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Background: Iron refractory iron deficiency anemia (IRIDA) is a rare autosomal recessive type of anemia characterized by unresponsiveness to oral iron therapy and partial response to parenteral iron therapy. In this article, we report the clinical presentation of four patients with IRIDA admitted to our clinic, including their laboratory values at admission and after oral and parenteral iron treatment, and the analysis of their mutation(s) in TMPRSS6 gene., Case: Four patients from different families, aged between 3 and 14 years, two girls and two boys, two of whom were from consanguineous marriages, who were diagnosed with iron deficiency anemia in primary health care institutions and referred to our clinic because of inadequate response to oral iron treatment were included. Patients were evaluated for the differential diagnosis of microcytic, hypochromic anemia and investigated for the etiology of IDA. Homozygous or compound heterozygous mutations causing defective matriptase-2 protein expression were detected in the TMPRSS6 gene; these mutations included four frameshift mutations-two of which were the same in two cases and causing premature terminal stop codons-and a nonsense mutation, all of which were previously demonstrated in the literature. The response to parental iron therapy ranged from complete non-response to mild to good response in hemoglobin levels, but none of the patients showed improvement in iron parameters., Conclusions: Increased awareness of IRIDA and keeping it in mind in the differential diagnosis in the presence of hypochromic microcytic anemia that does not respond to iron treatment will be crucial in improving the diagnosis and treatment of the disease and ultimately enhancing the quality of care for affected individuals., Competing Interests: The authors declare that there is no conflict of interest.

The crosstalk between cuproptosis and the tumor immune microenvironment (TIME) is vital during clear cell renal cell carcinoma (ccRCC) malignant progression. However, the underlying molecular mechanisms regulate this cross-talk remain elusive. Through tailored machine learning, we analyze clinical ccRCC data from The Cancer Genome Atlas (TCGA) to explore the critical factors that regulate the interaction among cuproptosis, TIME, and tumor progression. We found that rhomboid-like 2 (RHBDL2), critical gene affecting this process, might inhibit cuproptosis-related genes (CRGs) and promotes ccRCC progression through the Wnt/β-catenin pathway. Next, knocking down RHBDL2 expression increased the cuproptosis-related genes ferredoxin 1 (FDX1) and lipoic acid synthase (LIAS) levels but reduced forkhead box P3 (FOXP3) levels and tumor growth in vivo and in vitro models. By employing HLY78, Wnt/β-catenin pathway activator, we rescued the expression of CRGs and the malignant proliferation and metastasis capacity in ccRCC cells with RHBDL2 knockdown. Mechanistically, RHBDL2 inhibits cuproptosis and promotes malignant progression of ccRCC through the Wnt/β-catenin pathway. Abnormal RHBDL2 expression may cause the suppressive TIME formation by regulating Treg-cell infiltration, thus triggering immune escape. In summary, our results indicated that RHBDL2 is an oncogene that induces tumorigenesis and targeting RHBDL2 may be novel therapeutic direction for metastatic ccRCC., (© 2024. The Author(s).)

Secreted protein Reelin is implicated in neuropsychiatric disorders and its supplementation ameliorates neurological symptoms in mouse disease models. Recombinant human Reelin protein may be useful for the treatment of human diseases, but its properties remain uncharacterized. Here, we report that full-length human Reelin was well secreted from transfected cells and was able to induce Dab1 phosphorylation. Unexpectedly, the central fragment of human Reelin was much less secreted than that of mouse Reelin. Three residues in the sixth Reelin repeat contributed to the secretion inefficiency, and their substitutions with mouse residues increased the secretion without affecting its biological activity. Our findings help efficient production of human Reelin protein for the supplementation therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mutations in various type II transmembrane serine protease (TMPRSS) family members are associated with non-syndromic hearing loss, with some mechanisms still unclear. For instance, the mechanism underlying profound hearing loss and tectorial membrane (TM) malformations in hepsin/TMPRSS1 knockout (KO) mice remains elusive. In this study, we confirmed significantly elevated hearing thresholds and abnormal TM morphology in hepsin KO mice, characterized by enlarged TM with gaps and detachment from the spiral limbus. Transgenic mouse lines were created to express either wild-type or a serine protease-dead mutant of human hepsin in the KO background. The Tg68;KO line, expressing moderate levels of wild-type human hepsin in the cochlea, showed partial restoration of hearing function. Conversely, the Tg5;KO or TgRS;KO lines, with undetectable hepsin or protease-dead hepsin, did not show such improvement. Histological analyses revealed that Tg68;KO mice, but not Tg5;KO or TgRS;KO mice, had a more compact TM structure, partially attached to the spiral limbus. These results indicate that hepsin expression levels correlate with improvements in hearing and TM morphology, and its protease activity is critical for these effects. Hepsin's role was further examined by studying its relationship with α-tectorin (TECTA) and β-tectorin (TECTB), non-collagenous proteins crucial for TM formation. Hepsin was co-expressed with TECTA and TECTB in the developing cochlear epithelium. Immunostaining showed decreased levels of TECTA and TECTB in hepsin KO TM, partially restored in Tg68;KO mice. These findings suggest that hepsin is essential for proper TM morphogenesis and auditory function, potentially by proteolytic processing/maturation of TECTA and TECTB and their incorporation into the TM., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Manduca sexta hemolymph protease-6 (HP6) plays a central role in coordinating antimicrobial responses, such as prophenoloxidase (PPO) activation and Toll signaling. Our previous studies indicated that HP5 and GP6 activate proHP6 in larval hemolymph and extraembryonic tissues, respectively. Here, we report the characterization of HP17b as another HP6 activating enzyme and its regulation by multiple serpins in hemolymph. The precursor of HP17b expressed in baculovirus infected Sf9 cells became spontaneously cleaved at two sites, and these products were purified together in one preparation named HP17b', a mixture of proHP17b, a 35 kDa intermediate, and HP17b. HP17b' converted proHP6 to HP6. As reported before, HP6 converted precursors of PPO activating protease-1 (PAP1) and HP8 to their active forms. HP8 activates proSpӓtzle-1 to turn on Toll signaling. We found HP17b' directly activated proSPHI and II to form a cofactor for PPO activation by PAP1. Supplementation of larval hemolymph with HP17b', HP17b, or proHP17b significantly increased PPO activation. Adding Micrococcus luteus to the reactions did not enhance PPO activation in the reactions containing HP17b', HP17b, or proHP17b. Using HP17b antibodies, we isolated from induced plasma HP17b fragments and associated proteins (e.g., serpin-4). Serpin-1A, 1J, 1J', 4, 5, or 6 reduced the activation of proHP6 by HP17b' through formation of covalent complexes with active HP17b. We detected an activity for proHP17b cleavage in hemolymph from bar-stage pharate pupae but failed to purify the protease due to its high instability. Other known HPs did not activate proHP17b in vitro. Together, these results suggest that HP17b is a clip-domain protease activated by an unknown endopeptidase in response to a danger signal and regulated by multiple serpins., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/β)-hydrolase superfamily containing a well-conserved α/β-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min -1 ) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0-8.0), and has great stability over a broad range of pH (5.0-10.0), and temperature (40-85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca 2+ , Mn 2+ , and Mg 2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg -1 ) and olive oil (1182.05 U mg -1 ). The values of K m (0.576 mM), V max (4216 μmol mg -1  min -1 ), V max K m -1 (7319.44 min -1 ), k cat (1106.74 s -1 ), and k cat K m -1 (1921.42 mM -1  s -1 ) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH 2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca 2+ and Na + increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.

