Transmembrane serine protease 4 expression in the prognosis of radical resection for biliary tract cancer

BACKGROUND Recent advancements in biliary tract cancer(BTC)treatment have expanded beyond surgery to include adjuvant therapy,yet the prognosis remains poor.Identifying prognostic biomarkers could enhance the assessment of patients who have undergone radical resection for BTC.AIM To determine transmembrane serine protease 4(TMPRSS4)utility as a prognostic biomarker of radical resection for BTC.METHODS Medical records of patients who underwent radical resection for BTC,excluding intrahepatic cholangiocarcinoma,were retrospectively reviewed.The associations between TMPRSS4 expression and clinicopathological factors,overall survival,and recurrence-free survival were analyzed.RESULTS Among the 85 patients undergoing radical resection for BTC,46(54%)were TMPRSS4-positive.The TMPRSS4-positive group exhibited significantly higher preoperative carbohydrate antigen 19-9(CA19-9)values and greater lymphatic invasion than the TMPRSS4-negative group(P=0.019 and 0.039,respectively).Postoperative overall survival and recurrence-free survival were significantly worse in the TMPRSS4-positive group(median survival time:25.3 months vs not reached,P<0.001;median survival time:28.7 months vs not reached,P=0.043,respectively).Multivariate overall survival analysis indicated TMPRSS4 positivity,pT3/T4,and resection status R1 were independently associated with poor prognosis(P=0.032,0.035 and 0.030,respectively).TMPRSS4 positivity correlated with preoperative CA19-9 values≥37 U/mL and pathological tumor size≥30 mm(P=0.016 and 0.038,respectively).CONCLUSION TMPRSS4 is a potential prognostic biomarker of radical resection for BTC.

Characteristics of Mycobacterium tuberculosis serine protease Rv1043c in enzymology and pathogenicity in mice

The serine proteases of Mycobacteria tuberculosis(Mtb)are important contributors to the process of bacterial invasion and its pathogenesis.In the present study,we systematically characterized the role of the Rv1043c protein in Mycobacterium infection by purifying the Rv1043c protein in Escherichia coli and constructing a Mycobacterium smegmatis(Msg)strain overexpressing Rv1043c(Msg_Rv1043c).We found that Rv1043c had serine protease activity and localized to the surface of Mtb.We determined that the optimal pH and temperature for the Rv1043c serine protease were 9.0 and 45°C,respectively.Moreover,the serine protease activity of Rv1043c was enhanced by divalent metal ions of Ca^(2+)and Mg^(2+).Site-directed mutagenesis studies demonstrated that the serine 279 residue in Rv1043c plays a catalytic role.Additionally,mouse model studies confirmed that Rv1043c significantly enhanced the survival of Msg in vivo,induced pulmonary injury and lung cell apoptosis,and promoted the release of pro-inflammatory cytokines interleukin-1βand interleukin-6 in mice.This study presents novel insights into the relationship between mycobacterial serine protease and the pathogenesis of the disease.

Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis(Meyen)via the Toll pathway

Female adults of the migratory locust,Locusta migratoria manilensis(Meyen),can sense seasonal photoperiod changes,which induces embryonic diapause as a key strategy to overwinter.Serine protease inhibitor genes(SPNs)were thought to play key roles during diapause,while few SPNs were functionally characterized.LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs;however,its biological function remained to be explored.So,we conducted RNAi knockdown of LmSPN2,resulting in a significant decrease of the egg diapause rate by 29.7%.Using yeast two-hybrid assays,co-immunoprecipitation,and pull-down methods,we found an interaction between LmSPN2 and LmSPN3,which was proved to be mediated by a glutamate(E331)binding site of LmSPN2.RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%,indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation.Double knockdown of two SPN genes resulted in a 26.4%reduction in diapause rate,indicating that LmSPN2 was the dominant regulatory signal.Moreover,we found four Toll pathway genes(easter,spätzle,pelle,and dorsal)upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3.Therefore,we speculate that two SPNs regulate diapause through the Toll pathway.Our results indicated that LmSPN2 positively regulates locust egg entry into diapause,while LmSPN3 is a negative regulator of embryonic commitment to diapause.Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway.This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population:Role of serine protease inhibitor Kazal 1type and alcohol

