal that IGFBP7, which is a cytokine downstream of TGF-β and secreted from failing cardiomyocytes, is the most predictable marker of advanced heart failure. These findings highlight the roles of cardiac fibroblasts in regulating cardiomyocyte homeostasis and cardiac fibrosis through the Htra3-TGF-β-IGFBP7 pathway, which would be a therapeutic target for heart failure., (© 2022. The Author(s).)

Purpose: To analyze the correlations between age-related macular degeneration (AMD) and genetic and environmental risk factors for in a Brazilian population., Design: Cross-sectional study with a control group., Methods: We collected data on 236 participants 50 years of age or older (141 with AMD and 95 controls without the disease). Data was obtained using a questionnaire and included information on demographics, ocular and medical history, family history of AMD, lifestyle, and smoking and drinking habits. Genetic evaluations included direct sequencing for the LOC387715 (rs10490924) variant, as well as PCR and enzymatic digestion for the CFH Y402H (rs1061170) and HTRA1 (rs11200638) variants. We performed a risk assessment of environmental risk factors and genetic variants associated with AMD and determined correlations between AMD and the data collected using multiple linear regression analysis., Results: Of the 141 AMD cases, 99 (70%) had advanced AMD in at least one eye (57% neovascular AMD and 13% geographic atrophy), and 42 (30%) had not-advanced AMD. Family history of AMD (OR: 6.58; 95% CI: 1.94-22.31), presence of cardiovascular disease (CVD) (OR: 2.39; 95% CI: 1.08-5.28), low physical activity level (OR: 1.39; 95% CI: 0.82-2.37), and high serum cholesterol (OR: 1.49; 95% CI: 0.84-2.65) were associated with an increased risk for AMD. There was a significant association between CVD and incidence of advanced AMD (OR: 2.29; 95% CI 0.81-6.44). The OR for the risk allele of the LOC387715 gene, the CFH gene and the HTRA1 gene were 2.21 (95% CI: 1.47-3.35), 2.27 (95% CI: 1.52-3.37), and 2.76 (95% CI: 1.89-4.03), respectively. In the stepwise multiple linear regression analyses, the HTRA1 and CFH risk alleles, family history of AMD, the LOC387715 risk allele, and CVD were associated with an increased risk of AMD for a total of 25.6% contribution to the AMD phenotype., Conclusions: The analysis correlating environmental and genetic risk factors such as family history of AMD, and CVD and the variants of HTRA1, CFH, and LOC387715 genes showed an expressive contribution for the development of AMD among this admixed population., Competing Interests: The authors have declared that no competing interests exist.

To better understand the genetics of hearing loss, we performed a genome-wide association meta-analysis with 125,749 cases and 469,497 controls across five cohorts. We identified 53/c loci affecting hearing loss risk, including common coding variants in COL9A3 and TMPRSS3. Through exome sequencing of 108,415 cases and 329,581 controls, we observed rare coding associations with 11 Mendelian hearing loss genes, including additive effects in known hearing loss genes GJB2 (Gly12fs; odds ratio [OR] = 1.21, P = 4.2 × 10 -11 ) and SLC26A5 (gene burden; OR = 1.96, P = 2.8 × 10 -17 ). We also identified hearing loss associations with rare coding variants in FSCN2 (OR = 1.14, P = 1.9 × 10 -15 ) and KLHDC7B (OR = 2.14, P = 5.2 × 10 -30 ). Our results suggest a shared etiology between Mendelian and common hearing loss in adults. This work illustrates the potential of large-scale exome sequencing to elucidate the genetic architecture of common disorders where both common and rare variation contribute to risk., (© 2022. The Author(s).)

Proprotein Convertase Subtilisin/Kexin-type 9 (PCSK9) is a circulating negative regulator of hepatic low-density lipoprotein receptor (LDLR), which clears cholesterol from blood. Gain-of-function genetic mutations that amplify PCSK9 activity have been found to cause potentially lethal familial hypercholesterolemia. Inversely, reduction of its activity through loss-of-function genetics or with pharmaceuticals was shown to increase hepatic LDLR, to lower blood cholesterol, and to protect against cardiovascular diseases. New epidemiological and experimental evidence suggests that this reduction could also attenuate inflammation, reinforce cancer immunity, provide resistance to infections, and protect against liver pathologies. In this review, we question the relevance of this protein under normal physiology. We propose that PCSK9 is an important, but nonessential, modulator of cholesterol metabolism and immunity, and that its pathogenicity results from its chronic overexpression.

Aim: To investigate the clinical role of transmembrane protease serine 3 (TMPRSS3) in radioresistance and prognosis of colorectal cancer (CRC). Methods: Standardized mean difference (SMD) and summary area under the curve (AUC) of TMPRSS3 were calculated by combining all available high-throughput data globally. The prognostic significance of TMPRSS3 was determined by Kaplan-Meier and Cox regression analyses. Results: TMPRSS3 was remarkably upregulated in 198 CRC radioresistant cases compared with nonradioresistance (SMD = 0.38, AUC = 0.71). Overexpression of TMPRSS3 was observed in 1601 CRC patients compared with control subjects without CRC. TMPRSS3 was a risk factor for disease-free survival of CRC with the summarized hazard ratio 1.28. Conclusion: TMPRSS3 contributes to the radioresistance and unfavorable prognosis of CRC.

Objectives: The oral cavity is one of the main entry sites for SARS-CoV-2. Gingival keratinocytes express transmembrane serine protease 2 (TMPRSS2), responsible for priming the SARS-CoV-2 spike protein. We investigated whether periodontitis increased the expression of TMPRSS2., Methods: To investigate gene expression in periodontitis, we analyzed the expression of specific genes from (1) the Gene Expression Omnibus (GEO) dataset of 247 human gingival tissues and (2) an experimentally-induced periodontitis mouse model. Human gingival tissues with or without periodontitis were immunohistochemically stained using an anti-TMPRSS2 antibody. Analysis of the TMPRSS2 promoter was performed using a ChIP-Atlas dataset. TMPRSS2 expression was detected in cultured human keratinocytes using quantitative reverse transcription (qRT)-PCR and Western blot analysis., Results: GEO dataset analysis and an experimentally-induced periodontitis model revealed increased expression of TMPRSS2 in periodontitis gingiva. The keratinocyte cell membrane in periodontitis gingiva was strongly immunohistochemically stained for TMPRSS2. Using ChIP-Atlas and GEO datasets, we screened for transcription factors that bind to the TMPRSS2 promoter region. We found one candidate, estrogen receptor 1 (ESR1), highly expressed in periodontitis gingiva. Analysis of the GEO dataset revealed a correlation between ESR1 and TMPRSS2 expression in gingival tissues. An ESR1 ligand induced TMPRSS2 expression in cultured keratinocytes., Conclusions: Periodontitis increases TMPRSS2 expression in the cell membrane of gingival keratinocytes., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, β-, and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α-ENaC and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αβγ-ENaC heterologously expressed in Xenopus laevis oocytes. We found that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically inactive TMPRSS2 mutant and was associated with fully cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C-Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated that TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2 knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

