Transmembrane Serine Protease Research Articles

A functional interplay between the two BMP-SMAD pathway inhibitors TMPRSS6 and FKBP12 regulates hepcidin expression in vivo.

The Activin A Receptor type I (ALK2) is a critical component of BMP-SMAD signaling that, in the presence of ligands, phosphorylates cytosolic SMAD1/5/8 and modulates important biological processes, including bone formation and iron metabolism. In hepatocytes, the BMP-SMAD pathway controls the expression of hepcidin, the liver peptide hormone that regulates body iron homeostasis via the BMP receptors ALK2 and ALK3, and the hemochromatosis proteins. The main negative regulator of the pathway in the liver is transmembrane serine protease 6 (TMPRSS6), which downregulates hepcidin by cleaving the BMP coreceptor hemojuvelin. ALK2 function is inhibited also by the immunophilin FKBP12, which maintains the receptor in an inactive conformation. FKBP12 sequestration by tacrolimus or its silencing upregulates hepcidin in primary hepatocytes and in vivo in acute but not chronic settings. Interestingly, gain-of-function mutations in ALK2 that impair FKBP12 binding to the receptor and activate the pathway cause a bone phenotype in patients affected by Fibrodysplasia Ossificans Progressiva but not hepcidin and iron metabolism dysfunction. This observation suggests that additional mechanisms are active in the liver to compensate for the increased BMP-SMAD signaling. Here we demonstrate that Fkbp12 downregulation in hepatocytes by antisense oligonucleotide treatment upregulates the expression of the main hepcidin inhibitor Tmprss6, thus counteracting the ALK2-mediated activation of the pathway. Combined downregulation of both Fkbp12 and Tmprss6 blocks this compensatory mechanism. Our findings reveal a previously unrecognized functional cross talk between FKBP12 and TMPRSS6, the main BMP-SMAD pathway inhibitors, in the control of hepcidin transcription.NEW & NOTEWORTHY This study uncovers a previously unrecognized mechanism of hepcidin and BMP-SMAD pathway regulation in hepatocytes mediated by the immunophilin FKBP12 and the transmembrane serine protease TMPRSS6.

ACE2 and TMPRSS2 distribution in the respiratory tract of different animal species and its correlation with SARS-CoV-2 tissue tropism.

A wide range of animal species show variable susceptibility to SARS-CoV-2; however, host factors associated with varied susceptibility remain to be defined. Here, we examined whether susceptibility to SARS-CoV-2 and virus tropism in different animal species are dependent on the expression and distribution of the virus receptor angiotensin-converting enzyme 2 (ACE2) and the host cell factor transmembrane serine protease 2 (TMPRSS2). We cataloged the upper and lower respiratory tract of multiple animal species and humans in a tissue-specific manner and quantitatively evaluated the distribution and abundance of ACE2 and TMPRSS2 mRNA in situ. Our results show that: (i) ACE2 and TMPRSS2 mRNA are abundant in the conduction portion of the respiratory tract, (ii) ACE2 mRNA occurs at a lower abundance compared to TMPRSS2 mRNA, (iii) co-expression of ACE2-TMPRSS2 mRNAs is highest in those species with the highest susceptibility to SARS-CoV-2 infection (i.e., cats, Syrian hamsters, and white-tailed deer), and (iv) expression of ACE2 and TMPRSS2 mRNA was not altered following SARS-CoV-2 infection. Our results demonstrate that while specific regions of the respiratory tract are enriched in ACE2 and TMPRSS2 mRNAs in different animal species, this is only a partial determinant of susceptibility to SARS-CoV-2 infection.IMPORTANCESARS-CoV-2 infects a wide array of domestic and wild animals, raising concerns regarding its evolutionary dynamics in animals and potential for spillback transmission of emerging variants to humans. Hence, SARS-CoV-2 infection in animals has significant public health relevance. Host factors determining animal susceptibility to SARS-CoV-2 are vastly unknown, and their characterization is critical to further understand susceptibility and viral dynamics in animal populations and anticipate potential spillback transmission. Here, we quantitatively assessed the distribution and abundance of the two most important host factors, angiotensin-converting enzyme 2 and transmembrane serine protease 2, in the respiratory tract of various animal species and humans. Our results demonstrate that while specific regions of the respiratory tract are enriched in these two host factors, they are only partial determinants of susceptibility. Detailed analysis of additional host factors is critical for our understanding of the underlying mechanisms governing viral susceptibility and reservoir hosts.

Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth

Transmembrane serine protease 6 (Tmprss6) has been correlated with the occurrence and progression of tumors, but any specific molecular mechanism linking the enzyme to oncogenesis has remained elusive thus far. In the present study, we found that Tmprss6 markedly inhibited mouse neuroblastoma N2a (neuro-2a) cell proliferation and tumor growth in nude mice. Tmprss6 inhibits Smad1/5/8 phosphorylation by cleaving the bone morphogenetic protein (BMP) co-receptor, hemojuvelin (HJV). Ordinarily, phosphorylated Smad1/5/8 binds to Smad4 for nuclear translocation, which stimulates the expression of hepcidin, ultimately decreasing the export of iron through ferroportin 1 (FPN1). The decrease in cellular iron levels in neuro-2a cells with elevated Tmprss6 expression limited the availability of the metal forribo nucleotide reductase activity, thereby arresting the cell cycle prior to S phase. Interestingly, Smad4 promoted nuclear translocation of activating transcription factor 3 (ATF3) to activate the p38 mitogen-activated protein kinases signaling pathway by binding to ATF3, inducing apoptosis of neuro-2a cells and inhibiting tumor growth. Disruption of ATF3 expression significantly decreased apoptosis in Tmprss6 overexpressed neuro-2a cells. Our study describes a mechanism whereby Tmprss6 regulates the cell cycle and apoptosis. Thus, we propose Tmprss6 as a candidate target for inhibiting neuronal tumor growth.

Expression of the SARS-CoV-2 Receptor ACE2 and Protease TMPRSS2 in Ocular Hypertension Eyes of Nonhuman Primate and Human

Purpose Coronavirus disease 2019 is a disease caused by the novel severe acute respiratory syndrome coronavirus 2. The double-positive of angiotensin-converting enzyme 2 and transmembrane protease serine type 2 have a higher risk of being infected by severe acute respiratory syndrome coronavirus 2. The susceptibility of coronavirus disease 2019 in patients with chronic diseases, especially in different tissues of ocular hypertension eyes like glaucoma, is not yet known. Methods An ocular hypertension model was established by laser photocoagulation in rhesus monkeys. The expression of angiotensin-converting enzyme 2 and transmembrane protease serine type 2 in three ocular hypertension eyes and the three control eyes were analyzed using immunofluorescence. Results No difference was observed between ocular hypertension and control eyes in the expression of angiotensin-converting enzyme 2 and transmembrane protease serine type 2 in the conjunctival epithelium, corneal epithelium, and ciliary muscle. In ocular hypertension eyes and control eyes, angiotensin-converting enzyme 2 and transmembrane protease serine type 2 expression were both observed in the retina. Angiotensin-converting enzyme 2 staining of retinal ganglion cells was found to be significantly higher in ocular hypertension eyes than in control eyes. However, there was no difference in angiotensin-converting enzyme 2 and transmembrane protease serine type 2 expression in retinal vessels and choroidal vessels between ocular hypertension and control eyes. In our study, the expression and distribution of angiotensin-converting enzyme 2 and TMPREE2 in human retina were similar to that of non-human primates as expected. Conclusion Our study confirmed that angiotensin-converting enzyme 2 and transmembrane protease serine type 2 were expressed widely in rhesus monkey eyes. When compared with controls eyes, the expression of angiotensin-converting enzyme 2 was higher in the retinal ganglion cells in ocular hypertension eyes, suggesting that high ocular pressure may affect the patients’ ocular susceptibility to severe acute respiratory syndrome coronavirus 2 infection.