Background and Aims: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection., Methods: We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed., Results: We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome., Conclusion: We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19., (© 2024 The Author(s). Liver International published by John Wiley & Sons Ltd.)

A major challenge in prevention and early treatment of organ fibrosis is the lack of valuable tools to assess the evolving profibrotic maladaptive repair after injury in vivo in a non-invasive way. Here, using acute kidney injury (AKI) as an example, we tested the utility of fibroblast activation protein (FAP) imaging for dynamic assessment of maladaptive repair after injury. The temporospatial pattern of kidney FAP expression after injury was first characterized. Single-cell RNA sequencing and immunostaining analysis of patient biopsies were combined to show that FAP was specifically upregulated in kidney fibroblasts after AKI and was associated with fibroblast activation and chronic kidney disease (CKD) progression. This was corroborated in AKI mouse models, where a sustained and exaggerated kidney FAP upregulation was coupled to persistent fibroblast activation and a fibrotic outcome, linking kidney FAP level to post-insult maladaptive repair. Furthermore, using positron emission tomography (PET)/CT scanning with FAP-inhibitor tracers ([ 18 F]FAPI-42, [ 18 F]FAPT) targeting FAP, we demonstrated the feasibility of non-invasively tracking of maladaptive repair evolution toward kidney fibrosis. Importantly, a sustained increase in kidney [ 18 F]FAPT (less hepatobiliary metabolized than [ 18 F]FAPI-42) uptake reflected persistent kidney upregulation of FAP and characterized maladaptive repair after AKI. Kidney [ 18 F]FAPT uptake at hour 2-day 7 correlated with kidney fibrosis 14 days after AKI. Similar changes in [ 18 F]FAPI-42 PET/CT imaging were observed in patients with AKI and CKD progression. Thus, persistent kidney FAP upregulation after AKI was associated with maladaptive repair and a fibrotic outcome. Hence, FAP-specific PET/CT imaging enables dynamic visualization of maladaptive repair after AKI and prediction of kidney fibrosis within a clinically actionable window., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Deficits in social behavior are found in neurodevelopmental disorders, including autism spectrum disorders (ASDs). Since abnormalities in cerebellar morphology and function are observed in ASD patients, the cerebellum is thought to play a role in social behavior. However, it remains unknown whether the cerebellum is involved in social behavior in other animals and how cerebellar circuits control social behavior. To address this issue, we employed zebrafish stereotyped orienting behavior as a model of social behaviors, in which a pair of adult zebrafish in two separate tanks approach each other, with one swimming at synchronized angles (orienting angles) with the other. We harnessed transgenic zebrafish that express botulinum toxin, which inhibits the release of neurotransmitters, in either granule cells or Purkinje cells (PCs), and zebrafish mutants of reelin , which is involved in the positioning of cerebellar neurons, including PCs. These zebrafish, deficient in the function or formation of cerebellar neural circuits, showed a significantly shorter period of orienting behavior compared with their control siblings. We found an increase in c- fos and egr1 expression in the cerebellum after the orienting behavior. These results suggest that zebrafish cerebellar circuits play an important role in social orienting behavior., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Hosaka et al.)

Background: Cardiovascular function and metabolic homeostasis are closely linked, but the underlying mechanisms are not fully understood. Corin is a protease that activates atrial natriuretic peptide (ANP), an essential hormone for normal blood pressure and cardiac function. The goal of this study is to investigate a potential corin and ANP function in regulating liver glycogen metabolism and glucose homeostasis., Methods: Liver glycogen and blood glucose levels were analyzed in Corin or Nppa (encoding ANP) knockout (KO) mice. ANP signaling was examined in livers from Corin and Nppa KO mice and in cultured human and mouse hepatocytes by western blotting., Results: We found that Corin and Nppa KO mice had reduced liver glycogen contents and increased blood glucose levels. By analyzing conditional KO mice lacking either cardiac or renal Corin, we showed that cardiac corin and ANP act in an endocrine manner to enhance cGMP-protein kinase G (PKG)-AKT-GSK3 signaling in hepatocytes. In cultured hepatocytes, ANP treatment stimulated PKG signaling, glucose uptake, and glycogen production, which could be blocked by small molecule PKG and AKT inhibitors., Conclusions: Our results indicate that corin and ANP are important regulators in liver glycogen metabolism and glucose homeostasis, suggesting that defects in the corin and ANP pathway may contribute to both cardiovascular and metabolic diseases., (© 2024. The Author(s).)