AIM： To test the hypothesis that calcium sensing receptor （CASR） polymorphisms are associated with chronic pancreatitis （CP）, and to determine whether serine protease inhibitor Kazal 1type （SPINK1） N34S or alcohol are necessary co-factors in its etiology. METHODS： Initially, 115 subjects with pancreatitis and 66 controls were evaluated, of whom 57 patients and 21 controls were predetermined to carry the high-risk SPINK1 N34S polymorphism. We sequenced CASR gene exons 2, 3, 4, 5 and 7, areas containing the majority of reported polymorphisms and novel mutations. Based on the initial results, we added 223 patients and 239 controls to analyze three common nonsynonymous single nucleotide polymorphisms （SNPs） in exon 7 （A986S, R990G, and Q1011E）. RESULTS： The CASR exon 7 R990G polyrnorphism was significantly associated with CP （OR, 2.01; 95% CI, 1.12-3.59; P = 0.015）. The association between CASR R990G and CP was stronger in subjects who reported moderate or heavy alcohol consumption （OR, 3.12; 95% CI, 1.14-9.13; P = 0.018）. There was no association between the various CASR genotypes and SPINK1 N34S in pancreatitis. None of the novel CASR polymorphisms reported from Germany and India was detected. CONCLUSION： Our United States-based study confirmed an association of CASR and CP and for the first time demonstrated that CASR R990G is a significant risk factor for CP. We also conclude that the risk of CP with CASR R990G is increased in subjects with moderate to heavy alcohol consumption.

Serine protease HtrA1 expression in human hepatocellular carcinoma

BACKGROUND: HtrA1, a serine protease, is down-regulated in various human solid tumors. Overexpression of HtrA1 in human cancer cells inhibits cell growth and proliferation in vitro and in vivo, suggesting its possible role as a tumor suppressor. METHODS: Immunohistochemistry was used to determine the expression of HtrA1 in 50 hepatocellular carcinoma specimens and adjacent liver tissues. The correlation between the expression of HtrA1 and the clinico-pathologic data were analyzed. RESULTS: The levels of HtrA1 were lower in tumor tissues than in their adjacent liver tissues. Moreover, an inverse relationship was found between HtrA1 expression and the differentiation of hepatocellular carcinoma. Loss of HtrA1 was more frequently found in tumors in Edmondson grade especially in those with venous invasion, compared to tumors in Edmondson grade I-II. Most importantly, patients with higher HtrA1 expression had a better survival rate. CONCLUSION: All these data suggest an important role of HtrA1 in hepatocellular carcinoma development and progression, which may be a new target for its treatment.

Effects of Dietary Soybean Stachyose and Phytic Acid on Gene Expressions of Serine Proteases in Japanese Flounder(Paralichthys olivaceus)

Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

Cloning,Expression and Activity Analysis of a Bacterial Serine Protease

In the study, a serine protease gene from Pantoea ananatis was cloned and expressed in prokaryotic cells. The activity of recombinant serine protease was analyzed. The results showed that the recombinant serine protease gene was 1 062 bp, encoding 352 amino acids ; the optimal reaction temperature for recombinant serine protease was 50 ℃, and the optimal pH was 5. 0. The serine protease could be developed into a new tool enzyme in biological engineering and food processing.

Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation

Objective： To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus, and to analyze their serine protease activities. Methods： The point mutation plasmid constructs were generated by using the QuickChange site-directed mutagenesis kit with the backbone of M-H05-5 （AI-1）, and were named as subgroup A1-2, A2-1, A2-2, BI-1, B1-2, B2-1, and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results： The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The immunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/SBAC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion： The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different secondary structure of the 120-aa sequence at N-terminus of NS3.

Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease from Arenicola cristata

The full-length c DNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of c DNA ends technique and sequenced. The size of the c DNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity(< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

Transmembrane serine protease 2 and angiotensin-converting enzyme 2 anti-inflammatory receptors for COVID-19/inflammatory bowel diseases treatment

long-term,and relapsing inflammatory disorders.IBD may spontaneously grow in the colon,and in severe cases may result in tumor lesions such as invasive carcinoma in inflamed regions of the intestine.Recent epidemiological reports indicate that old age and underlying diseases such as IBD contribute to severity and mortality in patients with coronavirus disease 2019(COVID-19).Currently,the ongoing COVID-19 pandemic caused serious morbidity and mortality worldwide.It has also been shown that the transmembrane serine protease 2 is an essential factor for viral activation and viral engulfment.Generally,viral entry causes a'cytokine storm'that induces excessive generation of proinflammatory cytokines/chemokines including interleukin(IL)-6,IL-2,IL-7,tumor necrosis factor-α,and interferon-γ.Future research could concentrate on developing inflammatory immunological responses that are efficient to encounter COVID-19.Current analysis elucidates the role of inflammation and immune responses during IBD infection with COVID-19 and provides a list of possible targets for IBD-regulated therapies in particular.Data from clinical,in vitro,and in vivo studies were collected in English from PubMed,Google Scholar,Scopus,and the Cochrane library until May 2021.