Genome-wide bioinformatics analysis of human protease capacity for proteolytic cleavage of the SARS-CoV-2 spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) primarily enters the cell by binding the virus's spike (S) glycoprotein to the angiotensin-converting enzyme 2 receptor on the cell surface, followed by proteolytic cleavage by host proteases. Studies have identified furin and transmembrane protease serine 2 proteases in priming and triggering cleavages of the S glycoprotein, converting it into a fusion-competent form and initiating membrane fusion, respectively. Alternatively, SARS-CoV-2 can enter the cell through the endocytic pathway, where activation is triggered by lysosomal cathepsin L. However, other proteases are also suspected to be involved in both entry routes. In this study, we conducted a genome-wide bioinformatics analysis to explore the capacity of human proteases in hydrolyzing peptide bonds of the S glycoprotein. Predictive models of sequence specificity for 169 human proteases were constructed and applied to the S glycoprotein together with the method for predicting structural susceptibility to proteolysis of protein regions. After validating our approach on extensively studied S2' and S1/S2 cleavage sites, we applied our method to each peptide bond of the S glycoprotein across all 169 proteases. Our results indicate that various members of the proprotein convertase subtilisin/kexin type, type II transmembrane family serine protease, and kallikrein families, as well as specific coagulation factors, are capable of cleaving S2' or S1/S2 sites. We have also identified a potential cleavage site of cathepsin L at the K790 position within the S2' loop. Structural analysis suggests that cleavage of this site induces conformational changes similar to the cleavage at the R815 (S2') position, leading to the exposure of the fusion peptide and subsequent fusion with the membrane. Other potential cleavage sites and the influence of mutations in common SARS-CoV-2 variants on proteolytic efficiency are discussed.IMPORTANCEThe entry of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) into the cell, activated by host proteases, is considerably more complex in coronaviruses than in most other viruses and is not fully understood. There is evidence that other proteases beyond the known furin and transmembrane protease serine 2 can activate the spike protein. Another example of uncertainty is the cleavage site for the alternative endocytic route of SARS-CoV-2 entrance, which is still unknown. Bioinformatics methods, modeling protease specificity and estimating the structural susceptibility of protein regions to proteolysis, can aid in studying this topic by predicting the involved proteases and their cleavage sites, thereby substantially reducing the amount of experimental work. Elucidating the mechanisms of spike protein activation is crucial for preventing possible future coronavirus pandemics and developing antiviral drugs.

CAMP-phosphodiesterase 4D7 (PDE4D7) forms a cAMP signalosome complex with DHX9 and is implicated in prostate cancer progression.

A robust body of work has demonstrated that a reduction in cAMP-specific 3',5'-cyclic phosphodiesterase 4D isoform 7 (PDE4D7) is linked with negative prostate cancer outcomes; however, the exact molecular mechanism that underpins this relationship is unknown. Epigenetic profiling has shown that the PDE4D gene can be hyper-methylated in transmembrane serine protease 2 (TMPRSS2)-ETS transcriptional regulator ERG (ERG) gene-fusion-positive prostate cancer (PCa) tumours, and this inhibits messenger RNA (mRNA) expression, leading to a paucity of cellular PDE4D7 protein. In an attempt to understand how the resulting aberrant cAMP signalling drives PCa growth, we immunopurified PDE4D7 and identified binding proteins by mass spectrometry. We used peptide array technology and proximity ligation assay to confirm binding between PDE4D7 and ATP-dependent RNA helicase A (DHX9), and in the design of a novel cell-permeable disruptor peptide that mimics the DHX9-binding region on PDE4D7. We discovered that PDE4D7 forms a signalling complex with the DExD/H-box RNA helicase DHX9. Importantly, disruption of the PDE4D7-DHX9 complex reduced proliferation of LNCaP cells, suggesting the complex is pro-tumorigenic. Additionally, we have identified a novel protein kinase A (PKA) phosphorylation site on DHX9 that is regulated by PDE4D7 association. In summary, we report the existence of a newly identified PDE4D7-DHX9 signalling complex that may be crucial in PCa pathogenesis and could represent a potential therapeutic target.

Evidence of Placental Villous Inflammation and Apoptosis in Third-Trimester Symptomatic SARS-CoV-2 Maternal Infection.

In view of conflicting reports on the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to infect placental tissue, this study aimed to further evaluate the impact of inflammation and placental damage from symptomatic third-trimester maternal COVID-19 infection. This case-control study included 32 placenta samples each from symptomatic COVID-19 pregnancy and normal non-COVID-19 pregnancy. The villous placental area's inflammatory expression [angiotensin converting enzyme-2 (ACE-2), transmembrane protease serine-2 (TMPRSS2), interferon-γ (IFN-γ), interleukin-6 (IL-6), and SARS-CoV-2 spike protein] and apoptotic rate were examined using immunohistochemistry and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. Comparison and correlation analysis were used based on COVID-19 infection, placental SARS-CoV-2 spike protein evidence, and maternal severity status. Higher expressions of TMPRSS2, IFN-γ, and trophoblast apoptotic rate were observed in the COVID-19 group (p<0.001), whereas ACE-2 and IL-6 expressions were not significantly different from the control group (p>0.05). Additionally, SARS-CoV-2 spike protein was detected in 8 (25%) placental samples of COVID-19 pregnancy. COVID-19 subgroup analysis revealed increased IFN-γ, trophoblast, and stromal apoptosis (p<0.01). Moreover, the results of the current study revealed no correlation between maternal COVID-19 severity and placental inflammation as well as the apoptotic process. The presence of SARS-CoV-2 spike protein as well as altered inflammatory and apoptotic processes may indicate the presence of placental disturbance in third-trimester maternal COVID-19 infection. The lack of correlation between placental disruption and maternal severity status suggests the need for more research to understand the infection process and any potential long-term impacts on all offsprings born to COVID-19-infected pregnant women.