Background: Osteoarthritis (OA) is a common joint disease that leads to chronic pain and functional limitations. Recent research has revealed soluble fibroblast activation protein (FAP) secreted from OA synovium could degrade type II collagen (Col2) in cartilage to promote the progression of OA. This study aimed to reveal the role of FAP from chondrocytes in OA and develop a novel lipid nanoparticle (LNP)-FAP siRNA delivery system for OA treatment., Methods: The expression of FAP in the cartilage of knee OA patients was investigated using [68 Ga]Ga-FAPI-04 PET in vivo and immunofluorescence, western blotting, and RT-qPCR in vitro. Cell senescence was determined by senescence-associated β-galactosidase (SA-β-Gal) assay after FAP overexpressing or knockdown in chondrocytes. An OA model with chondrocyte-specific FAP knockout mice was applied to investigate the role of FAP in chondrocyte senescence and OA development. The therapeutic effects of lipid nanoparticle (LNP) @FAP siRNA on cartilage degeneration were evaluated in the rat OA model., Results: Our study found that higher [68 Ga]Ga-FAPI-04 uptake was detected in knee OA patients by PET/CT scan. FAP mRNA and protein levels were highly expressed in OA-damaged cartilage. Moreover, we found that overexpression of FAP promotes chondrocyte senescence, and the genetic knockout of FAP in chondrocytes alleviates OA. Knockdown FAP by siRNA could alleviate chondrocyte senescence and suppress the NF-κB pathway to reduce the senescence-associated secretory phenotype (SASP). In the rat model of OA, intraarticular injection of LNP@FAP siRNA can reduce senescent cells and ameliorate cartilage destruction., Conclusion: FAP-positive chondrocytes play a significant role in the pathogenesis of OA. Targeting these cells selectively has the potential to mitigate the progression of the disease. Our study provides valuable insights into the intraarticular injection of LNP@FAP siRNA as a promising strategy for the treatment of OA., (© 2024. The Author(s).)

Corin is a protease that activates atrial natriuretic peptide (ANP), a hormone in cardiovascular homeostasis. Structurally, ANP is similar to C-type natriuretic peptide (CNP) crucial in bone development. Here, we examine the role of corin and ANP in chondrocyte differentiation and bone formation. We show that in Corin and Nppa (encoding ANP) knockout (KO) mice, chondrocyte differentiation is impaired, resulting in shortened limb long bones. In adult mice, Corin and Nppa deficiency impairs bone density and microarchitecture. Molecular studies in cartilages from newborn Corin and Nppa KO mice and in cultured chondrocytes indicate that corin and ANP act in chondrocytes via cGMP-dependent protein kinase G signaling to inhibit mitogen-activated protein kinase phosphorylation and stimulate glycogen synthase kinase-3β phosphorylation and β-catenin upregulation. These results indicate that corin and ANP signaling regulates chondrocyte differentiation in bone development and homeostasis, suggesting that enhancing ANP signaling may improve bone quality in patients with osteoporosis., (© 2024. The Author(s).)

The cell culture-based isolation of novel coronavirus SARS-CoV-2 from clinical specimens obtained from patients with suspected COVID-19 is important not only for laboratory diagnosis but also for obtaining live virus to characterize emerging variants. Previous studies report that monkey kidney-derived VeroE6/TMPRSS2 cells allow efficient isolation of SARS-CoV-2 from clinical specimens because these cells show stable expression of the receptor molecule monkey ACE2 and the serine-protease TMPRSS2. Here, we demonstrated that VeroE6 cells overexpressing human ACE2 and TMPRSS2 (Vero E6-TMPRSS2-T2A-ACE2 cells) are superior to VeroE6/TMPRSS2 for isolating SARS-CoV-2 from clinical specimens. These cells showed a 1.6-fold increase in efficiency in SARS-CoV-2 isolation, and were particularly effective for clinical specimens with a relatively low viral load (< 10 6 copies/mL). When using vesicular stomatitis virus (VSV) pseudoviruses (VSV/SARS-2pv) bearing the spike proteins of all of the tested SARS-CoV-2 strains, Vero E6-TMPRSS2-T2A-ACE2 cells showed a 2- to fourfold increase in infectivity. Furthermore, the results of virus titration and neutralization antibody assays using Vero E6-TMPRSS2-T2A-ACE2 cells were different from those using VeroE6/TMPRSS2, highlighting the importance of selecting appropriate cell culture systems to determine SARS-CoV-2 infectivity., (© 2024. The Author(s).)

