-
Marine Drugs May 2024Brown seaweeds of the genus represent a rich source of natural antiviral products. In this study, a hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of...
Brown seaweeds of the genus represent a rich source of natural antiviral products. In this study, a hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme's proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease's active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.
Topics: Coronavirus 3C Proteases; Antiviral Agents; Ligands; Fucus; Protease Inhibitors; SARS-CoV-2; Plant Extracts; Peptides; Molecular Docking Simulation; Humans; Seaweed
PubMed: 38921555
DOI: 10.3390/md22060244 -
Marine Drugs May 2024Antarctica, one of the most extreme environments on Earth, hosts diverse microbial communities. These microbes have evolved and adapted to survive in these hostile...
Antarctica, one of the most extreme environments on Earth, hosts diverse microbial communities. These microbes have evolved and adapted to survive in these hostile conditions, but knowledge on the molecular mechanisms underlying this process remains limited. The Italian Collection of Antarctic Bacteria ( (CIBAN)), managed by the University of Messina, represents a valuable repository of cold-adapted bacterial strains isolated from various Antarctic environments. In this study, we sequenced and analyzed the genomes of 58 marine Gammaproteobacteria strains from the CIBAN collection, which were isolated during Italian expeditions from 1990 to 2005. By employing genome-scale metrics, we taxonomically characterized these strains and assigned them to four distinct genera: , , and . Genome annotation revealed a previously untapped functional potential, including secondary metabolite biosynthetic gene clusters and antibiotic resistance genes. Phylogenomic analyses provided evolutionary insights, while assessment of cold-shock protein presence shed light on adaptation mechanisms. Our study emphasizes the significance of CIBAN as a resource for understanding Antarctic microbial life and its biotechnological potential. The genomic data unveil new horizons for insight into bacterial existence in Antarctica.
Topics: Antarctic Regions; Phylogeny; Genome, Bacterial; Gammaproteobacteria; Genomics; Psychrobacter; Pseudoalteromonas; Multigene Family
PubMed: 38921549
DOI: 10.3390/md22060238 -
Frontiers in Immunology 2024Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood...
INTRODUCTION
Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations.
METHODS
Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice).
RESULTS
Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits.
CONCLUSION
We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.
Topics: Complement Factor H; Animals; Humans; Mice; Complement C3; Mice, Knockout; Disease Models, Animal; Proof of Concept Study; Mice, Inbred C57BL
PubMed: 38919628
DOI: 10.3389/fimmu.2024.1334151 -
Virology Journal Jun 2024The genus Jeilongvirus comprises non-segmented negative-stranded RNA viruses that are classified within the Paramyxoviridae family by phylogeny. Jeilongviruses are found...
The genus Jeilongvirus comprises non-segmented negative-stranded RNA viruses that are classified within the Paramyxoviridae family by phylogeny. Jeilongviruses are found in various reservoirs, including rodents and bats. Rodents are typical viral reservoirs with diverse spectra and zoonotic potential. Little is currently known about jeilongviruses in rodents from central China. The study utilized high-throughput and Sanger sequencing to obtain jeilongvirus genomes, including those of two novel strains (HBJZ120/CHN/2021 (17,468 nt) and HBJZ157/CHN/2021 (19,143 nt)) and three known viruses (HBXN18/CHN/2021 (19,212 nt), HBJZ10/CHN/2021 (19,700 nt), HBJM106/CHN/2021 (18,871 nt)), which were characterized by genome structure, identity matrix, and phylogenetic analysis. Jeilongviruses were classified into three subclades based on their topology, phylogeny, and hosts. Based on the amino acid sequence identities and phylogenetic analysis of the L protein, HBJZ120/CHN/2021 and HBJZ157/CHN/2021 were found to be strains rather than novel species. Additionally, according to specific polymerase chain reaction screening, the positive percentage of Beilong virus in Hubei was 6.38%, suggesting that Beilong virus, belonging to the Jeilongvirus genus, is likely to be widespread in wild rodents. The identification of novel strains further elucidated the genomic diversity of jeilongviruses. Additionally, the prevalence of jeilongviruses in Hubei, China, was profiled, establishing a foundation for the surveillance and early warning of emerging paramyxoviruses.
