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Microbiology Spectrum May 2024The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene...
The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait.
PubMed: 38808977
DOI: 10.1128/spectrum.00159-24 -
Nature Communications May 2024Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we...
Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer's disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms, providing insight into the human genome evolution. In addition, some of the isoforms are expressed in a cell-type specific manner, whose alternative 3'-UTRs usage contributes to their specificity. Further, we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing.
Topics: Humans; Alternative Splicing; Protein Isoforms; Quantitative Trait Loci; Genome-Wide Association Study; 3' Untranslated Regions; Alzheimer Disease; Genome, Human; Mitochondrial Precursor Protein Import Complex Proteins
PubMed: 38806455
DOI: 10.1038/s41467-024-48615-4 -
Nature Communications May 2024Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains...
Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.
Topics: Protons; Bacterial Proteins; Staphylococcus aureus; Cryoelectron Microscopy; Multidrug Resistance-Associated Proteins; Anti-Bacterial Agents; Models, Molecular; Biological Transport; Binding Sites; Hydrogen Bonding; Protein Conformation
PubMed: 38802368
DOI: 10.1038/s41467-024-48759-3 -
BioRxiv : the Preprint Server For... May 2024Mitochondria carry out essential functions in eukaryotic cells. The mitochondrial genome encodes factors critical to support oxidative phosphorylation and mitochondrial...
Mitochondria carry out essential functions in eukaryotic cells. The mitochondrial genome encodes factors critical to support oxidative phosphorylation and mitochondrial protein import necessary for these functions. However, organisms like budding yeast can readily lose their mitochondrial genome, yielding respiration-deficient mutants. The fission yeast is petite-negative, but some nuclear mutations enable the loss of its mitochondrial genome. Here, we characterize the classical -positive mutation as a loss of function allele of the proteasome 19S regulatory subunit component , involved in the Ubiquitin-dependent degradation pathway. The mutation results in an altered oxidative stress response, with increased levels of oxidized glutathione, and increased levels of mitochondrial and cytoplasmic chaperones. We propose that Ubiquitin-proteasome regulation of chaperones involved in the Unfolded Protein Response and mitochondrial protein import underlies petite-negativity in fission yeast.
PubMed: 38798546
DOI: 10.1101/2024.05.09.593392 -
BioRxiv : the Preprint Server For... May 2024Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70...
Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human mitochondria, mitochondrial Hsp70 (mortalin) and the nucleotide exchange factor (GrpEL1) work synergistically to stabilize proteins, assemble protein complexes, and facilitate protein import. However, our understanding of the molecular mechanisms guiding these processes is hampered by limited structural information. To elucidate these mechanistic details, we used cryoEM to determine the first structures of full-length human mortalin-GrpEL1 complexes in previously unobserved states. Our structures and molecular dynamics simulations allow us to delineate specific roles for mortalin-GrpEL1 interfaces and to identify steps in GrpEL1-mediated nucleotide and substrate release by mortalin. Subsequent analyses reveal conserved mechanisms across bacteria and mammals and facilitate a complete understanding of sequential nucleotide and substrate release for the Hsp70 chaperone system.
PubMed: 38798347
DOI: 10.1101/2024.05.10.593630 -
Redox Report : Communications in Free... Dec 2024Deficiency of TOM5, a mitochondrial protein, causes organizing pneumonia (OP) in mice. The clinical significance and mechanisms of TOM5 in the pathogenesis of OP remain...
Deficiency of TOM5, a mitochondrial protein, causes organizing pneumonia (OP) in mice. The clinical significance and mechanisms of TOM5 in the pathogenesis of OP remain elusive. We demonstrated that TOM5 was significantly increased in the lung tissues of OP patients, which was positively correlated with the collagen deposition. In a bleomycin-induced murine model of chronic OP, increased TOM5 was in line with lung fibrosis. In vitro, TOM5 regulated the mitochondrial membrane potential in alveolar epithelial cells. TOM5 reduced the proportion of early apoptotic cells and promoted cell proliferation. Our study shed light on the roles of TOM5 in OP.
Topics: Animals; Alveolar Epithelial Cells; Mice; Humans; Membrane Potential, Mitochondrial; Mitochondrial Precursor Protein Import Complex Proteins; Male; Apoptosis; Female; Cell Proliferation; Mice, Inbred C57BL; Disease Models, Animal; Cryptogenic Organizing Pneumonia; Organizing Pneumonia
PubMed: 38794801
DOI: 10.1080/13510002.2024.2354625 -
Viruses May 2024The L 1 region of bovine adenovirus (BAdV)-3 encodes a multifunctional protein named protein VII. Anti-protein VII sera detected a protein of 26 kDa in transfected or...