This study investigated the role of TMPRSS6 C > T polymorphism (TMPRP) on the effects of chronic aerobic training on main hematological parameters in male soccer referees, which is yet unknown. Two groups composed of total of 45 healthy male soccer referees and 42 sedentary were compared for hemogram, serum hepcidin, ferritin, and iron levels. TMPRP was determined from genomic DNA samples. Participants' physical and physiological (Yoyo endurance level-2 test) measurements were carried out. The athletic T carrier (Tc = TT + TC) group RBC count was significantly higher than the control (p < 0.01), whereas the athletic CC homozygous group serum iron and transferrin saturation (TS) were lower than the control depending on the TMPRP. The ferritin and iron values of the athletic Tc group were higher than of the athletic CC group (29.2% and 14.1%, respectively; p > 0.05) although the control Tc group RBC (p < 0.05) and iron (23.8%, p > 0.05) values were lower than the control CC due to genetic tendency. The training did not change hepcidin levels. These results suggest that the TMPRP can modify the endurance training effects on iron and TS levels and RBC count (in the CC and Tc groups) respectively. The CC group may be adversely affected for iron and TS from endurance trainings. It may be recommended that the training programs should be organized according to phenotype characteristics., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The transcription factor RpoS of Escherichia coli controls many genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by azidothymidine. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp and are regulated by regulatory inactivation of DnaA, dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays, we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced regulatory inactivation of DnaA. We also demonstrate that the RNA-polymerase associated factor, stringent starvation protein A, induced by the accumulation of ppGpp, also affects iraD expression, with a positive effect on constitutive expression and a negative effect on azidothymidine-induced expression., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although males and females are at equivalent risk of infection, males are more prone to develop a higher severity disease, regardless of age. The factors that mediate susceptibility to SARS-CoV-2 and transmission are still under investigation. A potential role has been attributed to differences in the immune systems response to viral antigens between males and females as well as to different regulatory actions played by sex-related hormones on the two crucial molecular effectors for SARS-CoV-2 infection, TMPRSS2 and ACE2. While few and controversial data about TMPRSS2 transcript regulation in lung cells are emerging, no data on protein expression and activity of TMPRSS2 have been reported. Aim of the present study was to search for possible modulatory actions played by sex-related hormones on TMPRSS2 and ACE2 expression in Calu-3 cells, to test the effects of sex-steroids on the expression of the 32kDa C-term fragment derived from autocatalitic cleavage of TMPRSS2 and its impact on priming of transiently transfected spike protein. Cells were stimulated with different concentrations of methyltrienolone (R1881) or estradiol for 30 h. No difference in mRNA and protein expression levels of full length TMPRSS2 was observed. However, the 32 kDa cleaved serine protease domain was increased after 100 nM R1881 (+2.36 ± 1.13 fold-increase vs control untreated cells, p < 0.05) and 10 nM estradiol (+1.90 ± 0.64, fold-increase vs control untreated cells, p < 0.05) treatment. Both R1881 and estradiol significantly increased the activating proteolytic cleavage of SARS-CoV-2 Spike (S) transfected in Calu-3 cells (+1.76 ± 0.18 and +1.99±,0.76 increase in S cleavage products at R1881 100nM and 10 nM estradiol treatment, respectively, p < 0.001 and p < 0.05 vs control untreated cells, respectively). Finally, no significant differences in ACE2 expression were observed between hormones-stimulated cells and untreated control cells. Altogether, these data suggest that both male and female sex-related hormones are able to induce a proteolityc activation of TMPRSS2, thus promoting viral infection, in agreement with the observation that males and females are equally infected by SARS-CoV-2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Treppiedi, Marra, Di Muro, Catalano, Mangili, Esposito, Barbieri, Arosio, Mantovani and Peverelli.)

By the end of December 2021, coronavirus disease 2019 (COVID-19) produced more than 271 million cases and 5.3 million deaths. Although vaccination is an effective strategy for pandemic control, it is not yet equally available in all countries. Therefore, identification of prognostic biomarkers remains crucial to manage COVID-19 patients. The aim of this study was to evaluate predictors of COVID-19 severity previously proposed. Clinical and demographic characteristics and 120 single-nucleotide polymorphisms were analyzed from 817 patients with COVID-19, who attended the emergency department of the Hospital Universitario de La Princesa during March and April 2020. The main outcome was a modified version of the 7-point World Health Organization (WHO) COVID-19 severity scale (WHOCS); both in the moment of the first hospital examination (WHOCS-1) and of the severest WHOCS score (WHOCS-2). The TMPRSS2 rs75603675 genotype (OR = 0.586), dyslipidemia (OR = 2.289), sex (OR = 0.586), and the Charlson Comorbidity Index (OR = 1.126) were identified as the main predictors of disease severity. Consequently, these variables might influence COVID-19 severity and could be used as predictors of disease development., (© 2022 Villapalos-García et al.)

Genome wide association studies (GWAS) have focused on elucidating the genetic architecture of complex traits by assessing single variant effects in additive genetic models, albeit explaining a fraction of the trait heritability. Epistasis has recently emerged as one of the intrinsic mechanisms that could explain part of this missing heritability. We conducted epistasis analysis for genome-wide body mass index (BMI) associated SNPs in Alzheimer's Disease Neuroimaging Initiative (ADNI) and followed up top significant interacting SNPs for replication in the UK Biobank imputed genotype dataset. We report two pairwise epistatic interactions, between rs2177596 (RHBDD1) and rs17759796 (MAPK1), rs1121980 (FTO) and rs6567160 (MC4R), obtained from a consensus of nine different epistatic approaches. Gene interaction maps and tissue expression profiles constructed for these interacting loci highlights co-expression, co-localisation, physical interaction, genetic interaction, and shared pathways emphasising the neuronal influence in obesity and implicating concerted expression of associated genes in liver, pancreas, and adipose tissues insinuating to metabolic abnormalities characterized by obesity. Detecting epistasis could thus be a promising approach to understand the effect of simultaneously interacting multiple genetic loci in disease aetiology, beyond single locus effects., (© 2022. The Author(s).)

