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Microbial Biotechnology Jun 2024Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a...
Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a biodegradable commodity. Enzymatic recycling is one strategy for ensuring circularity of PLLA, but this approach requires a thorough understanding of enzymatic mechanisms and protein engineering strategies to enhance activity. In this study, we engineer PLLA depolymerizing subtilisin enzymes originating from Bacillus species to elucidate the molecular mechanisms dictating their PLLA depolymerization activity and to improve their function. The surface-associated amino acids of two closely related subtilisin homologues originating from Bacillus subtilis (BsAprE) and Bacillus pumilus (BpAprE) were compared, as they were previously engineered to have nearly identical active sites, but still varied greatly in PLLA depolymerizing activity. Further analysis identified several surface-associated amino acids in BpAprE that lead to enhanced PLLA depolymerization activity when engineered into BsAprE. In silico protein modelling demonstrated increased enzyme surface hydrophobicity in engineered BsAprE variants and revealed a structural motif favoured for PLLA depolymerization. Experimental evidence suggests that increases in activity are associated with enhanced polymer binding as opposed to substrate specificity. These data highlight enzyme adsorption as a key factor in PLLA depolymerization by subtilisins.
Topics: Polyesters; Adsorption; Polymerization; Bacillus; Subtilisins; Bacillus subtilis; Models, Molecular; Protein Engineering; Bacterial Proteins
PubMed: 38877615
DOI: 10.1111/1751-7915.14473 -
Journal of Neuroinflammation Jun 2024Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key...
Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.
Topics: Animals; Recovery of Function; Male; Plasma Kallikrein; Mice; Mice, Inbred C57BL; Infarction, Middle Cerebral Artery; Blood-Brain Barrier; Stroke; Thrombosis; Ischemic Stroke; Inflammation
PubMed: 38872149
DOI: 10.1186/s12974-024-03149-w -
PloS One 2024It has been postulated from a combination of evidence that a sudden increase in COVID-19 cases among pediatric patients after onset of the Omicron wave was attributed to...
It has been postulated from a combination of evidence that a sudden increase in COVID-19 cases among pediatric patients after onset of the Omicron wave was attributed to a reduced requirement for TMPRSS2-mediated entry in pediatric airways with lower expression levels of TMPRSS2. Epidemic strains were isolated from the indigenous population in an area, and the levels of TMPRSS2 required for Delta and Omicron variants were assessed. As a result, Delta variants proliferated fully in cultures of TMPRSS2-positive Vero cells but not in TMPRSS2-negative Vero cell culture (350-fold, Delta vs 9.6-fold, Omicron). There was no obvious age-dependent selection of Omicron strains affected by the TMPRSS2 (9.6-fold, Adults vs. 12-fold, Children). A phylogenetic tree was generated and Blast searches (up to 100 references) for the spread of strains in the study area showed that each strain had almost identical homology (>99.5%) with foreign isolates, although indigenous strains had obvious differences from each other. This suggested that the differences had been present abroad for a long period. Therefore, the lower requirement for TMPRSS2 by Omicron strains might be applicable to epidemic strains globally. In conclusion, the property of TMPRSS2-independent cleavage makes Omicron proliferate with ease and allows epidemics among children with fewer TMPRSS2 on epithelial surfaces of the respiratory organs.
Topics: SARS-CoV-2; Humans; Serine Endopeptidases; Chlorocebus aethiops; COVID-19; Vero Cells; Child; Animals; Child, Preschool; Phylogeny; Adult; Infant; Male; Female
PubMed: 38870131
DOI: 10.1371/journal.pone.0299445 -
Journal of Virology Jun 2024Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine...
Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3C) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3C selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3C cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3C partially differ from previously reported classical motifs recognized by other picornaviral 3C, highlighting the distinct characteristics of SVA 3C. Together, these results reveal a mechanism by which SVA 3C antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3C.IMPORTANCESenecavirus A (SVA), the only member in the genus within the family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3C, a protease of SVA, functions as an antagonist for the IFN response. 3C utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3C as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.