The Role of Hydrogen Bond in Catalytic Triad of Serine Proteases

In order to investigate the origin of catalytic power for serine proteases,the role of the hydrogen bond in the catalytic triad was studied in the proteolysis process of the peptides chymotrypsin inhibitor 2(CI2),MCTI-A,and a hexapeptide(SUB),respectively.We first calculated the free energy profile of the proton transfer between His and Asp residues of the catalytic triad in the enzyme-substrate state and transition state by employing QM/MM molecular dynamics simulations.The results show that a low-barrier hydrogen bond(LBHB)only forms in the transition state of the acylation of CI2,while it is a normal hydrogen bond in the acylation of MCTI-A or SUB.In addition,the change of the hydrogen bond strength is much larger in CI2 and SUB systems than in MCTI-A system,which decreases the acylation energy barrier significantly for CI2 and SUB.Clearly,a LBHB formed in the transition state region helps accelerate the acylation reaction.But to our surprise,a normal hydrogen bond can also help to decrease the energy barrier.The key to reducing the reaction barrier is the increment of hydrogen bond strength in the transition state state,whether it is a LBHB or not.Our studies cast new light on the role of the hydrogen bond in the catalytic triad,and help to understand the catalytic triad of serine proteases.

Expressions and clinical significances of mannose-binding lectin(MBL) and MBL-associated serine protease 2(MASP-2) in patients with thyroid neoplasm

Objective： The aim of the study was to detect the levels of mannose-binding lectin （MBL）, MBL-associated serine protease 2 （MASP-2） and explore the clinical significances of them in patients with primary thyroid neoplasms. Methods： By using ELISA method, we detected the serum levels of MBL and MASP-2 in 26 patients with papillary thyroid carcinoma （PTC）, 30 patients with thyroid adenoma （TA） and 26 healthy people, respectively. Results： Serum MBL level was （565.23 ± 76.70） μg/L in PTCs higher than （324.267 ±24.74） μg/L in TAs, and （152.69± 16.95） IJg/L in healthy of controlling group. There was statistical significance between PTC and TA （P 〈 0.05）, however there was no difference between TA and healthy （P 〉 0.05）. Serum MASP-2 level was （726.153± 78.88） pg/L in PTCs higher than （379.266 ± 30.26） μg/L in TAs, and （203.846 ± 29.09） μg/L in healthy. Serum MASP-2 level was higher in PTCs than TAs, and the difference had statistical significance （P 〈 0.01）. But no difference was observed between in TAs and healthy. Conclusion： These findings might reflect inflammatory processes induced by defense mechanisms, in response to the development of the turnout. MBL may also be involved in the elimination of possible tumourigenic pathogens.

Crystal Structures of the Serine Protease Domain of Murine Plasma Kallikrein

Plasma kallikrein（PK）, a serine protease in the trypsin clan（SA）, plays critical roles in many physiological and pathological pathways. Regulating the abnormal activity of PK has been successfully used in the clinical therapy of hereditary angioedema. In this study, the serine protease domain of murine plasma kallikrein（m PK） was expressed in the pichia pastoris system. The recombinant protein was a glycosylated active enzyme after purification by the cation exchange and size-exclusion chromatography, and was crystallized at the precipitant condition of 25% PEG 3350, 0.1 M Tris-HCl pH 8.5 and 0.1 M Na Cl. The crystal structure of m PK was determined at 2.6 . This is the first published crystal structure of m PK, showing some distinctive features at S2＇ and S3＇ pockets when compared to its human analogue（human plasma kallikrein, h PK）. In addition, m PK show unique structural features in the non-conservative 67-72 and 76-81 loops when compared to other serine proteases. These results provide insights for the design of potent and selective PK inhibitors.