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8 + T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lin, Tien, Kuo, Lin, Tsai, Chen, Tsai, Su, Pathak, Yang, Yu, Chuang, Wu, Tsai, Dai, Liao, Chai, Su, Chuang, Liu, Chen, Dou, Chen, Chen, Liao and Yu.)

Background: The human protein transmembrane protease serine type 2 (TMPRSS2) plays a key role in SARS-CoV-2 infection, as it is required to activate the virus' spike protein, facilitating entry into target cells. We hypothesized that naturally-occurring TMPRSS2 human genetic variants affecting the structure and function of the TMPRSS2 protein may modulate the severity of SARS-CoV-2 infection., Methods: We focused on the only common TMPRSS2 non-synonymous variant predicted to be damaging (rs12329760 C>T, p.V160M), which has a minor allele frequency ranging from 0.14 in Ashkenazi Jewish to 0.38 in East Asians. We analysed the association between the rs12329760 and COVID-19 severity in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units recruited as part of the GenOMICC (Genetics Of Mortality In Critical Care) study. Logistic regression analyses were adjusted for sex, age and deprivation index. For in vitro studies, HEK293 cells were co-transfected with ACE2 and either TMPRSS2 wild type or mutant (TMPRSS2 V160M ). A SARS-CoV-2 pseudovirus entry assay was used to investigate the ability of TMPRSS2 V160M to promote viral entry., Results: We show that the T allele of rs12329760 is associated with a reduced likelihood of developing severe COVID-19 (OR 0.87, 95%CI:0.79-0.97, p = 0.01). This association was stronger in homozygous individuals when compared to the general population (OR 0.65, 95%CI:0.50-0.84, p = 1.3 × 10 -3 ). We demonstrate in vitro that this variant, which causes the amino acid substitution valine to methionine, affects the catalytic activity of TMPRSS2 and is less able to support SARS-CoV-2 spike-mediated entry into cells., Conclusion: TMPRSS2 rs12329760 is a common variant associated with a significantly decreased risk of severe COVID-19. Further studies are needed to assess the expression of TMPRSS2 across different age groups. Moreover, our results identify TMPRSS2 as a promising drug target, with a potential role for camostat mesilate, a drug approved for the treatment of chronic pancreatitis and postoperative reflux esophagitis, in the treatment of COVID-19. Clinical trials are needed to confirm this., Competing Interests: Declaration of Competing Interest Dr. David reports grants from Wellcome Trust during the conduct of the study; Dr. Parkinson reports grants from Wellcome Trust during the conduct of the study; Dr. Peacock reports grants from MRC/UKRI, grants from BBSRC during the conduct of the study; Dr. Pairo-Castineira has nothing to disclose. Dr. Khanna reports grants from BBSRC during the conduct of the study; Dr. Cobat has nothing to disclose. Dr. Tenesa reports grants from BBSRC, grants from Health Data Research UK during the conduct of the study. Dr. Sancho-Shimizu reports grants from UKRI Future Leader's Fellowship during the conduct of the study; Dr. Casanova reports other from Howard Hughes Medical Institute, other from Rockefeller University, other from St. Giles Foundation, other from Fisher centre for Alzheimer's Research Foundation, other from Meyer Foundation, other from Square Foundation, other from Grandir - Fonds de solidarité pour l'enfance, other from SCOR Corporate Foundation for Science, other from Institut National de la Santé et de la Recherche Médicale (INSERM), other from University of Paris, other from National Institutes of Health (NIH), other from French Foundation for Medical Research (FRM), other from FRM and French National Research Agency (ANR) GENCOVID project during the conduct of the study; Dr. Abel reports other from Agence Nationale de la Recherche during the conduct of the study; Dr. Barclay reports grants from BBSRC during the conduct of the study; Dr. Baillie has nothing to disclose. Dr. Sternberg reports grants from Wellcome Trust, grants from BBSRC, during the conduct of the study., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: Hereditary hearing loss exhibits high degrees of genetic and clinical heterogeneity. To elucidate the population-specific and age-related genetic and clinical spectra of hereditary hearing loss, we investigated the sequencing data of causally associated hearing loss genes in a large cohort of hearing-impaired probands with a balanced age distribution from a single center in Southwest Germany., Design: Genetic testing was applied to 305 hearing-impaired probands/families with a suspected genetic hearing loss etiology and a balanced age distribution over a period of 8 years (2011-2018). These individuals were representative of the regional population according to age and sex distributions. The genetic testing workflow consisted of single-gene screening (n = 21) and custom-designed hearing loss gene panel sequencing (n = 284) targeting known nonsyndromic and syndromic hearing loss genes in a diagnostic setup. Retrospective reanalysis of sequencing data was conducted by applying the current American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines., Results: A genetic diagnosis was established for 75 (25%) of the probands that involved 75 causal variants in 35 genes, including 16 novel causal variants and 9 medically significant variant reclassifications. Nearly half of the solved cases (47%; n = 35) were related to variants in the five most frequently affected genes: GJB2 (25%), MYO15A, WFS1, SLC26A4, and COL11A1 (all 5%). Nearly one-quarter of the cases (23%; n = 17) were associated with variants in seven additional genes (TMPRSS3, COL4A3, LOXHD1, EDNRB, MYO6, TECTA, and USH2A). The remaining one-third of single cases (33%; n = 25) were linked to variants in 25 distinct genes. Diagnostic rates and gene distribution were highly dependent on phenotypic characteristics. A positive family history of autosomal-recessive inheritance in combination with early onset and higher grades of hearing loss significantly increased the solve rate up to 60%, while late onset and lower grades of hearing loss yielded significantly fewer diagnoses. Regarding genetic diagnoses, autosomal-dominant genes accounted for 37%, autosomal-recessive genes for 60%, and X-linked genes for 3% of the solved cases. Syndromic/nonsyndromic hearing loss mimic genes were affected in 27% of the genetic diagnoses., Conclusions: The genetic epidemiology of the largest German cohort subjected to comprehensive targeted sequencing for hereditary hearing loss to date revealed broad causal gene and variant spectra in this population. Targeted hearing loss gene panel analysis proved to be an effective tool for ensuring an appropriate diagnostic yield in a routine clinical setting including the identification of novel variants and medically significant reclassifications. Solve rates were highly sensitive to phenotypic characteristics. The unique population-adapted and balanced age distribution of the cohort favoring late hearing loss onset uncovered a markedly large contribution of autosomal-dominant genes to the diagnoses which may be a representative for other age balanced cohorts in other populations., Competing Interests: B.S., S.F., M.S., F.B., and S.B. are current or former employees of Praxis für Humangenetik, Tübingen, and/or CeGaT GmbH, Tübingen. The employment relationship had no impact on data collection or interpretation or the writing of the manuscript. The other authors have no conflicts of interest to disclose., (Copyright © 2021 The Authors. Ear & Hearing is published on behalf of the American Auditory Society, by Wolters Kluwer Health, Inc.)