PubMed: 38869319
DOI: 10.1128/jvi.00585-24 -
Croatian Medical Journal Jun 2024To assess the association of single nucleotide polymorphisms (SNPs) in the ACE2 and TMPRSS2 genes with COVID-19 severity and key biomarkers.
AIM
To assess the association of single nucleotide polymorphisms (SNPs) in the ACE2 and TMPRSS2 genes with COVID-19 severity and key biomarkers.
METHODS
The study involved 750 COVID-19 patients from Bosnia and Herzegovina, divided into three groups: mild, moderate, and severe cases. Genetic variations within the ACE2 (rs2285666) and TMPRSS2 (rs2070788) genes were examined with real-time polymerase chain reaction. Biochemical markers were determined with standard procedures.
RESULTS
There was a significant difference in the rs2070788 genotype distribution between patients with mild and moderate symptoms, but not between other groups. For the rs2285666 polymorphism, no significant difference in genotype distribution was found. In patients with mild symptoms, carriers of the GG genotype of rs2070788 had significantly higher total bilirubin levels than carriers of the AA genotype. Similarly, carriers of the TT genotype of rs2285666 had significantly higher activated partial thromboplastin time and international normalized ratio, and lower lactate dehydrogenase levels compared with the CC genotype. Among patients with severe symptoms, carriers of the GG genotype showed significantly higher potassium levels than carriers of the AA genotype, while carriers of the TT genotype showed significantly higher erythrocyte count as well as hemoglobin and hematocrit levels compared with the CC genotype.
CONCLUSION
This study highlights the role of genetic factors, particularly SNPs in the ACE2 and TMPRSS2 genes, in determining COVID-19 severity, aiding patient risk assessment and prognosis.
Topics: Humans; Serine Endopeptidases; COVID-19; Angiotensin-Converting Enzyme 2; Male; Female; Bosnia and Herzegovina; Middle Aged; Polymorphism, Single Nucleotide; Severity of Illness Index; Biomarkers; SARS-CoV-2; Adult; Aged; Genotype
PubMed: 38868968
DOI: 10.3325/cmj.2024.65.220 -
Scientific Reports Jun 2024Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of...
Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of non-coding-RNA in cancer proliferation and carcinogenesis has attracted much attention in the last decade; however, microRNAs (miRNAs), as non-coding RNA, are considered master mediators in various cancer progressions. Yet the role of miR-141 as a modulator for specific cellular processes in liver cancer cell proliferation is still unclear. This study identified the role of miR-141 and its potential functions in liver carcinogenesis. The level of miR-141 in HepG2 and HuH7 cells was assessed using quantitative real-time PCR (qRT-PCR) and compared with its expression in normal hepatocytes. A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HepG2 cells. The protein profile of the kallikrein-related peptidase 10 (KLK10) and tumor necrosis factor TNFSF-15 was investigated in HepG2 cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced inflammatory cytokines from transfected HepG2 cells. Our findings showed that the expression of miR-141 significantly increased in HepG2 and HuH7 cells compared to the normal hepatocytes. Transfection of HepG2 cells with an inhibitor, antagonist miR-141, showed a significant reduction of HepG2 cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HepG2 cells overexpressed miR-141 provided a hundred downregulated genes, including KLK10 and TNFSF-15. Furthermore, the expression profile of KLK10 and TNFSF-15 markedly depleted in HepG2 cells transfected with miR-141 overexpression accompanied by a decreasing level of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α), indicating the role of miR-141 in HepG2 cell proliferation and programmed cell death. Interestingly, the experimental rats with liver cancer induced by Diethylnitrosamine injection further confirmed the upregulation of miR-141 level, IL-10, and TNF-α and the disturbance in KLK10 and TNFSF-15 gene expression compared with their expression in normal rats. The in-silico online tools, IntaRNA and miRWalk were used to confirm the direct interaction and potential binding sites between miR-141 and identified genes. Thus, the seeding regions of potential targeted sequences was cloned upstream of luciferase reporter gene in pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HepG2 cells pre-transfected with miR-141 overexpression vector, while increasing in cells pre-transfected with miR-141 specific inhibitor. In summary, these data suggest the crucial role of miR-141 in liver cancer development via targeting KLK10 and TNFSF-15 and provide miR-141 as an attractive candidate in liver cancer treatment and protection.