A novel serine protease SNC19 associated with human colorectal cancer

Abstract:Objective To study the structure and function of a novel serine protease gene associated with human colorectal cancer SNC19.Methods The cDNA sequence was determined by both manual and automatic sequencing techniques. The full length cDNA sequence was obtained by the 5'-Rapid Amplification of cDNA Ends technique and web-based analysis. Open reading frame analysis and protein function prediction were also performed. Northern blot was used to detect the expression of SNC19 in various human normal tissues and tumor cell lines. Fluorescent in situ hybridization combined with fluorescent R-banding technique was employed to map the SNC19 gene on human chromosome.Results Full length SNC19 cDNA, size 3152 bp, encodes a protein highly homologous to a mouse serine protease epithin. In normal human tissues, high SNC19 expression levels were observed in the kidney, pancreas, prostate, small intestine and colon; moderate SNC19 expression levels were observed in the placenta, lung, liver, spleen thymus, testis and peripheral blood lymphocytes; and extremely low expression levels were observed in the heart, brain, skeletal muscle and ovary. In tumor cell lines, colorectal cancer cells SW480, SW620, SW1116 and Colo205, breast cancer cell Bcap37 and gastric cancer cells MKN28 and SGC7901 showed high levels of SNC19 expression; cervical cancer cell HeLa-S3, lung cancer PAA, oral epithelial cancer cell KB and lymphoma cell Raji showed moderate levels of SNC19 expression; and tongue squamous cancer cell Tca8113, leukemia cells HL-60, K562, MOLT-4, lung cancer cell A549 and melanoma cell G361 showed very low levels of SNC19 expression. SNC19 was mapped to human chromosome 11q24-25. Conclusion SNC19 encodes a novel human serine protease with 855 amino acid residues. As a novel serine protease associated with human colorectal cancer, the expression of SNC19 in various tissues and cell lines may have very important impact on their phenotypes and biological behaviors.

A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori

Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells ofBombyx mori. BmSP36 contains an intact catalytic triad （H57, D102 and S195） and a conserved substrate-binding site （G189, H216 and G226）, suggesting that it is a serine protease with chymotrypsin- like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down- regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development.

Identification of two clip domain serine proteases involved in the pea aphid's defense against bacterial and fungal infection

Phenoloxidases(POs)are required for the pea aphid s defense against bacterial and fungal infection.Prophenoloxidases(PPOs)are protcolytically converted to its active form PO through a clip domain serine protease cascade.In this study,we identified five clip domain serine proteases in the pea aphids.The messenger RNA levels of two of them,Ap.SPLP and Ap_VP,were upregulated by Gram-positive bacterium Staphylococcus aureus and fungus Beauveha bassiana infections.Double-stranded RNA-based expression knockdown of these two genes resulted in reduced PO activity of the aphid hemolymph,higher loads of S.aureus and B.bassiana in the aphids,and lower survival rates of the aphids after infections.Our data suggest that Ap.SPLP and Ap_VP are involved in PPO activation pathway in the pea aphid.

Cloning, expression and localization of a trypsin-like serine protease in the spruce budworm, Choristoneura fumiferana

A trypsin-like molting-related serine protease cDNA （CfMRSP） was cloned from the spruce budworm, Choristoneurafumiferana. The full-length CfMRSP complementary DNA （cDNA） encoded a 43 kDa protein that contained a trypsin-like serine protease catalytic domain, but no clip domain. The C-terminal extension contained five cystein residues, which may allow the protein to form a homodimer through interchain disulfide bonds and regulate the activity of CfMRSE Phylogenetic tree analysis showed that CfMRSP clusters with lepidopteran homologues such as serine protease 1 of Lonomia obliqua, hemolymph proteinase 20 （HP20）, pattern recognition serine proteinase precursor （ProHP14） and a trypsin-like protein ofManduca sexta. Northern blot analysis of developmental expression of CfMRSP indicated that its transcripts were found primarily in the epidermis and were produced during all of the tested stadia, from 4th instar larvae to pupae, but increased levels of CfMRSP transcripts were always found after each molt. A high level of the protein was found in the epidermis by immunohistochemistry analysis. Altogether these data suggest that CfMRSP plays a role in the epidermis during molting and metamorphosis.