NASOPHARYNGEAL ACE2, CD147, AND TMPRSS2 EXPRESSIONS IN MIS-C PATIENTS

Objective: Multisystem Inflammatory Syndrome in Children (MIS-C) is a disease developed after about eight weeks of the new coronavirus disease (COVID-19) infection in children. The underlying reason for MIS-C is still unknown. This study aims to assess the nasopharyngeal expressions of Angiotensin-converting enzyme 2 (ACE2), the differentiation 147 receptor cluster (CD147), and the transmembrane serine protease 2 (TMPRSS2) receptors in the MIS-C population. Since these receptors are related to COVID-19 infection, children with previous COVID-19 history were included as a control group. This study is important for future studies to see if there is any significant expression difference between these receptors in control and MIS-C populations. Methods: The prospective cohort study took place at XXXXX University. The participants of this study consisted of patients aged 0-18 years who were diagnosed with MIS-C due to COVID-19 infection or patients with only previous COVID-19 pneumonia but without MIS-C development. A total of 79 cases were registered in this study. Nasopharyngeal samples of children were collected. Total RNA was isolated from all samples. ACE2, CD147, and TMPRSS2 genes’ expression was accessed by real-time quantitative PCR (RT-qPCR). Results: Nasopharyngeal expression of CD147 and TMPRSS2 were not changed in COVID-19 (n=42) and MIS-C (n=37) cases. Expression of ACE2 was increased in the under 10-year-old MIS-C group (n=23) (p=0.004381). Conclusion: Future studies may exclude the nasopharyngeal expression of CD147 and TMPRSS2 to develop MIS-C. Further investigations are required to learn how ACE2 expression contributes to MIS-C development.

Preventing SARS-CoV-2 infection using Fv-antibodies targeting the proprotein convertase (PPC) cleavage site.

Fv-antibodies targeting the proprotein convertase (PPC) region of the SARS-CoV-2 spike protein (SP) were screened from an Fv-antibody library to inhibit SARS-CoV-2 infection. Two selected Fv-antibodies were expressed as soluble recombinant proteins, and their binding affinities were assessed using a surface plasmon resonance biosensor. The binding regions of these Fv-antibodies corresponded to the cleavage sites of furin (S1/S2) and transmembrane serine protease 2 (TMPRSS2, S2'). The neutralizing activities of the two Fv-antibodies were demonstrated using a cell-based infection assay with pseudo-viruses carrying the SP of four different SARS-CoV-2 variants: wild-type (D614), delta (B.1.617.2), omicron (BA.2), and omicron (BA.4/5).

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

The Y498T499-SARS-CoV-2 spike (S) protein interacts poorly with rat ACE2 and does not affect the rat lung.

The rat is a useful laboratory model for respiratory diseases. SARS-CoV-2 proteins, such as the spike (S) protein, can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change, described in the S-protein of the mouse-adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lungs. A real-time S-ACE2 quantitative fusion assay shows that ancestral and L452R S-proteins fuse with human but not rat ACE2 expressed on HEK293 (human embryonic kidney-293) cells. The QP-YT S-protein retains the ability to fuse with human ACE2 and increases the binding to rat ACE2. Although lower lung of the rat contains both ACE2 and TMPRSS2 (transmembrane serine protease 2) target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however, with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed, and the QP-YT S-protein pseudotyped lentivirus poorly infected HEK293 cells expressing rat ACE2. Analysis of the amino acid changes across the S-ACE2 interface highlights not only the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2, and the QP-YT S-protein variant can bind to rat ACE2, but this does not result in infection or stimulate responses in the lung. Further, amino acid changes in S-protein may enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving lung inflammation.