Topics: MicroRNAs; Humans; Liver Neoplasms; Gene Expression Regulation, Neoplastic; Hepatoblastoma; Hep G2 Cells; Cell Proliferation; Kallikreins; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor
PubMed: 38866875
DOI: 10.1038/s41598-024-63223-4 -
Scientific Reports Jun 2024Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The incidence of cSCC is increasing globally and the prognosis of metastatic disease...
Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The incidence of cSCC is increasing globally and the prognosis of metastatic disease is poor. Currently there are no specific targeted therapies for advanced or metastatic cSCC. We have previously shown abundant expression of the complement classical pathway C1 complex components, serine proteases C1r and C1s in tumor cells in invasive cSCCs in vivo, whereas the expression of C1r and C1s was lower in cSCCs in situ, actinic keratoses and in normal skin. We have also shown that knockdown of C1s expression results in decreased viability and growth of cSCC cells by promoting apoptosis both in culture and in vivo. Here, we have studied the effect of specific IgG2a mouse monoclonal antibodies TNT003 and TNT005 targeting human C1s in five primary non-metastatic and three metastatic cSCC cell lines that show intracellular expression of C1s and secretion of C1s into the cell culture media. Treatment of cSCC cells with TNT003 and TNT005 significantly inhibited their growth and viability and promoted apoptosis of cSCC cells. These data indicate that TNT003 and TNT005 inhibit cSCC cell growth in culture and warrant further investigation of C1s targeted inhibition in additional in vitro and in vivo models of cSCC.
Topics: Humans; Carcinoma, Squamous Cell; Skin Neoplasms; Cell Proliferation; Cell Line, Tumor; Apoptosis; Animals; Mice; Antibodies, Monoclonal; Cell Survival
PubMed: 38866870
DOI: 10.1038/s41598-024-64088-3 -
Respiratory Research Jun 2024TMPRSS2, a key molecule for SARS-CoV-2 invading human host cells, has an association with cancer. However, its association with lung cancer remains insufficiently...
BACKGROUND
TMPRSS2, a key molecule for SARS-CoV-2 invading human host cells, has an association with cancer. However, its association with lung cancer remains insufficiently unexplored.
METHODS
In five bulk transcriptomics datasets, one single-cell RNA sequencing (scRNA-seq) dataset and one proteomics dataset for lung adenocarcinoma (LUAD), we explored associations between TMPRSS2 expression and immune signatures, tumor progression phenotypes, genomic features, and clinical prognosis in LUAD by the bioinformatics approach. Furthermore, we performed experimental validation of the bioinformatics findings.
RESULTS
TMPRSS2 expression levels correlated negatively with the enrichment levels of both immune-stimulatory and immune-inhibitory signatures, while they correlated positively with the ratios of immune-stimulatory/immune-inhibitory signatures. It indicated that TMPRSS2 levels had a stronger negative correlation with immune-inhibitory than with immune-stimulatory signatures. TMPRSS2 downregulation correlated with increased proliferation, stemness, genomic instability, tumor progression, and worse survival in LUAD. We further validated that TMPRSS2 was downregulated with tumor progression in the LUAD cohort we collected from Jiangsu Cancer Hospital, China. In vitro and in vivo experiments verified the association of TMPRSS2 deficiency with increased tumor cell proliferation and invasion and antitumor immunity in LUAD. Moreover, in vivo experiments demonstrated that TMPRSS2-knockdown tumors were more sensitive to BMS-1, an inhibitor of PD-1/PD-L1.
CONCLUSIONS
TMPRSS2 is a tumor suppressor, while its downregulation is a positive biomarker of immunotherapy in LUAD. Our data provide a potential link between lung cancer and pneumonia caused by SARS-CoV-2 infection.