Phenoloxidase (PO) is a crucial component of the insect immune response against microbial infection. In the tobacco hornworm Manduca sexta, PO is generated from its precursor proPO by prophenoloxidase activating proteases (PAPs) in the presence of two noncatalytic serine protease homologs (SPHs). cDNA cloning and genome analysis indicate that SPH1a (formerly known as SPH1), SPH1b, SPH4, SPH101, and SPH2 contain a clip domain, a linker, and a protease-like domain (PLD). The first 22 residues of the SPH1b, SPH4, and SPH101 PLDs are identical, and differ from SPH1a only at position 4, Thr 154 substituted with Asn 154 in SPH1a. While the sequence from Edman degradation was used to establish PAP cofactor as a high M r complex of SPH1a and SPH2, this assignment needed further validation, especially because SPH1b mRNA levels are much higher than SPH1a's and better correlate with SPH2 transcription. Thus, here we determined expression profiles of these SPH genes in different tissues from various developmental stages using highly specific primers. High levels of SPH1b and SPH2 proteins, low SPH4, and no SPH1a or SPH101 were detected in hemolymph from larvae in the feeding, wandering and bar stages, pupae, and adults by targeted LC-MS/MS analysis, based on unique peptides from the trypsin-treated SPHs. We expressed the five proSPHs in baculovirus-infected Sf9 cells for use as standards to identify and quantify their counterparts in plasma samples. Moreover, we tested their cleavage by PAP3 and efficacy of the SPH1a, 1b, 4, and 101 as SPH2 partners in PAP3-mediated proPO activation. PAP3 processed proSPH1b and 101 more readily than proSPH1a and 4; PAP3 activated proPO more efficiently in the presence of SPH2 with SPH101 or SPH1b than with SPH1a or SPH4. These results generally agree with their order of appearance or sequence similarity: SPH101 > SPH1b (98%) > SPH1a (90%) > SPH4 (83%). In other words, likely due to positive selection, products of the newly duplicated genes (SPH1b and SPH101) are more favorable substrates of PAP3 and better SPH2 partners in forming a high M r cofactor than SPH1a or SPH4 is. Electrophoresis on native gel and immunoblot analysis further indicated that SPH101 or 1b form high M r complexes more readily than SPH1a or 4 does. In comparison, SPH2 showed a small mobility decrease and then increase on native gel after PAP3 cleavage at the first site. Since the natural cofactor in bar-stage hemolymph is complexes of SPH1 and 2 with an average M r of 790 kDa, PAP3-activated SPH2 may associate with the higher M r SPH1b scaffolds to form super-complexes. Their structures and formation in relation to cleavage of SPH1b at different sites await further exploration., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Clip domain serine proteinases participate in invertebrate innate immunity by acting as crucial enzymes in the signaling cascade involved in shrimp immunity. To functionally characterize its role in Fenneropenaeus merguiensis, FmclipSP cDNA was cloned and characterized. The FmclipSP gene comprised 1353 bp with an open reading frame of 1110 bp and encoded 369 amino acids. The protein contained clip and serine protease domains. FmClipSP mRNA is highly expressed in hemocytes, and its expression was significantly upregulated by bacterial or viral pathogen challenge. Furthermore, FmClipSP recombinant protein (rFmClipSP) was produced and possessed protease activity, stimulating prophenoloxidase activity. Additionally, rFmClipSP exhibited antibacterial activity against pathogens and nonpathogens. ELISA results demonstrated the binding ability of rFmClipSP to a recombinant protein of VP28 (rVP28). Interestingly, the binding significantly inhibited prophenoloxidase activity. Altogether, we partially characterized the function of FmclipSP and demonstrated its association with VP28. This study indicates the importance of clipSP as a component of F. merguiensis innate immunity. However, the role of clipSP in crustaceans remains unclear and requires further investigation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses angiotensin-converting enzyme-2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2) as a primary receptor for invasion. Cell entry by the virus requires the co-expression of these molecules in the host cells., Objective: We investigated ACE2 and TMPRSS2 expression and localization in paranasal epithelium of eosinophilic chronic rhinosinusitis (ECRS) patients (n = 38), non-ECRS (n = 31), and healthy controls (n = 25). CRS inflammatory patterns are characterized by the type of cytokines; we investigated whether inflammatory endotypes are associated with cell-entry molecules, as this could be linked to susceptibility to SARS-CoV-2 infection., Methods: The ACE2, TMPRSS2, and other inflammatory cytokine mRNA levels were assessed by quantitative RT-PCR. The localizations of ACE2- and TMPRSS2-positive cells were examined with immunofluorescent double-staining using laser scanning confocal microscopy (LSCM)., Results: The non-ECRS patients showed significantly increased ACE2 and TMPRSS2 mRNA expressions compared to the ECRS patients. The CRS patients' ACE2 and TMPRSS2 mRNA levels were positively correlated with IFN-γ ( r  = 0.3227 and r  = 0.3264, respectively) and TNF-α ( r  = 0.4008, r  = 0.3962, respectively). ACE2 and TMPRSS2 were negatively correlated with tissue eosinophils ( r  =  -0.3308, r  =  -0.3112, respectively), but not with IL-13. ACE2 mRNA levels were positively correlated with TMPRSS2 ( r  = 0.7478). ACE2 and TMPRSS2 immunoreactivities were localized mainly in the epithelial ciliated cells, as confirmed by co-staining with TMPRSS2 and acetylated α-tubulin, a cilia organelle marker. Using LSCM imaging, we observed higher expressions of these molecules in the non-ECRS patients versus the ECRS patients., Conclusion: ECRS patients with type 2 inflammation showed decreased ACE2 and TMPRSS2 expressions in their sinus mucosa. ACE2 and TMPRSS2 regulation seems to be positively related to IFN-γ and TNF-α production in CRS patients; ACE2 and TMPRSS2 were co-expressed in the ciliated epithelium of their paranasal mucosa, implicating the paranasal epithelium as a portal for initial infection and transmission.