From intramuscular to nasal: unleashing the potential of nasal spray vaccines against coronavirus disease 2019.

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected 700 million people worldwide since its outbreak in 2019. The current pandemic strains, including Omicron and its large subvariant series, exhibit strong transmission and stealth. After entering the human body, the virus first infects nasal epithelial cells and invades host cells through the angiotensin-converting enzyme 2 receptor and transmembrane serine protease 2 on the host cell surface. The nasal cavity is an important body part that protects against the virus. Immunisation of the nasal mucosa produces immunoglobulin A antibodies that effectively neutralise viruses. Saline nasal irrigation, a type of physical therapy, can reduce the viral load in the nasal cavity and prevent viral infections to some extent. As a commonly used means to fight SARS-CoV-2, the intramuscular (IM) vaccine can induce the human body to produce a systemic immune response and immunoglobulin G antibody; however, the antibody is difficult to distribute to the nasal mucosa in time and cannot achieve a good preventive effect. Intranasal (IN) vaccines compensate for the shortcomings of IM vaccines, induce mucosal immune responses, and have a better effect in preventing infection. In this review, we discuss the nasal defence barrier, the harm caused by SARS-CoV-2, the mechanism of its invasion into host cells, nasal cleaning, IM vaccines and IN vaccines, and suggest increasing the development of IN vaccines, and use of IN vaccines as a supplement to IM vaccines.

The comparative utility of FAPI-based PET radiotracers over [18F]FDG in the assessment of malignancies.

Fibroblast activation protein (FAP) is a type II transmembrane serine protease overexpressed in cancer-associated fibroblasts (CAFs) and has been associated with poor prognosis. PET/CT imaging with radiolabeled FAP inhibitors (FAPI) is currently being studied for various malignancies. This review identifies the uses and limitations of FAPI PET/CT in malignancies and compares the advantages and disadvantages of FAPI and 18F-fluorodeoxyglucose ([18F]FDG). Due to high uptake, rapid clearance from the circulation, and limited uptake in normal tissue, FAPI tumor-to-background contrast ratios are equivalent to or better than [18F]FDG in most applications. In several settings, FAPI has shown greater uptake specificity than [18F]FDG and improved sensitivity in detecting lymph node, bone, and visceral tissue metastases. Therefore, FAPI PET/CT may be complementary in distinguishing pathological lesions with conventional imaging, determining the primary site of malignancy, improving tumor staging, and detecting disease recurrence, especially in patients with inconclusive [18F]FDG PET/CT findings. Nevertheless, FAPI has limitations, including certain settings with non-specific uptake, modified uptake with age and menopause status, challenges with clinical access, and limited clinical evidence.

Sensitive detection of dipeptidyl peptidase based on DNA-peptide conjugates and double signal amplification of CHA and DNAzymes.

Dipeptidyl peptidase IV (DPPIV) is an enzyme belonging to the type II transmembrane serine protease family that has gained wide interest in the fields of hematology, immunology, and cancer biology. Moreover, DPPIV has emerged as a promising target for therapeutic intervention in type II diabetes. Due to its biological limitations, traditional strategies cannot meet the requirements of low abundance DPPIV analysis in complex environments. In this work, combining the high programmability of DNA and the chemical diversity of peptides, we designed DNA-peptide conjugates that can be specifically recognized, polypeptides as specific substrates for target DPPIV and DNA probes as primers for catalytic hairpin assembly (CHA), recycling a large amount of DNAzymes by triggering CHA amplification. The DNAzyme substrate modified with the FAM fluorescent group was immobilized on the surface of gold nanoparticles by S-Au chemical bonds to form a signal output probe. The DNAzymes enzyme cleaved the substrate of the signal outputs probe, yielding a double-amplified fluorescence signal. This method has a detection limit as low as 0.18 mU mL-1 and a linear range of 0-5 mU mL-1 in serum samples, showing high stability and good potential for practical applications.

Interaction Between Genetic Susceptibility and COVID-19 Pathogenesis in Pediatric Multisystem Inflammatory Disorders: The Role of Immune Responses.

