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Coinfection of HIV and multidrug-resistant tuberculosis (MDR-TB) presents significant challenges in terms of the treatment and prognosis of tuberculosis, leading to complexities in managing the disease and impacting the overall outcome for TB patients. This study presents a remarkable case of a patient with MDR-TB and HIV coinfection who survived for over 8 years, despite poor treatment adherence and comorbidities. Whole genome sequencing (WGS) of the infecting Mycobacterium tuberculosis (Mtb) strain revealed a unique genomic deletion, spanning 18 genes, including key genes involved in hypoxia response, intracellular survival, immunodominant antigens, and dormancy. This deletion, that we have called "Del-X," potentially exerts a profound influence on the bacterial physiology and its virulence. Only few similar deletions were detected in other non-related Mtb genomes worldwide. In vivo evolution analysis identified drug resistance and metabolic adaptation mutations and their temporal dynamics during the patient's treatment course.
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The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.
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The Salmonella Pathogenicity Island-2 (i.e. SPI-2) encodes a unique type III secretion system that delivers effector proteins from the Salmonella-containing vacuole (SCV) into the host cell. The SPI-2 locus also encodes translocated effectors as well as a two-component system - termed SpiR/SsrB - that is essential for the expression of SPI-2 genes. Transcription of the horizontally acquired SPI-2 genes requires the ancestral nucleoid-associated proteins (i.e. NAPs) IHF and Fis, the regulatory protein SlyA, and the two-component systems PhoP/PhoQ and OmpR/EnvZ, as well as the DNA binding protein HilD encoded in a different pathogenicity island. Some of these positive SPI-2 regulators act to antagonize the robust silencing promoted by the NAPs H-NS, Hha, and YdgT.
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Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Membrana/genéticaRESUMO
Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming, labor-intensive and require much experience for accurate interpretation. Here we took advantage of the Fluorescence Activated Cell Sorter (FACS) to quantify the turnover of GFP-LC3 as an assay to measure autophagic activity in living mammalian cells. We showed that during induction of autophagy by rapamycin, tunicamycin or starvation to amino acids, fluorescence intensity of GFP-LC3 is reduced in a time-dependent manner. This decrease occurred specifically in wild type LC3, but not in mutant LC3(G120A), and was inhibited by autophagic or lysosomal inhibitors, indicating that this signal is specific to selective autophagy-mediated delivery of LC3 into lysosomes. By utilizing this assay, we tested the minimal nutrient requirement for the autophagic process and determined its induction by deprivation of specific single amino acids. We conclude that this approach can be successfully applied to different cell-lines as a reliable and simple method to quantify autophagic activity in living mammalian cells.
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Autofagia/fisiologia , Separação Celular , Citometria de Fluxo , Animais , Biomarcadores/metabolismo , Células CHO , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismoRESUMO
LC3 belongs to a novel ubiquitin-like protein family that is involved in different intracellular trafficking processes, including autophagy. All members of this family share a unique three-dimensional structure composed of a C-terminal ubiquitin core and two N-terminal alpha-helices. Here, we focus on the specific contribution of these regions to autophagy induced by amino acid deprivation. We show that the ubiquitin core by itself is sufficient for LC3 processing through the conjugation machinery and for its consequent targeting to the autophagosomal membrane. The N-terminal region was found to be important for interaction between LC3 and p62/SQSTM1 (hereafter termed p62). This interaction is dependent on the first 10 amino acids of LC3 and on specific residues located within the ubiquitin core. Knockdown of LC3 isoforms and overexpression of LC3 mutants that fail to interact with p62 blocked the incorporation of p62 into autophagosomes. The accumulation of p62 was accompanied by elevated levels of polyubiquitylated detergent-insoluble structures. p62, however, is not required for LC3 lipidation, autophagosome formation and targeting to lysosomes. Our results support the proposal that LC3 is responsible for recruiting p62 into autophagosomes, a process mediated by phenylalanine 52, located within the ubiquitin core, and the N-terminal region of the protein.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Sequência de Aminoácidos , Animais , Autofagia/fisiologia , Células CHO , Cricetinae , Cricetulus , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina/fisiologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Proteína Sequestossoma-1 , Ubiquitinação/fisiologiaRESUMO
Atg8, a member of an evolutionarily conserved ubiquitin-like protein family, is involved in multiple membrane trafficking pathways including autophagy. In a recent study, we have identified two functional sites in the yeast Saccharomyces cerevisiae Atg8, one involving residues Tyr49 and Leu50, and the other--located on the opposite side of the molecule--residues Phe77 and Phe79. Here we extended our studies to the mammalian system and report that in LC3 residues Phe80 and Leu82, the equivalents of Phe77 and Phe79 in Atg8, are essential for its C-terminal cleavage. We propose that these residues are part of the Atg4 recognition site.