Background: Long non-coding RNAs (lncRNAs) play vital roles in tumorigenesis. Here, we explored how lncRNA HOXA11-AS functions in the progression of breast cancer (BC)., Methods: HOXA11-AS and miR-125a-5p levels were measured by a quantitative real-time polymerase chain reaction, whereas western blotting determined TMPRSS4 levels in BC tumor tissues, adjacent normal tissues and BC cell lines. The roles of HOXA11-AS, miR-125a-5p and TMPRSS4 in BC proliferation were investigated using cell counting kit-8, colony formation and flow cytometry assays, whereas scratch and transwell assays were used to measure metastasis. RNA pull-down assays and dual-luciferase assays assessed direct interactions between HOXA11-AS and miR-125a-5p. The effects of HOXA11-AS in vivo were investigated in a BC xenograft model., Results: HOXA11-AS was upregulated in tumor tissues of 56 BC patients compared to adjacent non-tumor tissues, with high levels being associated with worse overall survival. Silencing of HOXA11-AS inhibited the proliferation and metastasis of BC cells, leading to cell cycle arrest in G0/G1 and induction of apoptosis. We identified miR-125a-5p as a target of HOXA11-AS, with miR-125a-5p inhibitors partially restoring the reduction of cell proliferation and metastasis induced by HOXA11-AS silencing. We also determined that miR-125a-5p targeted TMPRSS4 mRNA, with HOXA11-AS knockdown and miR-125a-5p mimics suppressing TMPRSS4. Overexpression of TMPRSS4 partially compensated for the reduction of cell proliferation and metastasis induced by HOXA11-AS silencing. Finally, we confirmed the mechanism of HOXA11-AS in the regulation of tumorigenesis in the mouse model., Conclusions: HOXA11-AS regulates the tumorigenic ability of BC via the miR-125a-5p/TMPRSS4 axis. This provides insights for regulatory mechanisms involved in BC progression, and may enable new treatment strategies in the clinical setting., (© 2022 John Wiley & Sons, Ltd.)

Several comorbidities including diabetes, immune deficiency, and chronic respiratory disorders increase the risk of severe Covid-19 and fatalities among SARS-CoV-2 infected individuals. Severe Covid-19 risk among diabetes patients may reflect reduced immune response to viral infections. SARS-CoV-2 initially infects respiratory tract epithelial cells by binding to the host cell membrane ACE2, followed by proteolytic priming for cell entry by the host cell membrane serine protease TMPRSS2. Additionally, the protease FURIN facilitates cell exit of mature SARS-CoV-2 virions. Alpha-1 antitrypsin (AAT), the major plasma serine protease inhibitor, encoded by SERPINA1, is known to promote immune response to viral infections. AAT inhibits neutrophil elastase, a key inflammatory serine protease implicated in alveolar cell damage during respiratory infections, and AAT deficiency is associated with susceptibility to lung infections. AAT is implicated in Covid-19 as it inhibits TMPRSS2, a protease essential for SARS-CoV-2 cell entry. Here we show that treatment of A549 human lung epithelial cells for 7 days with 25 mM d-galactose, an inducer of diabetic-like and oxidative stress cellular phenotypes, leads to increased mRNA levels of ACE2, TMPRSS2, and FURIN, along with reduced SERPINA1 mRNA. Together, the dysregulated transcription of these genes following d-galactose treatment suggests that chronic diabetic-like conditions may facilitate SARS-CoV-2 infection of lung epithelial cells. Our findings may in part explain the higher severe Covid-19 risk in diabetes, and highlight the need to develop special treatment protocols for diabetic patients., (© 2021 Wiley Periodicals LLC.)

The COVID-19 pandemic caused by SARS-CoV-2 has lasted for more than two years. Despite the presence of very effective vaccines, the number of virus variants that escape neutralizing antibodies is growing. Thus, there is still a need for effective antiviral treatments that target virus replication independently of the circulating variant. Here, we show for the first time that deficiency or pharmacological inhibition of the cellular lysine-methyltransferase SMYD2 decreases TMPRSS2 expression on both mRNA and protein levels. SARS-CoV-2 uses TMPRSS2 for priming its spike protein to infect target cells. Treatment of cultured cells with the SMYD2 inhibitors AZ505 or BAY598 significantly inhibited viral replication. In contrast, treatment of Vero E6 cells, which do not express detectable amounts of TMPRSS2, had no effect on SARS-CoV-2 infection. Moreover, by generating a recombinant reporter virus that expresses the spike protein of the Delta variant of SARS-CoV-2, we demonstrate that BAY598 exhibits similar antiviral activity against this variant of concern. In summary, SMYD2 inhibition downregulates TMPRSS2 and blocks viral replication. Targeting cellular SMYD2 represents a promising tool to curtail SARS-CoV-2 infection.

Background: Hepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries., Methods: The full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI-MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease., Results: Fusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC 50 values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MS n analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC 50 value of 11.34 ± 3.83 µg/mL., Conclusions: A FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a., (© 2022. The Author(s).)

Understanding genetic causes of hearing loss can determine the pattern and course of a patient's hearing loss and may also predict outcomes after cochlear implantation. Our goal in this study was to evaluate genetic causes of hearing loss in a large cohort of adults and children with cochlear implants. We performed comprehensive genetic testing on all patients undergoing cochlear implantation. Of the 459 patients included in the study, 128 (28%) had positive genetic testing. In total, 44 genes were identified as causative. The top 5 genes implicated were GJB2 (20, 16%), TMPRSS3 (13, 10%), SLC26A4 (10, 8%), MYO7A (9, 7%), and MT-RNR1 (7, 5%). Pediatric patients had a higher diagnostic rate. This study lays the groundwork for future studies evaluating the relationship between genetic variation and cochlear implant performance.

Abstract: Projection neurons of the spinal cord dorsal horn which transmit pain, itch, and temperature information to the brain comprise the anterolateral system (AS). A recent molecular and genetic study showed that many developing AS neurons express the transcription factor Phox2a and provided insights into the mechanisms of their ontogeny and wiring of nociceptive neuronal circuits. Here, we show that the loss of the axonal guidance and neuronal migration signal netrin1 results in impaired migration of mouse Phox2a+ AS neurons into the spinal lamina I. Furthermore, we show that in the absence of Dab1, an intracellular transducer of the neuronal migration signal reelin, the migration of spinal lamina V and lateral spinal nucleus Phox2a+ AS neurons is impaired, in line with deficits in nociception seen in mice with a loss of reelin signaling. Together, these results provide evidence that netrin1 and reelin control the development of spinal nociceptive projection neurons, suggesting a mechanistic explanation for studies that link sequence variations in human genes encoding these neurodevelopmental signals and abnormal pain sensation., (Copyright © 2021 International Association for the Study of Pain.)