Numerous studies have highlighted the emergence of coronavirus disease (COVID-19) symptoms reminiscent of Kawasaki disease in children, including fever, heightened multisystem inflammation, and multiorgan involvement, posing a life-threatening complication. Consequently, extensive research endeavors in pediatric have aimed to elucidate the intricate relationship between COVID-19 infection and the immune system. COVID-19 profoundly impacts immune cells, culminating in a cytokine storm that particularly inflicts damage on the pulmonary system. The gravity and vulnerability to COVID-19 are closely intertwined with the vigor of the immune response. In this context, the human leukocyte antigen (HLA) molecule assumes pivotal significance in shaping immune responses. Genetic scrutiny of HLA has unveiled the presence of at least one deleterious allele in children afflicted with multisystem inflammatory syndrome in children (MIS-C). Furthermore, research has demonstrated that COVID-19 exploits the angiotensin-converting enzyme 2 (ACE-2) receptor, transmembrane serine protease type 2, and various other genes to gain entry into host cells, with individuals harboring ACE-2 polymorphisms being at higher risk. Pediatric studies have employed diverse genetic methodologies, such as genome-wide association studies (GWAS) and whole exome sequencing, to scrutinize target genes. These investigations have pinpointed two specific genomic loci linked to the severity and susceptibility of COVID-19, with the HLA locus emerging as a notable risk factor. In this comprehensive review article, we endeavor to assess the available evidence and consolidate data, offering insights into current clinical practices and delineating avenues for future research. Our objective is to advance early diagnosis, stabilization, and appropriate management strategies to mitigate genetic susceptibility's impact on the incidence of COVID-19 in pediatric patients with multisystem inflammation.

Inhibitor design for TMPRSS2: insights from computational analysis of its backbone hydrogen bonds using a simple descriptor

Transmembrane protease serine 2 (TMPRSS2) is an important drug target due to its role in the infection mechanism of coronaviruses including SARS-CoV-2. Current understanding regarding the molecular mechanisms of known inhibitors and insights required for inhibitor design are limited. This study investigates the effect of inhibitor binding on the intramolecular backbone hydrogen bonds (BHBs) of TMPRSS2 using the concept of hydrogen bond wrapping, which is the phenomenon of stabilization of a hydrogen bond in a solvent environment as a result of being surrounded by non-polar groups. A molecular descriptor which quantifies the extent of wrapping around BHBs is introduced for this. First, virtual screening for TMPRSS2 inhibitors is performed by molecular docking using the program DOCK 6 with a Generalized Born surface area (GBSA) scoring function. The docking results are then analyzed using this descriptor and its relationship to the solvent-accessible surface area term ΔGsa of the GBSA score is demonstrated with machine learning regression and principal component analysis. The effect of binding of the inhibitors camostat, nafamostat, and 4-guanidinobenzoic acid (GBA) on the wrapping of important BHBs in TMPRSS2 is also studied using molecular dynamics. For BHBs with a large increase in wrapping groups due to these inhibitors, the radial distribution function of water revealed that certain residues involved in these BHBs, like Gln438, Asp440, and Ser441, undergo preferential desolvation. The findings offer valuable insights into the mechanisms of these inhibitors and may prove useful in the design of new inhibitors.

Molecular characterization of chronic cutaneous wounds reveals subregion- and wound type-specific differential gene expression.

A limited understanding of the pathology underlying chronic wounds has hindered the development of effective diagnostic markers and pharmaceutical interventions. This study aimed to elucidate the molecular composition of various common chronic ulcer types to facilitate drug discovery strategies. We conducted a comprehensive analysis of leg ulcers (LUs), encompassing venous and arterial ulcers, foot ulcers (FUs), pressure ulcers (PUs), and compared them with surgical wound healing complications (WHCs). To explore the pathophysiological mechanisms and identify similarities or differences within wounds, we dissected wounds into distinct subregions, including the wound bed, border, and peri-wound areas, and compared them against intact skin. By correlating histopathology, RNA sequencing (RNA-Seq), and immunohistochemistry (IHC), we identified unique genes, pathways, and cell type abundance patterns in each wound type and subregion. These correlations aim to aid clinicians in selecting targeted treatment options and informing the design of future preclinical and clinical studies in wound healing. Notably, specific genes, such as PITX1 and UPP1, exhibited exclusive upregulation in LUs and FUs, potentially offering significant benefits to specialists in limb preservation and clinical treatment decisions. In contrast, comparisons between different wound subregions, regardless of wound type, revealed distinct expression profiles. The pleiotropic chemokine-like ligand GPR15L (C10orf99) and transmembrane serine proteases TMPRSS11A/D were significantly upregulated in wound border subregions. Interestingly, WHCs exhibited a nearly identical transcriptome to PUs, indicating clinical relevance. Histological examination revealed blood vessel occlusions with impaired angiogenesis in chronic wounds, alongside elevated expression of genes and immunoreactive markers related to blood vessel and lymphatic epithelial cells in wound bed subregions. Additionally, inflammatory and epithelial markers indicated heightened inflammatory responses in wound bed and border subregions and reduced wound bed epithelialization. In summary, chronic wounds from diverse anatomical sites share common aspects of wound pathophysiology but also exhibit distinct molecular differences. These unique molecular characteristics present promising opportunities for drug discovery and treatment, particularly for patients suffering from chronic wounds. The identified diagnostic markers hold the potential to enhance preclinical and clinical trials in the field of wound healing.