Topics: Humans; Adenocarcinoma of Lung; Lung Neoplasms; Down-Regulation; Serine Endopeptidases; Animals; Mice; COVID-19; Immunotherapy; Gene Expression Regulation, Neoplastic; Male; Genes, Tumor Suppressor; Female; Mice, Nude; Cell Line, Tumor; SARS-CoV-2
PubMed: 38862975
DOI: 10.1186/s12931-024-02870-7 -
Scientific Reports Jun 2024**Ischemic stroke remains a leading cause of morbidity and mortality globally. Despite the advances in thrombolytic therapy, notably recombinant tissue plasminogen...
**Ischemic stroke remains a leading cause of morbidity and mortality globally. Despite the advances in thrombolytic therapy, notably recombinant tissue plasminogen activator (rtPA), patient outcomes are highly variable. This study aims to introduce a novel predictive model, the Acute Stroke Thrombolysis Non-Responder Prediction Model (ASTN-RPM), to identify patients unlikely to benefit from rtPA within the critical early recovery window. We conducted a retrospective cohort study at Baoding No.1 Central Hospital including 709 adult patients diagnosed with acute ischemic stroke and treated with intravenous alteplase within the therapeutic time window. The ASTN-RPM was developed using Least Absolute Shrinkage and Selection Operator (LASSO) regression technique, incorporating a wide range of biomarkers and clinical parameters. Model performance was evaluated using Receiver Operating Characteristic (ROC) curves, calibration plots, and Decision Curve Analysis (DCA). ASTN-RPM effectively identified patients at high risk of poor response to thrombolysis, with an AUC of 0.909 in the training set and 0.872 in the validation set, indicating high sensitivity and specificity. Key predictors included posterior circulation stroke, high admission NIHSS scores, extended door to needle time, and certain laboratory parameters like homocysteine levels. The ASTN-RPM stands as a potential tool for refining clinical decision-making in ischemic stroke management. By anticipating thrombolytic non-response, clinicians can personalize treatment strategies, possibly improving patient outcomes and reducing the burden of ineffective interventions. Future studies are needed for external validation and to explore the incorporation of emerging biomarkers and imaging data.
Topics: Humans; Ischemic Stroke; Male; Biomarkers; Female; Thrombolytic Therapy; Aged; Middle Aged; Retrospective Studies; Tissue Plasminogen Activator; Fibrinolytic Agents; ROC Curve; Treatment Outcome
PubMed: 38862629
DOI: 10.1038/s41598-024-64413-w -
Theranostics 2024Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene...
Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene expression during murine APAP-induced ALI by 3'mRNA sequencing (massive analysis of cDNA ends, MACE), we observed splenic mRNA accumulation encoding for the neutrophil serine proteases cathepsin G, neutrophil elastase, and proteinase-3 - all are hierarchically activated by cathepsin C (CtsC). This, along with increased serum levels of these proteases in diseased mice, concurs with the established phenomenon of myeloid cell mobilization during APAP intoxication. In order to functionally characterize CtsC in murine APAP-induced ALI, effects of its genetic or pharmacological inhibition were investigated. We report on substantially reduced APAP toxicity in CtsC deficient mice. Alleviation of disease was likewise observed by treating mice with the CtsC inhibitor AZD7986, both in short-term prophylactic and therapeutic protocols. This latter observation indicates a mode of action beyond inhibition of granule-associated serine proteases. Protection in CtsC knockout or AZD7986-treated wildtype mice was unrelated to APAP metabolization but, as revealed by MACE, realtime PCR, or ELISA, associated with impaired expression of inflammatory genes with proven pathogenic roles in ALI. Genes consistently downregulated in protocols tested herein included , , and . Moreover, , a positive regulator of the toll-like receptor/interferon-axis, was reduced by targeting CtsC. This work suggests CtsC as promising therapeutic target for the treatment of ALI, among others paradigmatic APAP-induced ALI. Being also currently evaluated in phase III clinical trials for bronchiectasis, successful application of AZD7986 in experimental APAP intoxication emphasizes the translational potential of this latter therapeutic approach.
Topics: Animals; Acetaminophen; Cathepsin C; Mice; Chemical and Drug Induced Liver Injury; Mice, Inbred C57BL; Mice, Knockout; Male; Disease Models, Animal
PubMed: 38855187
DOI: 10.7150/thno.96092