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with noncanonical receptors and unidentified coreceptors; however, the effects of which are less understood. Using high-throughput tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics and gene set enrichment analysis (GSEA), we identified both shared and unique intracellular pathways activated by Reelin through its canonical and noncanonical signaling in primary murine neurons of either sex during dendritic growth and arborization. We observed pathway cross talk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the noncanonical Reelin pathway including protein translation, mRNA metabolic process, and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin-binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for dendritic development., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Background: Mouse models that recapitulate key features of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are important tools for understanding complex interactions between host genetics, immune responses, and SARS-CoV-2 pathogenesis. Little is known about how predominantly cellular (Th1 type) versus humoral (Th2 type) immune responses influence SARS-CoV-2 dynamics, including infectivity and disease course., Methods: We generated knock-in (KI) mice expressing human ACE2 (hACE2) and/or human TMPRSS2 (hTMPRSS2) on Th1-biased (C57BL/6; B6) and Th2-biased (BALB/c) genetic backgrounds. Mice were infected intranasally with SARS-CoV-2 Delta (B.1.617.2) or Omicron BA.1 (B.1.1.529) variants, followed by assessment of disease course, respiratory tract infection, lung histopathology, and humoral and cellular immune responses., Findings: In both B6 and BALB/c mice, hACE2 expression was required for infection of the lungs with Delta, but not Omicron BA.1. Disease severity was greater in Omicron BA.1-infected hTMPRSS2-KI and double-KI BALB/c mice compared with B6 mice, and in Delta-infected double-KI B6 and BALB/c mice compared with hACE2-KI mice. hACE2-KI B6 mice developed more severe lung pathology and more robust SARS-CoV-2-specific splenic CD8 T cell responses compared with hACE2-KI BALB/c mice. There were no notable differences between the two genetic backgrounds in plasma cell, germinal center B cell, or antibody responses to SARS-CoV-2., Interpretation: SARS-CoV-2 Delta and Omicron BA.1 infection, disease course, and CD8 T cell response are influenced by the host genetic background. These humanized mice hold promise as important tools for investigating the mechanisms underlying the heterogeneity of SARS-CoV-2-induced pathogenesis and immune response., Funding: This work was funded by NIH U19 AI142790-02S1, the GHR Foundation, the Arvin Gottleib Foundation, and the Overton family (to SS and EOS); Prebys Foundation (to SS); NIH R44 AI157900 (to KJ); and by an American Association of Immunologists Career Reentry Fellowship (FASB)., Competing Interests: Declaration of interests CC, NS, and KJ were employees of Synbal, Inc. DOC is employed by and holds stock in TransViragen Inc., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background/aim: The emergence of novel DNA damage repair (DDR) pathways in molecular-target therapy drugs (MTTD) has shown promising outcomes in treating patients with metastatic castration-resistant prostate cancer (mCRPC). About 25% of mCRPC patients have actionable deleterious aberrations in DDR genes, primarily in the homologous recombination (HR) pathway. However, the response rate in patients with BRCA1/2 or mutations in HRR-related genes is only 45%-55%, when exposed to poly ADP ribose polymerase (PARP) inhibitor-based therapy (PARPi). A frequent characteristic feature of prostate cancer (PC) is the occurrence of genomic rearrangement that affects the transmembrane protease serine 2 (TMPRSS2) and E26 transformation-specific (ETS)- transcription factor-related gene (ERG)., Materials and Methods: In this study, a total of 114 patients with mCRPC had their RNA and DNA sequenced using next-generation sequencing., Results: Based on their genetic profile of deleterious gene alterations of BRCA1/2 or ATM, six patients were selected for PARPi. Patients with TMPRSS2:ERG gene fusion and homozygous alteration in ATM or BRCA2 (n=2) or heterozygous alterations (BRCA1 or BRCA2) and lack of TMPRSS2:ERG gene fusion (n=2) did not show clinical benefit from PARPi (treatment duration <16 weeks). In contrast, patients (n=2) without TMPRSS2:ERG gene fusion and homozygous deleterious alterations in ATM or BRCA2 all had clinical benefit from PARPi (treatment duration ≥16 weeks)., Conclusion: The TMPRSS2:ERG transcript product might be used as a PARPi resistance biomarker., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study was to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition and dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compound's efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein-3 (NS3) might prove as a funamental target for Betanin and Glycine Betaine against Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 μM-10 μM) of selected phytochemicals. The dose-response assay illustrated downregulated expression of DENV-2 NS3 gene after administration of Betanin (IC50 = 4.35 μM) and Glycine Betaine (IC50 = 4.49 μM). Dose response analysis of Betanin (80 μM-10 μM) depicted the significant inhibition of NS3 protein expression as well. These results suggested downregulated expression of DENV-2 NS3 at mRNA and protein level portraying the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: The transmembrane serine protease 4 (TMPRSS4) gene is upregulated in various human cancers. However, its biological functions in pancreatic ductal adenocarcinoma remain unclear. We examined the expression of TMPRSS4 in pancreatic ductal adenocarcinoma tissues and its correlation with clinicopathological parameters in patients with pancreatic ductal adenocarcinoma who underwent surgery., Methods: The TMPRSS4 expression was immunohistochemically examined in 81 PDAC patients with pancreatic ductal adenocarcinoma. We analyzed the association between the TMPRSS4 expression and clinicopathological factors, the recurrence-free survival (RFS), and the overall survival (OS) and examined the effect of TMPRSS4 expression on cell migration and sensitivity to 5-fluorouracil., Results: The expression rate of TMPRSS4 in the samples was 62.9% (51/81). The TMPRSS4 expression was not correlated with any clinicopathological feature. The five-year overall and recurrence-free survival rates were significantly lower in the TMPRSS4-positive group than in the TMPRSS4-negative group. On a multivariate analysis, TMPRSS4 positivity, poorly differentiated histology, and non-adjuvant chemotherapy predicted a poor OS, while TMPRSS4 positivity and poorly differentiated histology predicted a poor RFS. TMPRSS4-silenced pancreatic ductal adenocarcinoma cells showed higher sensitivity to 5- fluorouracil than did the control siRNA-transfected cells., Conclusions: TMPRSS4 can be considered a prognostic factor and therapeutic target for pancreatic ductal adenocarcinoma., (© 2024. The Author(s) under exclusive licence to Springer Nature Singapore Pte Ltd.)

The TMPRSS2 protease plays a key role in the entry of the SARS-CoV-2 into cells. The TMPRSS2 gene is highly polymorphic in humans, and some polymorphisms may affect the susceptibility to COVID-19 or disease severity. rs75603675 (c.23G > T) is a missense variant that causes the replacement of glycine with valine at position 8 (p.G8V) in the TMPRSS2 isoform 1. According to GnomAD v4.0.0 database, the allele frequency of the rs75603675 on a global scale is 38.10 %, and range from 0.92 % in East Asian to 40.77 % in non-Finnish European (NFE) population. We analyzed the occurrence of the rs75603675 in two cohorts of patients, the first with severe/critical COVID-19 enrolled in a French hospital (42 patients), and the second with predominantly asymptomatic/pauci-symptomatic/mild COVID-19 enrolled in an Italian hospital (69 patients). We found that the TMPRSS2-c.23T minor allele frequency was similar in the two cohorts, 46.43 % and 46.38 %, respectively, and higher than the frequency in the NFE population (40.77 %). Chi-square test provided significant results (p < 0.05) when the genotype data (TMPRSS2-c.23T/c.23T homozygotes + TMPRSS2-c.23G/c.23T heterozygotes vs. TMPRSS2-c.23G/c.23G homozygotes) of the two patient groups were pooled and compared to the expected data for the NFE population, suggesting a possible pathogenetic mechanism of the p.G8V substitution. We explored the possible effects of the p.G8V substitution and found that the N-terminal region of the TMPRSS2 isoform 1 contains a signal for clathrin/AP-2-dependent endocytosis. In silico analysis predicted that the p.G8V substitution may increase the accessibility to the endocytic signal, which could help SARS-CoV-2 enter cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declarations of interest: none., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cancer-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in the tumor microenvironment, which play important roles in regulating tumor progression and therapy resistance by transferring exosomes to cancer cells. However, how CAFs modulate esophageal squamous cell carcinoma (ESCC) progression and radioresistance remains incompletely understood. The expression of fibroblast activation protein (FAP) in CAFs was evaluated by immunohistochemistry in 174 ESCC patients who underwent surgery and 78 pretreatment biopsy specimens of ESCC patients who underwent definitive chemoradiotherapy. We sorted CAFs according to FAP expression, and the conditioned medium (CM) was collected to culture ESCC cells. The expression levels of several lncRNAs that were considered to regulate ESCC progression and/or radioresistance were measured in exosomes derived from FAP + CAFs and FAP - CAFs. Subsequently, cell counting kit-8, 5-ethynyl-2'-deoxyuridine, transwell, colony formation, and xenograft assays were performed to investigate the functional differences between FAP + CAFs and FAP - CAFs. Finally, a series of in vitro and in vivo assays were used to evaluate the effect of AFAP1-AS1 on radiosensitivity of ESCC cells. FAP expression in stromal CAFs was positively correlated with nerve invasion, vascular invasion, depth of invasion, lymph node metastasis, lack of clinical complete response and poor survival. Culture of ESCC cells with CM/FAP + CAFs significantly increased cancer proliferation, migration, invasion and radioresistance, compared with culture with CM/FAP - CAFs. Importantly, FAP + CAFs exert their roles by directly transferring the functional lncRNA AFAP1-AS1 to ESCC cells via exosomes. Functional studies showed that AFAP1-AS1 promoted radioresistance by enhancing DNA damage repair in ESCC cells. Clinically, high levels of plasma AFAP1-AS1 correlated with poor responses to dCRT in ESCC patients. Our findings demonstrated that FAP + CAFs promoted radioresistance in ESCC cells through transferring exosomal lncRNA AFAP1-AS1; and may be a potential therapeutic target for ESCC treatment., (© 2024 Wiley Periodicals LLC.)