Homeodomain proteins POU-M2, antennapedia and abdominal-B are involved in regulation of the segment-specific expression of the clip-domain serine protease gene CLIP13 in the silkworm, Bombyx mori

Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development. Our previous studies indicated that CLIP13, an epidermis-specific gene, was involved in cuticle remodeling during molting and metamorphosis in the silkworm, Bombyx mori. However, the transcriptional regulatory mechanism and regulatory pathways of CLIP13 remained unclear. In the present study, we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm. At the transcriptional level, expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis;homeodomain transcription factors POU-M2, antennapedia (Antp), and abdominal-B (Abd-B) showed similar expression change trends as CLIP13 in the head capsule, thorax, and abdomen, respectively. Furthermore, results of cell transfection assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation demonstrated that POU-M2, Antp, and Abd-B were involved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter. RNA interference-mediated silencing of POU-M2, Antp, and Abd-B led to a decrease of CLIP13 expression in the head capsule, the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments, respectively. Consistent with CLIP13, 20E treatment significantly upregulated expression of POU-M2, Antp, and Abd-B in the silkworm epidermis. Taken together, these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2, Antp, and Abd-B in different regions of the silkworm epidermis during metamorphosis, thus affecting the molting process. Our findings provide new insight into the functions of homeodomain transcription factors in insect molting.

Functional interplay among the flavivirus NS3 protease, helicase, and cofactors

Flaviviruses are positive-sense RNA viruses, and many are important human pathogens. Nonstructural protein 2B and 3 of the flaviviruses(NS2BNS3) form an endoplasmic reticulum(ER) membrane-associated hetero-dimeric complex through the NS2B transmembrane region. The NS2BNS3 complex is multifunctional. The N-terminal region of NS3, and its cofactor NS2B fold into a protease that is responsible for viral polyprotein processing, and the C-terminal domain of NS3 possesses NTPase/RNA helicase activities and is involved in viral RNA replication and virus particle formation. In addition, NS2BNS3 complex has also been shown to modulate viral pathogenesis and the host immune response. Because of the essential functions that the NS2BNS3 complex plays in the flavivirus life cycle, it is an attractive target for antiviral development. This review focuses on the recent biochemical and structural advances of NS2BNS3 and provides a brief update on the current status of drug development targeting this viral protein complex.

Exploring the potential mechanisms of impairment on genitourinary system associated with coronavirus disease 2019 infection:Bioinformatics and molecular simulation analyses

Objective:The novel coronavirus(severe acute respiratory syndrome coronavirus 2)has been spreading worldwide since December 2019,posing a serious danger to human health and socioeconomic development.A large number of clinical trials have revealed that coronavirus disease 2019(COVID-19)results in multi-organ damage including the urogenital system.This study aimed to explore the potential mechanisms of genitourinary damage associated with COVID-19 infection through bioinformatics and molecular simulation analysis.Methods:We used multiple publicly available databases to explore the expression patterns of angiotensin-converting enzyme 2(ACE2),transmembrane serine protease 2(TMPRSS2),and CD147 in major organs in the healthy and disease-specific populations,particularly the genitourinary organs.Single-cell RNA sequencing was used to analyze the cell-specific expression patterns of ACE2,TMPRSS2,CD147,cytokine receptors,and cytokine interacting proteins in genitourinary organs,such as the bladder,kidney,prostate,and testis.Additionally,gene set enrichmentanalysis was used to investigate the relationship between testosterone levels and COVID-19 vulnerability in patients with prostate cancer.Results:The results revealed that ACE2,TMPRSS2,and CD147 were highly expressed in normal urogenital organs.Then,they were also highly expressed in multiple tumors and chronic kidney diseases.Additionally,ACE2,TMPRSS2,and CD147 were significantly expressed in a range of cells in urogenital organs according to single-cell RNA sequencing.Cytokine receptors and cytokine interacting proteins,especially CCL2,JUN,and TIMP1,were commonly highly expressed in urogenital organs.Finally,gene set enrichment analysis results showed that high testosterone levels in prostate cancer patients were significantly related to the JAK-STAT signaling pathway and the Toll-like receptor signaling pathway which were associated with COVID-19.Conclusion:Our study provides new insights into the potential mechanisms of severe acute respiratory syndrome coronavirus 2 damage to urogenital organs from multiple perspectives,which may draw the attention of urologists to COVID-19 and contribute to the development of targeted drugs.