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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 -
Emerging Infectious Diseases Jul 2024Few cases of hantavirus pulmonary syndrome have been reported in northeastern Argentina. However, neighboring areas show a higher incidence, suggesting underreporting....
Few cases of hantavirus pulmonary syndrome have been reported in northeastern Argentina. However, neighboring areas show a higher incidence, suggesting underreporting. We evaluated the presence of antibodies against orthohantavirus in small rodents throughout Misiones province. Infected Akodon affinis montensis and Oligoryzomys nigripes native rodents were found in protected areas of Misiones.
Topics: Animals; Argentina; Orthohantavirus; Antibodies, Viral; Hantavirus Infections; Rodentia; Rodent Diseases; Humans; Hantavirus Pulmonary Syndrome; Disease Reservoirs
PubMed: 38916725
DOI: 10.3201/eid3007.240183 -
BioRxiv : the Preprint Server For... Jun 2024Gangliosides are sialylated glycosphingolipids with essential but enigmatic functions in healthy and disease brains. GD3 is the predominant species in neural stem cells...
Gangliosides are sialylated glycosphingolipids with essential but enigmatic functions in healthy and disease brains. GD3 is the predominant species in neural stem cells (NSCs) and GD3-synthase (sialyltransferase II; ) knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits including cognitive impairment, depression-like phenotypes, and olfactory dysfunction. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal, followed by restored neuronal functions. Our group discovered that GD3 is involved in the maintenance of NSC fate determination by interacting with epidermal growth factor receptors (EGFRs), by modulating expression of cyclin-dependent kinase (CDK) inhibitors p27 and p21, and by regulating mitochondrial dynamics via associating a mitochondrial fission protein, the dynamin-related protein-1 (Drp1). Furthermore, we discovered that nuclear GM1 promotes neuronal differentiation by an epigenetic regulatory mechanism. GM1 binds with acetylated histones on the promoter of as well as on the in differentiated neurons. In addition, epigenetic activation of the GM2S gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. Interestingly, GM1 induced epigenetic activation of the gene, with recruitment of Nurr1 and PITX3, dopaminergic neuron-associated transcription factors, to the promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression, and it would modify Parkinson's disease. Multifunctional gangliosides significantly modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains. Maintaining proper ganglioside microdomains benefits healthy neuronal development and millions of senior citizens with neurodegenerative diseases. Here, we introduce how to isolate GD3 and GM1 and how to administer them into the mouse brain to investigate their functions on NSC fate determination and nerve cell specification.
PubMed: 38915682
DOI: 10.1101/2024.06.09.598109 -
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 -
Journal of Biological Engineering Jun 2024Breast cancer remains a challenge for physicians. Metformin, an antidiabetic drug, show promising anticancer properties against cancers. An emerging quantum dot (QD)...
BACKGROUND
Breast cancer remains a challenge for physicians. Metformin, an antidiabetic drug, show promising anticancer properties against cancers. An emerging quantum dot (QD) material improves therapeutic agents' anticancer and imaging properties. QD are nano-sized particles with extreme application in nanotechnology captured by cells and accumulated inside cells, suggesting bioimaging and effective anticancer outcomes. In this study, a simple one-pot hydrothermal method was used to synthesize fluorescent metformin-derived carbon dots (M-CDs) and then investigated the cytotoxic effects and imaging features on two human breast cancer cell lines including, MCF-7 and MDA-MB-231 cells.
RESULTS
Results showed that M-CDs profoundly decreased the viability of both cancer cells. IC50 values showed that M-CDs were more cytotoxic than metformin either 24-48 h post-treatment. Cancer cells uptake M-CDs successfully, which causes morphological changes in cells and increased levels of intracellular ROS. The number of Oil Red O-positive cells and the expression of caspase-3 protein were increased in M-CDs treated cells. Authophagic factors including, AMPK, mTOR, and P62 were down-regulated, while p-AMPK, Becline-1, LC3 I, and LC3 II were up-regulated in M-CDs treated cells. Finally, M-CDs caused a decrease in the wound healing rate of cells.
CONCLUSIONS
For the first, M-CDs were synthesized by simple one-pot hydrothermal treatment without further purification. M-CDs inhibited both breast cancer cells through modulating autophagy signalling.
PubMed: 38915025
DOI: 10.1186/s13036-024-00433-4 -
Scientific Reports Jun 2024As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this...
As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.
Topics: Robotics; Escherichia coli; Protein Engineering; High-Throughput Screening Assays; Hydrolases; Polyethylene Terephthalates; Reproducibility of Results
PubMed: 38914665
DOI: 10.1038/s41598-024-64938-0 -
Scientific Reports Jun 2024Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the...
Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the Staphylococcus aureus isolates recovered from subclinical mastitis animals in Pothohar region of the country. A total of 278 milk samples from 17 different dairy farms around two districts of the Pothohar region, Islamabad and Rawalpindi, were collected and screened for sub clinical mastitis using California Mastitis Test. Positive milk samples were processed for isolation of Staphylococcus aureus using mannitol salt agar. The recovered isolates were analyzed for their antimicrobial susceptibility and virulence genes using disc diffusion and PCR respectively. 62.2% samples were positive for subclinical mastitis and in total 70 Staphylococcus aureus isolates were recovered. 21% of these isolates were determined to be methicillin resistant, carrying the mecA gene. S. aureus isolates recovered during the study were resistant to all first line therapeutic antibiotics and in total 52% isolates were multidrug resistant. SCCmec typing revealed MRSA SCCmec types IV and V, indicating potential community-acquired MRSA (CA-MRSA) transmission. Virulence profiling revealed high prevalence of key genes associated with adhesion, toxin production, and immune evasion, such as hla, hlb, clfA, clfB and cap5. Furthermore, the Panton-Valentine leukocidin (PVL) toxin, that is often associated with recurrent skin and soft tissue infections, was present in 5.7% of isolates. In conclusion, the increased prevalence of MRSA in bovine mastitis is highlighted by this study, which also reveals a variety of virulence factors in S. aureus and emphasizes the significance of appropriate antibiotic therapy in combating this economically burdensome disease.
Topics: Animals; Cattle; Mastitis, Bovine; Female; Staphylococcal Infections; Pakistan; Virulence; Anti-Bacterial Agents; Staphylococcus aureus; Methicillin-Resistant Staphylococcus aureus; Virulence Factors; Microbial Sensitivity Tests; Milk; Bacterial Proteins
PubMed: 38914650
DOI: 10.1038/s41598-024-65448-9 -
Nature Communications Jun 2024Population-representative estimates of SARS-CoV-2 infection prevalence and antibody levels in specific geographic areas at different time points are needed to optimise...
Population-representative estimates of SARS-CoV-2 infection prevalence and antibody levels in specific geographic areas at different time points are needed to optimise policy responses. However, even population-wide surveys are potentially impacted by biases arising from differences in participation rates across key groups. Here, we used spatio-temporal regression and post-stratification models to UK's national COVID-19 Infection Survey (CIS) to obtain representative estimates of PCR positivity (6,496,052 tests) and antibody prevalence (1,941,333 tests) for different regions, ages and ethnicities (7-December-2020 to 4-May-2022). Not accounting for vaccination status through post-stratification led to small underestimation of PCR positivity, but more substantial overestimations of antibody levels in the population (up to 21 percentage points), particularly in groups with low vaccine uptake in the general population. There was marked variation in the relative contribution of different areas and age-groups to each wave. Future analyses of infectious disease surveys should take into account major drivers of outcomes of interest that may also influence participation, with vaccination being an important factor to consider.
Topics: Humans; COVID-19; United Kingdom; Adult; Middle Aged; Aged; Adolescent; SARS-CoV-2; Young Adult; Child; Male; Female; Prevalence; Child, Preschool; Spatio-Temporal Analysis; Antibodies, Viral; COVID-19 Vaccines; Infant; Vaccination; Aged, 80 and over
PubMed: 38914564
DOI: 10.1038/s41467-024-49201-4 -
PloS One 2024Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by...
Tick-borne encephalitis (TBE) is usually diagnosed based on the presence of TBE virus (TBEV)-specific IgM and IgG antibodies in serum. However, antibodies induced by vaccination or cross-reactivity to previous flavivirus infections may result in false positive TBEV serology. Detection of TBEV RNA may be an alternative diagnostic approach to detect viral presence and circumvent the diagnostic difficulties present when using serology. Viral RNA in blood is commonly detectable only in the first viremic phase usually lasting up to two weeks, and not in the second neurologic phase, when the patients contact the health care system and undergo diagnostic work-up. TBEV RNA has previously been detected in urine in a few retrospective TBE cases in the neurologic phase, and furthermore RNA of other flaviviruses has been detected in patient saliva. In this study, blood, saliva and urine were collected from 31 hospitalised immunocompetent patients with pleocytosis and symptoms of aseptic meningitis and/or encephalitis, suspected to have TBE. We wanted to pursue if molecular testing of TBEV RNA in these patient materials may be useful in the diagnostics. Eleven of the 31 study patients were diagnosed with TBE based on ELISA detection of TBEV specific IgG and IgM antibodies. None of the study patients had TBEV RNA detectable in any of the collected patient material.
Topics: Humans; Encephalitis, Tick-Borne; Encephalitis Viruses, Tick-Borne; Saliva; RNA, Viral; Male; Female; Middle Aged; Adult; Aged; Immunoglobulin M; Immunoglobulin G; Antibodies, Viral; Aged, 80 and over; Immunocompetence; Hospitalization
PubMed: 38913668
DOI: 10.1371/journal.pone.0305603