Low-grade serous ovarian carcinoma (LGSC) is a rare and lethal subtype of ovarian cancer. LGSC is pathologically, biologically, and clinically distinct from the more common high-grade serous ovarian carcinoma (HGSC). LGSC arises from serous borderline ovarian tumours (SBTs). The mechanism of transformation for SBTs to LGSC remains poorly understood. To better understand the biology of LGSC, we performed whole proteome profiling of formalin-fixed, paraffin-embedded tissue blocks of LGSC (n = 11), HGSC (n = 19), and SBTs (n = 26). We identified that the whole proteome is able to distinguish between histotypes of the ovarian epithelial tumours. Proteins associated with the tumour microenvironment were differentially expressed between LGSC and SBTs. Fibroblast activation protein (FAP), a protein expressed in cancer-associated fibroblasts, is the most differentially abundant protein in LGSC compared with SBT. Multiplex immunohistochemistry (IHC) for immune markers (CD20, CD79a, CD3, CD8, and CD68) was performed to determine the presence of B cells, T cells, and macrophages. The LGSC FAP + stroma was associated with greater abundance of Tregs and M2 macrophages, features not present in SBTs. Our proteomics cohort reveals that there are changes in the tumour microenvironment in LGSC compared with its putative precursor lesion, SBT. These changes suggest that the tumour microenvironment provides a supportive environment for LGSC tumourigenesis and progression. Thus, targeting the tumour microenvironment of LGSC may be a viable therapeutic strategy. © 2024 The Pathological Society of Great Britain and Ireland., (© 2024 The Pathological Society of Great Britain and Ireland.)

Dengue virus (DENV2) is the cause of dengue disease and a worldwide health problem. DENV2 replicates in the host cell using polyproteins such as NS3 protease in conjugation with NS2B cofactor, making NS3 protease a promising antiviral drug-target. This study investigated the efficacy of 'Niloticin' against NS2B/NS3-protease. In silico and in vitro analyses were performed which included interaction of niloticin with NS2B/NS3-protease, protein stability and flexibility, mutation effect, betweenness centrality of residues and analysis of cytotoxicity, protein expression and WNV NS3-protease activity. Similar like acyclovir, niloticin forms strong H-bonds and hydrophobic interactions with residues LEU149, ASN152, LYS74, GLY148 and ALA164. The stability of the niloticin-NS2B/NS3-protease complex was found to be stable compared to the apo NS2B/NS3-protease in structural deviation, PCA, compactness and FEL analysis. The IC 50 value of niloticin was 0.14 μM in BHK cells based on in vitro cytotoxicity analysis and showed significant activity at 2.5 μM in a concentration-dependent manner. Western blotting and qRT-PCR analyses showed that niloticin reduced DENV2 protein transcription in a dose-dependent manner. Besides, niloticin confirmed the inhibition of NS3-protease by the SensoLyte 440 WNV protease detection kit. These promising results suggest that niloticin could be an effective antiviral drug against DENV2 and other flaviviruses., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Le Pen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver damage, highlighting the limitations of current emergency treatments that primarily involve administering the glutathione precursor N-acetylcysteine and supportive therapy. This study highlights the essential protective role of the type II transmembrane serine protease (TTSP), hepsin, in mitigating acetaminophen-induced liver injury, particularly through its regulation of gap junction (GJ) abundance in response to reactive oxygen stress in the liver. We previously reported that reduced levels of activated hepatocyte growth factor and the c-Met receptor tyrosine kinase-both of which are vital for maintaining cellular redox balance-combined with increased expression of GJ proteins in hepsin-deficient mice. Here, we show that hepsin deficiency in mice exacerbates acetaminophen toxicity compared to wild-type mice, leading to more severe liver pathology, elevated oxidative stress, and greater mortality within 6 h after exposure. Administering hepsin had a protective effect in both mouse models, reducing hepatotoxicity by modulating GJ abundance. Additionally, transcriptome analysis and a functional GJ inhibitor have highlighted hepsin's mechanism for managing oxidative stress. Combining hepsin with relatively low doses of N-acetylcysteine had a synergistic effect that was more efficacious than high-dose N-acetylcysteine alone. Our results illustrate the crucial role of hepsin in modulating the abundance of hepatic GJs and reducing oxidative stress, thereby offering early protection against acetaminophen-induced hepatotoxicity and a new, combination approach. Emerging as a promising therapeutic target, hepsin holds potential for combination therapy with N-acetylcysteine, paving the way for novel approaches in managing drug-induced liver injury., (© 2024. The Author(s).)