The L 1 region of bovine adenovirus (BAdV)-3 encodes a multifunctional protein named protein VII. Anti-protein VII sera detected a protein of 26 kDa in transfected or BAdV-3-infected cells, which localizes to nucleus and nucleolus of infected/transfected cells. Analysis of mutant protein VII identified four redundant overlapping nuclear/nucleolar localization signals as deletion of all four potential nuclear/nucleolar localization signals localizes protein VII predominantly to the cytoplasm. The nuclear import of protein VII appears to use importin α (α-1), importin-β (β-1) and transportin-3 nuclear transport receptors. In addition, different nuclear transport receptors also require part of protein VII outside nuclear localization sequences for efficient interaction. Proteomic analysis of protein complexes purified from recombinant BAdV-3 expressing protein VII containing Strep Tag II identified potential viral and cellular proteins interacting with protein VII. Here, we confirm that protein VII interacts with IVa2 and protein VIII in BAdV-3-infected cells. Moreover, amino acids 91-101 and 126-137, parts of non-conserved region of protein VII, are required for interaction with IVa2 and protein VIII, respectively.
Topics: Animals; Cattle; Mastadenovirus; Viral Proteins; Protein Binding; Cell Line; Cell Nucleus; Proteomics; Host-Pathogen Interactions; Nuclear Localization Signals; Active Transport, Cell Nucleus; Humans
PubMed: 38793614
DOI: 10.3390/v16050732 -
Molecules (Basel, Switzerland) May 2024The European Union's (EU) agricultural self-sufficiency is challenged by its reliance on imported plant proteins, particularly soy from the Americas, contributing to...
The European Union's (EU) agricultural self-sufficiency is challenged by its reliance on imported plant proteins, particularly soy from the Americas, contributing to deforestation and greenhouse gas emissions. Addressing the EU's protein deficit, this study evaluates alternative protein sources for aquaculture, focusing on their nutritional value, elemental content, and polycyclic aromatic hydrocarbons (PAHs). Protein flours from gastropods (, , ) and their hepatopancreas, along with plant-based proteins from food industry by-products (oilcakes, coffee grounds, spent brewer's yeast), were analyzed. Results revealed that snail flour contained the highest protein content at 59.09%, significantly outperforming hepatopancreas flour at 42.26%. Plant-based proteins demonstrated substantial nutritional value, with coffee grounds flour exhibiting a remarkable protein content of 71.8% and spent brewer's yeast flour at 57.9%. Elemental analysis indicated high levels of essential minerals such as magnesium in hepatopancreas flour (5719.10 mg/kg) and calcium in slug flour (48,640.11 mg/kg). However, cadmium levels in hepatopancreas flour (11.45 mg/kg) necessitate caution due to potential health risks. PAH concentrations were low across all samples, with the highest total PAH content observed in hepatopancreas flour at 0.0353 µg/kg, suggesting minimal risk of PAH-related toxicity. The analysis of plant-based protein sources, particularly oilcakes derived from sunflower, hemp, flax, and pumpkin seeds, revealed that these by-products not only exhibit high protein contents but present a promising avenue for enhancing the nutritional quality of feed. This study underscores the potential of utilizing gastropod and plant-based by-products as sustainable and nutritionally adequate alternatives to conventional feeds in aquaculture, contributing to the EU's environmental sustainability goals.
Topics: Animals; Nutritive Value; Fishes; Animal Feed; Hepatopancreas; Plant Proteins; Polycyclic Aromatic Hydrocarbons; Aquaculture; Nutrients
PubMed: 38792193
DOI: 10.3390/molecules29102332 -
International Journal of Molecular... May 2024Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the...
Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress- and drug-resistance mechanisms drive post-therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress- and drug-resistance mechanisms in NBs include alternative splicing of the neurotrophin receptor tropomyosin-related kinase A (), which correlates with post-therapeutic relapse and advanced-stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII-expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and IncuCyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca-calmodulin, adenosine ribosylating factor (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by Omi/high temperature requirement protein A2 (HtrA2) then activated by a mechanism dependent upon calmodulin kinase II (CaMKII), alpha serine/threonine kinase (Akt), mitochondrial Ca uniporter and reactive oxygen species (ROS), involving inhibitory mitochondrial protein tyrosine phosphatase (PTPase) oxidation, resulting in phosphoinositide 3 kinase (PI3K) activation of mitochondrial Akt, which enhances stress resistance. This novel pro-survival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors and also by inhibitors of 78kDa glucose regulated protein (Grp78), heat shock protein 90 (Hsp90), Ca-calmodulin and PI3K. This identifies Grp78, Ca-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has the potential to enhance the stress-induced elimination of TrkAIII-expressing NB cells, with the potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation.
Topics: Humans; Endoplasmic Reticulum Chaperone BiP; Receptor, trkA; Neuroblastoma; Mitochondria; Cell Line, Tumor; Protein Folding; Signal Transduction; Stress, Physiological
PubMed: 38791513
DOI: 10.3390/ijms25105475 -
Microbial Cell Factories May 2024Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated...
Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum.
BACKGROUND
Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster.
RESULTS
Genetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD'EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw.
CONCLUSIONS
This study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.
Topics: Xylans; Lignin; Biomass; Coumaric Acids; Oligosaccharides; Clostridiales; Operon; Bacterial Proteins; Multigene Family; Acetylesterase; Carboxylic Ester Hydrolases
PubMed: 38789996
DOI: 10.1186/s12934-024-02423-z