Topics: Phylogeny; Animals; China; Genome, Viral; Rodentia; Animals, Wild; Paramyxovirinae; RNA, Viral; Paramyxoviridae Infections; High-Throughput Nucleotide Sequencing; Disease Reservoirs; Sequence Analysis, DNA
PubMed: 38918816
DOI: 10.1186/s12985-024-02417-8 -
BMC Oral Health Jun 2024Streptococcus mutans (S. mutans) is an important pathogenic bacterium that causes dental caries, while Streptococcus gordonii (S. gordonii) is a non-cariogenic bacterium...
BACKGROUND
Streptococcus mutans (S. mutans) is an important pathogenic bacterium that causes dental caries, while Streptococcus gordonii (S. gordonii) is a non-cariogenic bacterium that inhibits the growth of S. mutans. The SepM protein can promote the inhibitory ability of S. mutans against S. gordonii by cleaving CSP-21 and activating the ComDE two-component system. This study was designed to explore sepM mutation in S. mutans clinical isolates and related function in the regulation of interactions with S. gordonii.
METHODS
The S. mutans clinical strains that can inhibit the growth of S. gordonii constitute the inhibitory group. 286 C-serotype S. mutans strains were categorized into S. gordonii inhibitory (n = 114) and non-inhibitory bacteria (n = 172). We detected sanger sequencing of sepM gene, the expression levels of related genes and proteins in clinical isolates, obtained prokaryotic expression and purification of mutated proteins, and analyzed the effect of the target mutations on the binding between SepM and CSP-21.
RESULTS
We found that C482T, G533A, and G661A missense mutations were presented at significantly higher frequency in the inhibitory group relative to the non-inhibitory group. There was no significant difference in the expression of the sepM gene between selected clinical isolates harboring the G533A mutation and the control group. The expression levels of SepM, phosphorylated ComD, and ComE in the mutation group were significantly higher than those in the control group. SepM_control and SepM_D221N (G661A at the gene level) were found to contain two residues close to the active center while SepM_G178D (G533A at the gene level) contained three residues close to the active center. At 25 °C and a pH of 5.5, SepM_D221N (G661A) exhibited higher affinity for CSP-21 (KD = 8.25 µM) than did the SepM control (KD = 33.1 µM), and at 25 °C and a pH of 7.5, SepM_G178D (G533A) exhibited higher affinity (KD = 3.02 µM) than the SepM control (KD = 15.9 µM). It means that it is pH dependent.
CONCLUSIONS
Our data suggest that increased cleavage of CSP-21 by the the mutant SepM may be a reason for the higher inhibitory effect of S. mutans on S. gordonii .
Topics: Streptococcus mutans; Bacterial Proteins; Streptococcus gordonii; Humans; Mutation; Mutation, Missense; Dental Caries
PubMed: 38918777
DOI: 10.1186/s12903-024-04436-x -
Asian Pacific Journal of Cancer... Jun 2024Standard tools are not sensitive enough for hepatocellular carcinoma (HCC) early detection. This study aimed to evaluate the accuracy of dickkopf-1 (DKK1) and soluble...
BACKGROUND
Standard tools are not sensitive enough for hepatocellular carcinoma (HCC) early detection. This study aimed to evaluate the accuracy of dickkopf-1 (DKK1) and soluble Axl (sAxl) and their combined for early differentiating of HCC from premalignant benign liver diseases.
METHODS
A total of 210 chronic hepatitis C (CHC) patients (55 fibrotic, 45 cirrhotic and 110 HCC) were enrolled. Both DKK1 and sAxl were tested using ELISA for all participants.
RESULTS
HCC patients were accompanied by a significant increase (P<0.05) in DKK1 (5.38±2.05 ng/mL) and sAxl (178.02±49.39 ng/mL) compared to patients with fibrosis (2.16±0.6, 97.63±19.71 ng/mL, respectively) and cirrhosis (2.62±0.8, 121.84±34.66 ng/mL, respectively). Both DKK1 (AUC=0.852) and sAxl (AUC=0.882) had a good diagnostic accuracy in separating HCC from all non-HCC patients. Multiplying DKK1 with sAXL yielded values that significantly (P=0.0001) increased in patients who developed HCC (674.3 (434.2-1413.9)) versus fibrotic (204.9 (161.7-262)) and cirrhotic (254.4 (205.4-343.7)) patients. This model improves HCC diagnostic performances [AUC=0.921; sensitivity 90.9%, specificity 87%, PPV 88.5%, NPV 89.7% and efficiency 89.1%]. Elevated DKK1×sAxl values were associated with aggressive tumor features including multiple nodules, large size, Child-Pugh and BCLC late stages.