Serine protease 50 (PRSS50/TSP50) is highly expressed in spermatocytes. Our study investigated its role in testicular development and spermatogenesis. Initially, PRSS50 knockdown was observed to impair DNA synthesis in spermatocytes. To further explore this, we generated PRSS50 knockout ( Prss50 -/- ) mice ( Mus musculus ), which exhibited abnormal spermatid nuclear compression and reduced male fertility. Furthermore, dysplastic seminiferous tubules and decreased sex hormones were observed in 4-week-old Prss50 -/- mice, accompanied by meiotic progression defects and increased apoptosis of spermatogenic cells. Mechanistic analysis indicated that PRSS50 deletion resulted in increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and elevated levels of MAP kinase phosphatase 3 (MKP3), a specific ERK antagonist, potentially accounting for testicular dysplasia in adolescent Prss50 -/- mice. Taken together, these findings suggest that PRSS50 plays an important role in testicular development and spermatogenesis, with the MKP3/ERK signaling pathway playing a significant role in this process.

Soluble host factors in the upper respiratory tract can serve as the first line of defense against SARS-CoV-2 infection. In this study, we described the identification and function of a human airway trypsin-like protease (HAT), capable of reducing the infectivity of ancestral SARS-CoV-2. Further, in mouse models, HAT analogue expression was upregulated by SARS-CoV-2 infection. The antiviral activity of HAT functioned through the cleavage of the SARS-CoV-2 spike glycoprotein at R682. This cleavage resulted in inhibition of the attachment of ancestral spike proteins to host cells, which inhibited the cell-cell membrane fusion process. Importantly, exogenous addition of HAT notably reduced the infectivity of ancestral SARS-CoV-2 in vivo. However, HAT was ineffective against the Delta variant and most circulating Omicron variants, including the BQ.1.1 and XBB.1.5 subvariants. We demonstrate that the P681R mutation in Delta and P681H mutation in the Omicron variants, adjacent to the R682 cleavage site, contributed to HAT resistance. Our study reports what we believe to be a novel soluble defense factor against SARS-CoV-2 and resistance of its actions in the Delta and Omicron variants.

Background: Serum corin has been associated with stroke in observational studies, but the underlying causality is uncertain. This study examined the causal association between corin and stroke through Mendelian randomization study., Methods and Results: In the Gusu cohort, serum corin was assayed at baseline, and stroke incidents were prospectively obtained during 10 years of follow-up. Single-nucleotide polymorphisms (SNPs) in CORIN were genotyped by MassArray for 2310 participants (mean age, 53 years; 39% men). Seventeen SNPs passed the Hardy-Weinberg test and were considered the potential instruments. Only 1 SNP (rs2271037) determined variability of serum corin was significantly associated with stroke even after adjusting for conventional risk factors (hazard ratio [HR], 1.36 [95% CI, 1.00-1.85]). The weighted genetic risk score generated from the SNP-corin associations was significantly associated with stroke (HR, 2.01 [95% CI, 1.15-3.51]). Using this genetic risk score as the instrument, 1-sample Mendelian randomization analysis found a significant HR of stroke per-SD higher log 2 -transformed corin (HR, 1.37 [95% CI, 1.07-1.76]). The inverse variance-weighted analysis based on the SNP-corin and SNP-stroke associations found that the HR of stroke pre-SD higher log 2 -transformed corin was 5.92 (95% CI, 2.23-15.72). The effect estimates stayed consistent regardless of an individual SNP being removed from the instruments. An almost identical effect estimate was also confirmed by multiple other 2-sample Mendelian randomization methods., Conclusions: Genetically determined variations of serum corin concentration were significantly associated with the risk of stroke in Chinese adults. Elevated serum corin may be a risk factor for stroke.

Prolyl oligopeptidases from psychrophilic, mesophilic, and thermophilic organisms found in a range of natural environments are studied using a combination of protein structure prediction, atomistic molecular dynamics, and trajectory analysis to determine how the S9 protease family adapts to extreme thermal conditions. We compare our results with hypotheses from the literature regarding structural adaptations that allow proteins to maintain structure and function at extreme temperatures, and we find that, in the case of prolyl oligopeptidases, only a subset of proposed adaptations are employed for maintaining stability. The catalytic and propeller domains are highly structured, limiting the range of mutations that can be made to enhance hydrophobicity or form disulfide bonds without disrupting the formation of necessary secondary structure. Rather, we observe a pattern in which overall prevalence of bound interactions (salt bridges and hydrogen bonds) is conserved by using increasing numbers of increasingly short-lived interactions as temperature increases. This suggests a role for an entropic rather than energetic strategy for thermal adaptation in this protein family., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have described patients with deletions and nonsense variants in PREPL, but nothing is known about the effect of missense variants in the pathology of CMS22. In this study, we have functionally characterized missense variants in PREPL from 3 patients with CMS22, all with hallmark phenotypes. Biochemical evaluation revealed that these missense variants do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria and disease mechanism. Structural analysis showed that the variants affect regions most likely involved in intraprotein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the 3 variants. The importance of nonhydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines, which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the transgolgi network. In conclusion, these studies showed that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense variants that do not necessarily result in a loss of hydrolytic activity of PREPL.

This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections., (© 2024 Wiley Periodicals LLC.)