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Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Motivos de Aminoácidos , Animais , Família da Proteína 8 Relacionada à Autofagia , Humanos , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation-induced autophagy. We show that starvation stimulates formation of ROS, specifically H(2)O(2). These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H(2)O(2), and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process.
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Autofagia , Cisteína Endopeptidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Relacionadas à Autofagia , Células CHO , Cricetinae , Cricetulus , Cisteína/metabolismo , Cisteína Endopeptidases/química , Ativação Enzimática , Privação de Alimentos , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Oxirredução , Fosfatidilinositol 3-Quinases/metabolismo , FilogeniaRESUMO
Autophagy is a major catabolic pathway in eukaryotic cells whereby the lack of amino acids induces the formation of autophagosomes, double-bilayer membrane vesicles that mediate delivery of cytosolic proteins and organelles for lysosomal degradation. The biogenesis and turnover of autophagosomes in mammalian cells as well as the molecular mechanisms underlying induction of autophagy and trafficking of these vesicles are poorly understood. Here we utilized different autophagic markers to determine the involvement of microtubules in the autophagic process. We show that autophagosomes associate with microtubules and concentrate near the microtubule-organizing center. Moreover, we demonstrate that autophagosomes, but not phagophores, move along these tracks en route for degradation. Disruption of microtubules leads to a significant reduction in the number of mature autophagosomes but does not affect their life span or their fusion with lysosomes. We propose that microtubules serve to deliver only mature autophagosomes for degradation, thus providing a spatial barrier between phagophores and lysosomes.
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Autofagia , Lisossomos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Fagossomos/metabolismo , Animais , Células CHO , Cricetinae , Citosol/metabolismo , DNA/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Frações SubcelularesRESUMO
Heterochromatin protein 1 (HP1) controls heterochromatin formation in animal cells, at least partly through interaction with lysine 9 (Lys-9)-methylated histone H3. We aimed to determine whether a structurally conserved human HP1 protein exhibits conserved heterochromatin localization in plant cells and studied its relation to modified histone H3. We generated transgenic tobacco plants and cycling cells expressing the human HP1gamma fused to green fluorescent protein (GFP) and followed its association with chromatin. Plants expressing GFP-HP1gamma showed no phenotypic perturbations. We found that GFP-HP1gamma is preferentially associated with the transcriptionally "inactive" heterochromatin fraction, a fraction enriched in Lys-9-methylated histone H3. During mitosis GFP-HP1gamma is detached from chromosomes concomitantly with phosphorylation of histone H3 at serine 10 and reassembles as cells exit mitosis. However, this phosphorylation cannot directly account for the dissociation of GFP-HP1gamma from mitotic chromosomes inasmuch as phosphorylation does not interfere with binding to HP1gamma. It is, therefore, possible that phosphorylation at serine 10 creates a "code" that is read by as yet an unknown factor(s), eventually leading to detachment of GFP-HP1gamma from mitotic chromosomes. Together, our results suggest that chromatin organization in plants and animals is conserved, being controlled at least partly by the association of HP1 proteins with methylated histone H3.
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Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Histonas/metabolismo , Mitose , Nicotiana/genética , Núcleo Celular/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/análise , Humanos , Interfase , Metilação , Fosforilação , Serina/metabolismoRESUMO
Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.