Approximately 14.5 million Pakistani individuals have a hearing loss and half of these cases may be due to genetic causes. Though significant progress has been made in uncovering genetic variants for recessively inherited nonsyndromic deafness, Pendred syndrome, and Usher syndromes, the same is not true for dominantly inherited hearing loss, most syndromic cases and deafness with complex inheritance patterns. Variants of 57 genes have been reported to cause nonsyndromic recessive deafness in Pakistan, though most are rare. Variants of just five genes GJB2, HGF, MYO7A, SLC26A4, and TMC1 together explain 57% of profound deafness while those of GJB2, MYO15A, OTOF, SLC26A4, TMC1, and TMPRSS3 account for 47% of moderate to severe hearing loss. In contrast, although variants of at least 39 genes have been implicated in different deafness syndromes, their prevalence in the population and the spectrum of mutations have not been explored. Furthermore, research on genetics of deafness has mostly focused on individuals from the Punjab province and needs to be extended to other regions of Pakistan. Identifying the genes and their variants causing deafness in all ethnic groups is important as it will pinpoint rare as well as recurrent mutations. This information may ultimately help in offering genetic counseling and future treatments., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hearing impairment not etiologically associated with clinical signs in other organs (non-syndromic) is genetically heterogeneous, so that over 120 genes are currently known to be involved. The frequency of mutations in each gene and the most frequent mutations vary throughout populations. Here we review the genetic etiology of non-syndromic hearing impairment (NSHI) in Europe. Over the years, epidemiological data were scarce because of the large number of involved genes, whose screening was not cost-effective until implementation of massively parallel DNA sequencing. In Europe, the most common form of autosomal recessive NSHI is DFNB1, which accounts for 11-57% of the cases. Mutations in STRC account for 16% of the recessive cases, and only a few more (MYO15A, MYO7A, LOXHD1, USH2A, TMPRSS3, CDH23, TMC1, OTOF, OTOA, SLC26A4, ADGRV1 and TECTA) have contributions higher than 2%. As regards autosomal-dominant NSHI, DFNA22 (MYO6) and DFNA8/12 (TECTA) represent the most common forms, accounting for 21% and 18% of elucidated cases, respectively. The contribution of ACTG1 and WFS1 drops to 9% in both cases, followed by POU4F3 (6.5%), MYO7A (5%), MYH14 and COL11A2 (4% each). Four additional genes contribute 2.5% each one (MITF, KCNQ4, EYA4, SOX10) and the remaining are residually represented. X-linked hearing loss and maternally-inherited NSHI have minor contributions in most countries. Further knowledge on the genetic epidemiology of NSHI in Europe needs a standardization of the experimental approaches and a stratification of the results according to clinical features, familial history and patterns of inheritance, to facilitate comparison between studies., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Until now, the detection methods for serine proteases have been quite time-consuming or cannot indicate the "real" protease activity. Here, a rapid and simple method for determining the "real" activity of serine proteases toward AAPX (a kind of mixed polypeptide substrates, with X representing 20 standard amino acids) was developed. This AAPX method has high reliability, sensitivity, and repeatability and can be used for detecting the serine protease activity spectrophotometrically. Additionally, the site-directed saturation mutagenesis library of alkaline serine protease PRO (BcPRO) from Bacillus clausii was screened with this AAPX method. Three beneficial mutants S99R, S99H, and S99W were identified, and S99W displayed the highest activity. In comparison to wild-type BcPRO, S99W exhibited enhanced catalytic performance toward eight AAPX monomers, and the molecular dynamics simulation revealed the mechanism responsible for its improved activity toward AAPM. Consequently, this work provides an efficient method for detecting, characterizing, mining, and high-throughput screening of serine proteases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to have devastating consequences worldwide. Recently, great efforts have been made to identify SARS-CoV-2 host factors, but the regulatory mechanisms of these host molecules, as well as the virus per se, remain elusive. Here we report a role of RNA G-quadruplex (RG4) in SARS-CoV-2 infection. Combining bioinformatics, biochemical and biophysical assays, we demonstrate the presence of RG4s in both SARS-CoV-2 genome and host factors. The biological and pathological importance of these RG4s is then exemplified by a canonical 3-quartet RG4 within Tmprss2, which can inhibit Tmprss2 translation and prevent SARS-CoV-2 entry. Intriguingly, G-quadruplex (G4)-specific stabilizers attenuate SARS-CoV-2 infection in pseudovirus cell systems and mouse models. Consistently, the protein level of TMPRSS2 is increased in lungs of COVID-19 patients. Our findings reveal a previously unknown mechanism underlying SARS-CoV-2 infection and suggest RG4 as a potential target for COVID-19 prevention and treatment., (© 2022. The Author(s).)

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2 + human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.

The emergence of SARS-CoV-2 variants of concern with progressively increased transmissibility between humans is a threat to global public health. The Omicron variant of SARS-CoV-2 also evades immunity from natural infection or vaccines 1 , but it is unclear whether its exceptional transmissibility is due to immune evasion or intrinsic virological properties. Here we compared the replication competence and cellular tropism of the wild-type virus and the D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants in ex vivo explant cultures of human bronchi and lungs. We also evaluated the dependence on TMPRSS2 and cathepsins for infection. We show that Omicron replicates faster than all other SARS-CoV-2 variants studied in the bronchi but less efficiently in the lung parenchyma. All variants of concern have similar cellular tropism compared to the wild type. Omicron is more dependent on cathepsins than the other variants of concern tested, suggesting that the Omicron variant enters cells through a different route compared with the other variants. The lower replication competence of Omicron in the human lungs may explain the reduced severity of Omicron that is now being reported in epidemiological studies, although determinants of severity are multifactorial. These findings provide important biological correlates to previous epidemiological observations., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Treatments for coronavirus disease 2019, which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through interactions of its spike (S) protein with angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. We found that airway OM-85 administration inhibits Ace2 and Tmprss2 transcription in the mouse lung, suggesting that OM-85 might hinder SARS-CoV-2/host cell interactions., Objectives: We sought to investigate whether and how OM-85 treatment protects nonhuman primate and human epithelial cells against SARS-CoV-2., Methods: ACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein-pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung, and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2-transfected HEK293T cells treated with OM-85 in vitro., Results: OM-85 significantly downregulated ACE2 and TMPRSS2 transcription and surface ACE2 protein expression in epithelial cell lines and primary bronchial epithelial cells. OM-85 also strongly inhibited SARS-CoV-2 S1 protein binding to, SARS-CoV-2 S protein-pseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on SARS-CoV-2 receptor downregulation., Conclusions: OM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of coronavirus disease 2019., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Case report of malignant pleural mesothelioma with an ALK gene rearrangement, detected by FISH and confirmed by RNA-based next-generation sequencing. The co-occurrence of ALK gene fusions with the more common genetic alterations in CDKN2A, NF2 and BAP1 has, to our best knowledge, not yet been described in malignant mesothelioma. Furthermore, this unexpected finding could suggest a potential target for therapy in this subset of malignant mesotheliomas., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