Antimicrobial resistance (AMR) is a serious global threat demanding innovations for effective control of pathogens. The bacterial SOS response, regulated by the master regulators, LexA and RecA, contributes to AMR through advantageous mutations. Targeting the LexA/RecA system with a novel inhibitor could suppress the SOS response and potentially reduce the occurrence of AMR. RecA presents a challenge as a therapeutic target due to its conserved structure and function across species, including humans. Conversely, LexA which is absent in eukaryotes, can be potentially targeted, due to its involvement in SOS response which is majorly responsible for adaptive mutagenesis and AMR. Our studies combining bioinformatic, biochemical, biophysical, molecular, and cell-based assays present a unique inhibitor of mycobacterial LexA, wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA of Mycobacterium tuberculosis (Mtb), consequently hindering its cleavage, suppressing SOS response thereby reducing mutation frequency and AMR., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of the article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In layer II of the entorhinal cortex, the principal neurons that project to the dentate gyrus and the CA3/2 hippocampal fields markedly express the large glycoprotein reelin (Re + ECLII neurons). In rodents, neurons located at the dorsal extreme of the EC, which border the rhinal fissure, express the highest levels, and the expression gradually decreases at levels successively further away from the rhinal fissure. Here, we test two predictions deducible from the hypothesis that reelin expression is strongly correlated with neuronal metabolic rate. Since the mitochondrial turnover rate serves as a proxy for energy expenditure, the mitophagy rate arguably also qualifies as such. Because messenger RNA of the canonical promitophagic BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) is known to be highly expressed in the EC, we predicted that Bnip3 would be upregulated in Re + ECLII neurons, and that the degree of upregulation would strongly correlate with the expression level of reelin in these neurons. We confirm both predictions, supporting that the energy requirement of Re + ECLII neurons is generally high and that there is a systematic increase in metabolic rate as one moves successively closer to the rhinal fissure. Intriguingly, the systematic variation in energy requirement of the neurons that manifest the observed reelin gradient appears to be consonant with the level of spatial and temporal detail by which they encode information about the external environment., (© 2024. The Author(s).)

The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del × Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin, yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del × Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del × Ctrl-KO mice spontaneously progressed to chronic pancreatitis, whereas C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del × Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice; however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis; however, the minor isoform CTRL also contributes to an appreciable extent. NEW & NOTEWORTHY Chymotrypsins defend the pancreas against the inflammatory disorder pancreatitis by degrading harmful trypsinogen. This study demonstrates that mice devoid of pancreatic chymotrypsins are phenotypically normal but become sensitized to secretagogue hyperstimulation and exhibit increased intrapancreatic trypsin activation, more severe acute pancreatitis, and rapid progression to chronic pancreatitis. The observations confirm and extend the essential role of chymotrypsins in pancreas health.

Background: Major depressive disorder (MDD) is notably underdiagnosed and undertreated due to its complex nature and subjective diagnostic methods. Biomarker identification would help provide a clearer understanding of MDD aetiology. Although machine learning (ML) has been implemented in previous studies to study the alteration of microRNA (miRNA) levels in MDD cases, clinical translation has not been feasible due to the lack of interpretability (i.e. too many miRNAs for consideration) and stability., Methods: This study applied logistic regression (LR) model to the blood miRNA expression profile to differentiate patients with MDD (n = 60) from healthy controls (HCs, n = 60). Embedded (L1-regularised logistic regression) feature selector was utilised to extract clinically relevant miRNAs, and optimized for clinical application., Results: Patients with MDD could be differentiated from HCs with the area under the receiver operating characteristic curve (AUC) of 0.81 on testing data when all available miRNAs were considered (which served as a benchmark). Our LR model selected miRNAs up to 5 (known as LR-5 model) emerged as the best model because it achieved a moderate classification ability (AUC = 0.75), relatively high interpretability (feature number = 5) and stability (ϕ̂Z=0.55) compared to the benchmark. The top-ranking miRNAs identified by our model have demonstrated associations with MDD pathways involving cytokine signalling in the immune system, the reelin signalling pathway, programmed cell death and cellular responses to stress., Conclusion: The LR-5 model, which is optimised based on ML design factors, may lead to a robust and clinically usable MDD diagnostic tool., Competing Interests: Declaration of competing interest There are no conflict of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRR→AGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP&#95;AGTAA mutant entry. Furthermore, we show that the EBOV GP&#95;AGTAA mutant is cleaved into fusion-competent GP 2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

It is believed that DNA double-strand breaks induced by Zika virus (ZIKV) infection in pregnant women is a main reason of brain damage (e.g. microcephaly, severe brain malformation, and neuropathy) in newborn babies [1,2], but its underlying mechanism is poorly understood. In this study, we report that the depletion of ERp57, a member of the protein disulphide isomerase (PDI) family, leads to the limited production of ZIKV in nerve cells. ERp57 knockout not only suppresses viral induced reactive oxygen species (ROS) mediated host DNA damage, but also decreases apoptosis. Strikingly, DNA damage depends on ERp57-bridged complex formation of viral protein NS2B/NS3. LOC14, an ERp57 inhibitor, restricts ZIKV infection and virus-induced DNA damage. Our work reveals an important role of ERp57 in both ZIKV propagation and virus-induced DNA damage, suggesting a potential target against ZIKV infection.

Emerging viruses, such as novel influenza A viruses (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose a constant threat to animal and human health. Identification of host cell factors necessary for viral replication but dispensable for cellular survival might reveal novel, attractive targets for therapeutic intervention. Proteolytic activation of IAV hemagglutinin (HA) and SARS-CoV-2 spike protein (S) by the type II transmembrane serine protease (TTSPs), e.g. TMPRSS2 is sought to be critical for viral spread and pathogenesis. Here, we investigated the secondary structure of TMPRSS2 mRNA coding sequence and designed TMPRSS2-specific antisense oligonucleotides (ASOs). Several of these ASOs markedly reduced the TMPRSS2 expression and decreased IAV infection and SARS-CoV-2 entry into cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteolytic activation of the haemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro . IAV infection in vivo resulted in a significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to humans.

Fibrous dysplasia (FD) is a mosaic skeletal disorder involving the development of benign, expansile fibro-osseous lesions during childhood that cause deformity, fractures, pain, and disability. There are no well-established treatments for FD. Fibroblast activation protein (FAPα) is a serine protease expressed in pathological fibrotic tissues that has promising clinical applications as a biomarker and local pro-drug activator in several pathological conditions. In this study, we explored the expression of FAP in FD tissue and cells through published genetic expression datasets and measured circulating FAPα in plasma samples from patients with FD and healthy donors. We found that FAP genetic expression was increased in FD tissue and cells, and present at higher concentrations in plasma from patients with FD compared to healthy donors. Moreover, FAPα levels were correlated with skeletal disease burden in patients with FD. These findings support further investigation of FAPα as a potential imaging and/or biomarker of FD, as well as a pro-drug activator specific to FD tissue.