Comparative immunohistochemical evaluation of variable expression of ACE2 and TMPRSS2 in different age groups

Background and objectives: COVID-19 pandemic declared by World Health Organisation has drastically upsurged the death rate in the past three years. The specific host cell receptors for viral spike protein have been identified as Angiotensin Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2). The study aimed to evaluate the variation in the pattern of expression of ACE2 and TMPRSS2 by immunohistochemistry in the oral and nasopharyngeal mucosa of different age groups. Methods: Total of 40 patients were recruited for the study and segregated to four groups. Oral tissue samples from patients of age 18-40 years and 41-70 years were grouped as group I, group II respectively. Nasal tissue from 18-40 years was grouped as III and 41-70 years old as group IV. Immunohistochemical expression of ACE and TMPRSS2 were studied in the tissue samples. Scoring was done based on the intensity and percentage of staining and quantitative image analysis using Fiji image analysis software. Independent sample t‐test was done to compare the mean difference in pattern of expression among the age groups studied. Pearson correlation coefficient was done to correlate the expression with age. Statistical significance was set at value less than 0.05. Results: The mean difference in expression was significant for ACE2 (p=0.01) &amp; TMPRSS2 (p=0.02) expression in oral tissue. Both ACE2 and TMPRSS2 expression showed positive correlation between the groups. Conclusion: Age-specific variation might provide deeper understanding of clinical severity and elaborate the validation of therapeutic targets.

COVID-19 on Oral Health: A New Bilateral Connection for the Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission are generally known to be produced by respiratory droplets and aerosols from the oral cavity (O.C.) of infected subjects, as stated by the World Health Organization. Saliva also retains the viral particles and aids in the spread of COVID-19. Angiotensin-converting enzyme Type 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are two of the numerous factors that promote SARS-CoV-2 infection, expressed by O.C. structures, various mucosa types, and the epithelia of salivary glands. A systemic SARS-CoV-2 infection might result from viral replication in O.C. cells. On the other hand, cellular damage of different subtypes in the O.C. might be associated with various clinical signs and symptoms. Factors interfering with SARS-CoV-2 infection potential might represent fertile ground for possible local pharmacotherapeutic interventions, which may confine SARS-CoV-2 virus entry and transmission in the O.C., finally representing a way to reduce COVID-19 incidence and severity.

Eye Washing Downregulated Angiotensin-Converting Enzyme 2 in Conjunctival Tissue Samples from Smokers.

This study aimed to (1) determine whether the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 is increased in tobacco smokers, which potentially increases their susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and (2) assess whether eye rinsing can reduce susceptibility. This prospective study included 20 eyes of 10 smokers and 18 eyes of nine healthy non-smokers (control) for reverse-transcription polymerase chain reaction. This study also included 28 eyes of 14 smokers and 16 eyes of eight healthy non-smokers (control) for enzyme-linked immunosorbent assay. Tear and impression cytology samples were collected from the right eye of each patient. The left eye was then rinsed for 30 s, and after 5 min, the tear and impression cytology samples were collected in the same manner. The expression of the ACE2 gene was significantly higher in the conjunctiva of smokers (n = 17; median 3.07 copies/ng of total RNA) than in those of non-smokers (n = 17; median 1.92 copies/ng of total RNA, p = 0.003). Further, mRNA expression and protein levels of ACE2 were weakly correlated in smokers (r = 0.49). ACE2 protein levels in Schirmer's strip samples were significantly reduced from 5051 to 3202 pg/mL after eye washing (n = 10; p = 0.001). Ocular surface cells are susceptible to SARS-CoV-2 infection. Smoking may be a risk factor for SARS-CoV-2 infection, and eye rinsing may reduce the risk of infection.