CONCLUSIONS
combined use of DKK1×sAxl is simple and feasible HCC diagnostic model that could enhance HCC diagnostic accuracy and could replace AFP in follow up of patients with premalignant diseases.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Intercellular Signaling Peptides and Proteins; Male; Axl Receptor Tyrosine Kinase; Female; Middle Aged; Receptor Protein-Tyrosine Kinases; Biomarkers, Tumor; Proto-Oncogene Proteins; Hepatitis C, Chronic; Prognosis; Liver Cirrhosis; Follow-Up Studies; Adult; Hepacivirus; Early Detection of Cancer; Aged
PubMed: 38918682
DOI: 10.31557/APJCP.2024.25.6.2185 -
The Korean Journal of Gastroenterology... Jun 2024Toxocariasis, a zoonotic infection transmitted by (from dogs) and (from cats) larvae, poses rare but severe risks to humans. We present a case of hepatic visceral... (Review)
Review
Toxocariasis, a zoonotic infection transmitted by (from dogs) and (from cats) larvae, poses rare but severe risks to humans. We present a case of hepatic visceral larva migrans (VLM) caused by in a 21-year-old male with a history of close contact with a pet dog. Initial symptoms and imaging findings mimicked a pyogenic liver abscess. The initial laboratory investigations revealed neutrophilia and elevated levels of IgE. Despite broad-spectrum antibiotics, persistent fever prompted further investigation. Subsequent serological testing for Toxocara antibodies and histopathological analysis of liver tissue demonstrating eosinophil infiltrates and Charcot-Leyden crystals led to a confirmed diagnosis of a liver abscess caused by . Serological testing for Toxocara antibodies and histopathological analysis of liver tissue confirmed a -induced liver abscess. Albendazole treatment yielded significant clinical improvement. This case highlights the necessity of considering toxocariasis in liver abscess differentials, particularly in high-seroprevalence regions like Vietnam. Relying solely on serological tests may be insufficient, emphasizing the need for corroborative evidence, including invasive procedures like liver biopsy, for accurate hepatic toxocariasis diagnosis.
Topics: Humans; Toxocara canis; Larva Migrans, Visceral; Male; Animals; Young Adult; Albendazole; Tomography, X-Ray Computed; Dogs; Liver; Antibodies, Helminth; Ultrasonography; Liver Abscess; Toxocariasis; Immunoglobulin E; Anthelmintics
PubMed: 38918038
DOI: 10.4166/kjg.2024.051 -
PloS One 2024Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug...
Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.
Topics: Humans; Myeloblastin; Ligands; Recombinant Proteins; Animals; Sf9 Cells; Surface Plasmon Resonance; Protein Binding; Baculoviridae; Kinetics; Gene Expression; Spodoptera
PubMed: 38917138
DOI: 10.1371/journal.pone.0294827 -
BioRxiv : the Preprint Server For... Jun 2024Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF...
Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.
PubMed: 38915515
DOI: 10.1101/2024.06.13.598386 -
Frontiers in Immunology 2024Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza...
Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza hemagglutinin (HA) ignoring the neuraminidase (NA) as an important surface target. Vaccination with standardized NA concentration may offer broader and longer-lasting protection against influenza infection. In this regard, we aimed to compare the potency of a NA displayed on the surface of a VLP with a soluble NA. The baculovirus expression system (BEVS) and the novel virus-free Tnms42 insect cell line were used to express N2 NA on gag-based VLPs. To produce VLP immunogens with high levels of purity and concentration, a two-step chromatography purification process combined with ultracentrifugation was used. In a prime/boost vaccination scheme, mice vaccinated with 1 µg of the N2-VLPs were protected from mortality, while mice receiving the same dose of unadjuvanted NA in soluble form succumbed to the lethal infection. Moreover, NA inhibition assays and NA-ELISAs of pre-boost and pre-challenge sera confirm that the VLP preparation induced higher levels of NA-specific antibodies outperforming the soluble unadjuvanted NA.
Topics: Animals; Neuraminidase; Influenza Vaccines; Vaccines, Virus-Like Particle; Mice; Antibodies, Viral; Orthomyxoviridae Infections; Antibodies, Neutralizing; Female; Mice, Inbred BALB C; Recombinant Proteins; Vaccine Efficacy; Humans; Vaccination
PubMed: 38915410
DOI: 10.3389/fimmu.2024.1425842