Celiac disease (CD) is a common autoimmune disorder in which the patients are unable to digest gluten, which is present in foods made up of wheat, barley and rye. Whilst diagnosis happens late in 80% of the cases, avoidance of such foods appears to be the common solution. Alternative management strategies are required for the patients and their families since CD is also genetically carried over. Probiotic therapeutics and the consumption of appropriate enzymes, such as prolyloligopeptidases (POPs), from gut-friendly bacteria could reduce the disease burden and provide a better lifestyle for CD patients. We have examined around 5000 gut bacterial genomes and identified nearly 4000 non-redundant putative POPs. A select set of 10 gut bacterial POP sequences were subject to three-dimensional modelling, ligand docking and molecular dynamics simulations where stable interactions were observed between the POPs and gluten peptides. Our study provides sequence and structural analysis of potential POP enzymes in gut bacterial genomes, which form a strong basis to offer probiotic solutions to CD patients. In particular, these enzymes could be lead future therapeutics for this disease., (© 2024. The Author(s).)

West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.

Objective: To explore the role and molecular mechanism of cancer-associated fibroblasts (CAFs) in the tumor microenvironment of gastric cancer (GC)., Methods: The expression of CAFs in GC patients was first assessed for abundance, and survival analysis was performed. Subsequently, The Cancer Genome Atlas (TCGA) data were used for differential analysis, survival analysis, and EPIC analysis, while single-cell data (GSE183904) were downloaded for differential analysis of CAFs. Clinical data pooling, univariate and multivariate Cox analysis, and immunofluorescence were carried out on clinical GC tissue samples to explore RCN3 expression within patient CAFs. Western blot and quantitative polymerase chain reaction (qPCR) were used to detect the expression of RCN3. The relationship between RCN3 , PCSK6 , and STAT1 was explored by chromatin immunoprecipitation (CHIP) experiments, and the effects of the genes on macrophage polarization were detected by detecting biomarkers of biological M1/M2., Results: CAFs in GC were found to be significantly higher compared to the normal group. Revealing the results of TCGA differential analysis, it was observed that GC exhibited a substantial upregulation in the expression levels of RCN3. The clinical statistics indicate a positive correlation between an elevated level of RCN3 expression and the T-stage classification of tumor size. In addition, RCN3 was found to have a significant impact on the overall survival of patients with gastric cancer, acting as an independent prognostic indicator. Analysis of single-cell data showed high expression of PCSK6 in macrophages, and immunofluorescence staining of samples from GC patients showed increased expression of PCSK6 on the cell membranes of macrophages in GC tissues. The subsequent cellular experiments confirmed RCN3 protein can regulate the expression of PCSK6, and PCSK6 regulates macrophage polarization through STAT1., Conclusions: CAFs regulate macrophage polarization through the RCN3/PCSK6/STAT1 pathway in GC., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the β-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease., Competing Interests: The authors declare no conflict of interest.

Coronavirus disease 2019 (COVID-19) has led to a significant number of infections and deaths worldwide, yet its pathogenesis and severity remain incompletely understood. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), play crucial roles as receptors and molecules responsible for the virus's entry into host cells, initiating the infection process. Their polymorphisms have been extensively studied in relation to COVID-19 severity. The aim of this study was to examine the association of ACE2 (rs2074192) and TMPRSS2 (rs12329760) polymorphisms with COVID-19 outcome and severity. A prospective cohort study was conducted in 2022 at Haji Adam Malik Hospital, Medan, Indonesia. We randomly recruited hospitalized adult patients with COVID-19, confirmed by real-time polymerase chain reaction (RT-PCR). The baseline demographic data, disease severity, underlying disease, comorbidities, and COVID-19 vaccination status were collected. The single-nucleotide polymorphism (SNP) was assessed using TaqMan SNP genotyping assay, and the levels of TMPRSS2 and ACE2 proteins were measured using enzyme-linked immunosorbent assay (ELISA). A total of 151 COVID-19 patients were recruited and there were significant associations between age and severity with mortality outcomes. The age, kidney and lung diseases, and vaccination status were associated with severity levels. The results showed the CC genotype of ACE2 had the highest proportion, followed by TT and CT genotypes among patients, while CT was the most prevalent genotype, followed by CC and TT for TMPRSS2 . This study did not find a significant association between ACE2 and TMPRSS2 genetic variants with disease severity and outcome but highlighted a specific association of TMPRSS2 SNP with mortality within the group. In addition, ACE2 concentration was significant different between mild-moderate and severe-critical COVID-19 groups ( p =0.033)., Competing Interests: All the authors declare that there are no conflicts of interest., (© 2024 The Author(s).)

The serine protease CORIN catalyzes pro-atrial natriuretic peptide (pro-ANP) into an active ANP and maintains homeostasis of the internal environment. However, it is unclear whether CORIN participates in the regulation of tumor progression. We analyzed the expression profile of CORIN in gastric cancer tissues (GCs) and adjacent nontumoral tissues (NTs). We investigated the prognostic value of CORIN in GC patients. We characterized the in vitro and in vivo activity of CORIN in cultured GC cells with gain-of-function and loss-of-function experiments. The underlying mechanism was explored by using bioinformatics, a signaling antibody array, and confirmative western blot analyses, as well as rescue experiments with highly selective small-molecule inhibitors targeting the ERK1/2 MAPK signaling pathway. CORIN was upregulated in GCs than in NTs. Overexpression of CORIN was correlated with unfavorable prognoses in patients with GC. Ectopic expression of CORIN was promoted, whereas silencing of CORIN suppressed proliferation, colony formation, migration and invasion of GC cells, and tumor growth in vivo. Overexpression of CORIN-induced epithelial-mesenchymal transition (EMT) and activation of the ERK1/2 MAPK signaling pathway, while silencing of CORIN yielded opposite results. The in vitro tumor-promoting potency of CORIN could be antagonized by selective inhibitors targeting the ERK1/2 MAPK pathway. In conclusion, CORIN is a potential prognostic marker and therapeutic target for GC patients, which may promote tumor progression by mediating the ERK1/2 MAPK signaling pathway and EMT in GC cells., (© 2024 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.)

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)