SARS-CoV-2, the causative agent for COVID-19, an ongoing pandemic, engages the ACE2 receptor to enter the host cell through S protein priming by a serine protease, TMPRSS2. Variation in the TMPRSS2 gene may account for the disparity in disease susceptibility between populations. Therefore, in the present study, we have used next-generation sequencing (NGS) data of world populations from 393 individuals and analyzed the TMPRSS2 gene using a haplotype-based approach with a major focus on South Asia to study its phylogenetic structure and their haplotype sharing among various populations worldwide. Our analysis of phylogenetic relatedness showed a closer affinity of South Asians with the West Eurasian populations therefore, host disease susceptibility and severity particularly in the context of TMPRSS2 will be more akin to West Eurasian instead of East Eurasian. This is in contrast to our prior study on the ACE2 gene which shows South Asian haplotypes have a strong affinity towards West Eurasians. Thus ACE2 and TMPRSS2 have an antagonistic genetic relatedness among South Asians. Considering the significance of the TMPRSS2 gene in the SARS-CoV-2 pathogenicity, COVID-19 infection and intensity trends could be directly associated with increased expression therefore, we have also tested the SNPs frequencies of this gene among various Indian state populations with respect to the case fatality rate (CFR). Interestingly, we found a significant positive association between the rs2070788 SNP (G Allele) and the CFR among Indian populations. Further our cis eQTL analysis of rs2070788 shows that the GG genotype of the rs2070788 tends to have a significantly higher expression of TMPRSS2 gene in the lung compared to the AG and AA genotypes thus validating the previous observation and therefore it might play a vital part in determining differential disease vulnerability. We trust that this information will be useful in understanding the role of the TMPRSS2 variant in COVID-19 susceptibility and using it as a biomarker may help to predict populations at risk., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Aged males disproportionately succumb to increased COVID-19 severity, hospitalization, and mortality compared to females. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) facilitate SARS-CoV-2 viral entry and may have sexually dimorphic regulation. As viral load dictates disease severity, we investigated the expression, protein levels, and activity of ACE2 and TMPRSS2. Our data reveal that aged males have elevated ACE2 in both mice and humans across organs. We report the first comparative study comprehensively investigating the impact of sex and age in murine and human levels of ACE2 and TMPRSS2, to begin to elucidate the sex bias in COVID-19 severity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

RHBDD1 overexpression is found in various malignancies, including non-small cell lung cancer (NSCLC), and it is correlated with NSCLC patients' poor overall survival. This study aims to explore the function of RHBDD1 in regulating the progression of NSCLC and its potential molecular basis. qPCR, immunohistochemistry, and/or western blotting were used to evaluate the expression of RHBDD1 in NSCLC tissues and cell lines. RHBDD1 knockdown and overexpression were performed, CCK-8 assay and cell clone formation were applied to study the function of RHBDD1 in cell proliferation in vitro. Flow cytometry and immunofluorescence tests were employed to determine the regulation of apoptosis, cell cycle, and endoplasmic reticulum stress by RHBDD1. As a result, RHBDD1 was found significantly upregulated in NSCLC tissues and cells and associated with pathological tumor staging. RHBDD1 knockdown inhibited the proliferation of NSCLC cells both in vitro and in vivo, promoted their apoptosis, caused cell cycle arrest at G0/G1 phase, characterized with reduced CDK2, suppressed TGF-α secretion, and inhibited the EGFR/Raf/MEK/ERK signaling pathway. In contrast, RHBDD1 overexpression showed the opposite effects. These effects of the manipulated expression of RHBDD1 on NSCLC were restored by EGFR or MEK inhibitor. Additionally, RHBDD1 knockdown and overexpression resulted in decreased and increased BIK cleavage, respectively, but the effects could be blocked by a proteasome inhibitor. In conclusion, our research shows that RHBDD1 promotes the progression of NSCLC through enhancement of proliferation and induction of apoptosis by regulating the EGFR/Raf/MEK/ERK signaling pathway and the level of BIK protein level.

Background: The severity of COVID-19 after SARS-CoV-2 infection is unpredictable. Angiotensin-converting enzyme-2 (ACE2) is the receptor responsible for coronavirus binding, while subsequent cell entry relies on priming by the serine protease TMPRSS2 (transmembrane protease, serine 2). Although renin-angiotensin-aldosterone-system (RAAS) blockers have been suggested to upregulate ACE2, their use in COVID-19 patients is now considered well tolerated. The aim of our study was to investigate parameters that determine COVID-19 severity, focusing on RAAS-components and variation in the genes encoding for ACE2 and TMPRSS2., Methods: Adult patients hospitalized due to SARS-CoV-2 infection between May 2020 and October 2020 in the Haga Teaching Hospital were included, and soluble ACE2 (sACE2), renin, aldosterone (in heparin plasma) and polymorphisms in the ACE2 and TMPRSS2 genes (in DNA obtained from EDTA blood) were determined., Measurements and Main Results: Out of the 188 patients who were included, 60 were defined as severe COVID-19 (ICU and/or death). These patients more often used antidiabetic drugs, were older, had higher renin and sACE2 levels, lower aldosterone levels and a lower aldosterone/renin ratio. In addition, they displayed the TMPRSS2-rs2070788 AA genotype less frequently. No ACE2 polymorphism-related differences were observed. Multivariate regression analysis revealed independent significance for age, sACE2, the aldosterone/renin ratio, and the TMPRSS2 rs2070788 non-AA genotype as predictors of COVID-19 severity, together yielding a C-index of 0.79. Findings were independent of the use of RAAS blockers., Conclusion: High sACE2, a low aldosterone/renin ratio and having the TMPRSS2 rs2070788 non-AA genotype are novel independent determinants that may help to predict COVID-19 disease severity., Trial Registration: retrospectively registered., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Objective: To determine the effect of polymorphisms and mutations in angiotensin-converting enzyme 2 (ACE2) and Type 2 transmembrane serine proteases (TMPRSS2) genes on susceptibility to corona virus disease 2019 (COVID-19) and patient prognosis., Introduction: From December 2019 to the current time, an outbreak of epidemic of COVID-19, characterized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred around the world. It is now clear that SARS-CoV-2 binds to human ACE2 receptors, with expression of these receptors correlated with the rate of SARS-CoV-2 infection and mortality. Polymorphisms in individual patient factors, such as ACE2 and TMPRSS2 genes have been linked with an increase in negative outcomes, although evidence to affirm remains debatable., Methods: Here, we performed a systematic review, based on guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria, with the aim of assessing whether polymorphisms in ACE2 and TMPRSS2 genes affect the COVID-19 condition. We extensively searched PubMed, MEDLINE, Embase, the Cochrane Library, and Web of Science databases, for relevant articles and reports published in English between December 2019 and December 2021., Results: A total of 495 full-text articles were downloaded, of which 185 were excluded after preliminary examination as they were duplicates. Finally, 310 articles were evaluated, by reading their titles and abstracts, and 208 of them eliminated based on our selection criteria. Finally, 33 articles met our inclusion criteria and were included in the final assessment. Genetic data from 33,923 patients with COVID-19 drawn from the general population and deriving from over 160 regions and 50 countries, as well as approximately 560,000 samples from global-public genetic databases, were included in our analysis. Ultimately, we identified 10 SNPs and 21 mutations in the ACE2 gene, along with 13 SNPs and 12 variants in the TMPRSS2 gene, which may be associated with COVID-19., Conclusions: ACE2 and TMPRSS2 play vital roles in the onset, development, and prognosis of SARS-CoV-2 infection, and have both been strongly associated with vulnerability, intensity, and the clinical result of COVID-19. Overall, these genetic factors may have potential for future development of personalized drugs and vaccines against COVID-19., Trial Registration: CRD42021239400 in PROSPERO 2021., (© 2022. The Author(s).)

Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus-host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine's optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC 50 of 1.2 µM and EC 90 of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine's pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine's chemical structure could represent an orally available host-acting agent to inhibit virus entry.

Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the outcome of coronavirus disease 2019 (COVID-19) have been linked to underlying health conditions and the age of affected individuals. Here, we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18- to 39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is ∼32 PFU per animal, while in young animals it was estimated to be ∼100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. Similar to observations in humans, this was associated with higher transcription levels of two key viral entry factors, ACE2 and TMPRSS2, in the upper respiratory tract of aged ferrets. IMPORTANCE In humans, ACE2 and TMPRSS2 are expressed in various cells and tissues, and differential expression has been described in young and old people, with a higher level of expressing cells being detected in the nasal brushing of older people than young individuals. We described the same pattern occurring in ferrets, and we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2. Aged animals were more likely to get infected when exposed to lower infectious dose of the virus than young animals, and the viral replication in the upper respiratory tract and shedding are enhanced in aged ferrets. Together, these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated, at least in part, with transcription levels of ACE2 and TMPRSS2 at the sites of virus entry. The young and aged ferret model developed here may represent a great platform to assess age-related differences in SARS-CoV-2 infection dynamics and replication.

Pathogenic TMPRSS6 variants impairing matriptase-2 function result in inappropriately high hepcidin levels relative to body iron status, leading to iron refractory iron deficiency anemia (IRIDA). As diagnosing IRIDA can be challenging due to its genotypical and phenotypical heterogeneity, we assessed the transferrin saturation (TSAT)/hepcidin ratio to distinguish IRIDA from multi-causal iron deficiency anemia (IDA). We included 20 IRIDA patients from a registry for rare inherited iron disorders and then enrolled 39 controls with IDA due to other causes. Plasma hepcidin-25 levels were measured by standardized isotope dilution mass spectrometry. IDA controls had not received iron therapy in the last 3 months and C-reactive protein levels were <10.0 mg/L. IRIDA patients had significantly lower TSAT/hepcidin ratios compared to IDA controls, median 0.6%/nM (interquartile range, IQR, 0.4-1.1%/nM) and 16.7%/nM (IQR, 12.0-24.0%/nM), respectively. The area under the curve for the TSAT/hepcidin ratio was 1.000 with 100% sensitivity and specificity (95% confidence intervals 84-100% and 91-100%, respectively) at an optimal cut-off point of 5.6%/nM. The TSAT/hepcidin ratio shows excellent performance in discriminating IRIDA from TMPRSS6 -unrelated IDA early in the diagnostic work-up of IDA provided that recent iron therapy and moderate-to-severe inflammation are absent. These observations warrant further exploration in a broader IDA population.

The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte's bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated β-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted β-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown., (© 2022. The Author(s).)

Background: There are limited effective prophylactic/early treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Viral entry requires spike protein binding to the angiotensin-converting enzyme-2 receptor and cleavage by transmembrane serine protease 2 (TMPRSS2), a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is in clinical trials in early SARS-CoV-2 infection., Methods: We used differentiated primary human airway epithelial cells at the air-liquid interface to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection., Results: We first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-CoV-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, oral camostat is rapidly metabolised in the circulation, with poor airway bioavailability. We therefore modelled local airway administration by applying camostat to the apical surface of differentiated airway cultures. We demonstrated that a brief exposure to topical camostat effectively restricts SARS-CoV-2 infection., Conclusion: These experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial., (© 2022 Guo et al.)

Purpose: The aim of this study was to explore whether there are interactions between genetic (ARMS2/HTRA1) and environmental factors (cigarette smoking) in the pathogenesis of age-related macular degeneration (AMD)., Methods: Primary human retinal pigment epithelial (hRPE) cells were obtained from four donors' eyes with AMD high-risk ARMS2/HTRA1 alleles, and two donors' eyes with wild-type alleles were used as controls. The pooled serum from 32 smokers and 35 nonsmokers were collected and used separately to treat hRPE cells. The isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics was used to identify associated proteins and comparing the differences between AMD high-risk and low-risk HTRA1/ARMS2 alleles after exposure to smokers' serum., Results: After stimulation with the smokers' serum, 400 differentially expressed proteins (DEPs) were detected in the high-risk allele cells. Several DEPs are involved in neuronal protein degeneration and oxidative stress pathways. The smokers' serum stimulation or HTRA1 overexpression can both upregulate caveolin-1, which was one of the DEPs. Besides, the smokers' serum enhanced the phagocytosis of cultured human RPE cells., Conclusions: The study confirmed the AMD high-risk alleles, HTRA1, and cigarette smoking can promote AMD development by regulating caveolin-1 expression., Translational Relevance: AMD high-risk alleles and environmental risk factors can promote the occurrence and development of AMD by regulating caveolin-1 expression, upregulation of which will induce apoptotic cell death in response to cellular stress in early AMD conditions.