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Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1f/f; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1f/f; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.
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Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/patologia , Endorribonucleases/metabolismo , Macrófagos/fisiologia , Obesidade/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Diferenciação Celular/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Metabolismo Energético/genética , Humanos , Ativação de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.
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Colágeno Tipo IV , Peixe-Zebra , Animais , Humanos , Colágeno Tipo IV/genética , Células Endoteliais , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismo , Membrana Basal/metabolismoRESUMO
Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1S724A/S724A) and investigated the importance of phosphorylation at Ser724 within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser724 exacerbated ER stress-induced hepatic steatosis in tunicamycin-treated Ern1S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer-binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid ß-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser724 of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.
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Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Animais , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ativação Enzimática , Fígado Gorduroso/genética , Fibroblastos/metabolismo , Camundongos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
BACKGROUND: C1q/TNF-related protein 9 (CTRP9) and adiponectin (APN) have beneficial metabolic regulatory and vasoprotective effects. This study explored alteration of CTRP9 and APN multimers during onset of ischemic stroke and development, to provide novel clinical and experimental basis for recognition and prevention of ischemic stroke. METHODS: There were 269 patients with ischemic stroke and 182 control subjects included in this study. Serum levels of CTRP9 and APN multimers in different disease stages were measured. RESULTS: Serum CTRP9, total APN (tAPN), and high-molecular weight (HMW) APN decreased gradually in stage I (acute stage, within 72 h of onset) of ischemic stroke and increased during stage III (11th day to one month) and stage IV (1 month after), compared to control. In the non-hyperlipidemia group, serum CTRP9, tAPN, and HMW were decreased in ischemic stroke patients compared to control (P < 0.05). Serum CTRP9 is closely related to serum tAPN and HMW (r = 0.992, 0.991). Serum CTRP9 are protective against ischemic stroke (OR = 0.400, 95% CI 0.197-0.810, P < 0.05). CONCLUSIONS: Lower serum CTRP9, tAPN, LMW, and HMW are significantly associated with increased ischemic stroke risk in non-hyperlipidemia subjects. CTRP9, tAPN, and HMW isoforms may be valuable clinical indicators for patients with ischemic stroke.
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Adiponectina , AVC Isquêmico , Humanos , Adiponectina/metabolismo , Glicoproteínas/metabolismo , Peso MolecularRESUMO
Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen in addition to the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg in morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from cultured embryonic tissue experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the Laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen.
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Membrana Basal/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Glicoproteínas de Membrana/fisiologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Fertilidade/fisiologia , Masculino , Músculos/citologia , Músculos/metabolismo , Mutação , Especificidade de Órgãos/fisiologia , Organogênese/fisiologia , Domínios Proteicos/fisiologiaRESUMO
Obesity is associated with both endoplasmic reticulum (ER) stress and chronic metabolic inflammation. ER stress activates the unfolded protein response (UPR) and has been implicated in a variety of cancers, including hepatocellular carcinoma (HCC). It is unclear whether individual UPR pathways are mechanistically linked to HCC development, however. Here we report a dual role for inositol-requiring enzyme 1α (IRE1α), the ER-localized UPR signal transducer, in obesity-promoted HCC development. We found that genetic ablation of IRE1α in hepatocytes not only markedly reduced the occurrence of diethylnitrosamine (DEN)-induced HCC in liver-specific IRE1α knockout (LKO) mice when fed a normal chow (NC) diet, but also protected against the acceleration of HCC progression during high-fat diet (HFD) feeding. Irrespective of their adiposity states, LKO mice showed decreased hepatocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation, even in the face of increased hepatic apoptosis. Furthermore, IRE1α abrogation blunted obesity-associated activation of hepatic inhibitor of nuclear factor kappa B kinase subunit beta (IKKß)-nuclear factor kappa B (NF-κB) pathway, leading to reduced production of the tumor-promoting inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). Importantly, higher IRE1α expression along with elevated STAT3 phosphorylation was also observed in the tumor tissues from human HCC patients, correlating with their poorer survival rate. CONCLUSION: IRE1α acts in a feed-forward loop during obesity-induced metabolic inflammation to promote HCC development through STAT3-mediated hepatocyte proliferation. (Hepatology 2018).
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Carcinoma Hepatocelular/metabolismo , Endorribonucleases/metabolismo , Neoplasias Hepáticas/metabolismo , Obesidade/complicações , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Proliferação de Células , Citocinas/metabolismo , Dieta Hiperlipídica , Dietilnitrosamina/farmacologia , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Obesidade/veterinária , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
BACKGROUND & AIMS: As the main detoxifying organ of the body, the liver possesses a remarkable ability to regenerate after toxic injury, tissue resection or viral infection. A growing number of cellular signaling pathways have been implicated in orchestrating the process of liver regeneration. Here we investigated the role of inositol-requiring enzyme-1α (IRE1α), a key signal transducer of the unfolded protein response (UPR), in liver regeneration. METHODS: Using mice with hepatocyte-specific deletion of IRE1α, we examined the role of IRE1α in liver regeneration after challenges with carbon tetrachloride (CCl4) or hepatic surgery. We also investigated if IRE1α deficiency could affect the activation state of signal transducer and activator of transcription 3 (STAT3) in hepatocytes. Using co-immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we analyzed whether IRE1α could interact with STAT3 to regulate its phosphorylation. RESULTS: We found that in response to CCl4-induced liver damage or after two-thirds partial hepatectomy (PH), abrogation of IRE1α caused marked exacerbation of liver injury and impairment in regenerative proliferation of hepatocytes in mice. Furthermore, IRE1α deficiency resulted in dampened STAT3 activation, and restoration of IRE1α expression led to sustained phosphorylation of STAT3 in IRE1α-null hepatocytes. Additionally, IRE1α could directly and constitutively associate with STAT3, leading to elevated phosphorylation when stimulated by IL-6. CONCLUSIONS: These results suggest that IRE1α may promote liver regeneration through acting as a signaling platform to regulate the STAT3 pathway.
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Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/fisiologia , Regeneração Hepática/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Proliferação de Células/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/deficiência , Endorribonucleases/genética , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição de Fator Regulador X , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.
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Neoplasias Pulmonares , Linfócitos do Interstício Tumoral , Esferoides Celulares , Humanos , Linfócitos do Interstício Tumoral/imunologia , Esferoides Celulares/imunologia , Linhagem Celular Tumoral , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Receptor de Morte Celular Programada 1/imunologia , Imunoterapia Adotiva , Técnicas de Cocultura , Técnicas de Cultura de Células , Células Tumorais Cultivadas , Proliferação de CélulasRESUMO
The construction of reliable preclinical models is crucial for understanding the molecular mechanisms involved in gastric cancer and for advancing precision medicine. Currently, existing in vitro tumor models often do not accurately replicate the human gastric cancer environment and are unsuitable for high-throughput therapeutic drug screening. In this study, droplet microfluidic technology is employed to create novel gastric cancer assembloids by encapsulating patient-derived xenograft gastric cancer cells and patient stromal cells in Gelatin methacryloyl (GelMA)-Gelatin-Matrigel microgels. The usage of GelMA-Gelatin-Matrigel composite hydrogel effectively alleviated cell aggregation and sedimentation during the assembly process, allowing for the handling of large volumes of cell-laden hydrogel and the uniform generation of assembloids in a high-throughput manner. Notably, the patient-derived xenograft assembloids exhibited high consistency with primary tumors at both transcriptomic and histological levels, and can be efficiently scaled up for preclinical drug screening efforts. Furthermore, the drug screening results clearly demonstrated that the in vitro assembloid model closely mirrored in vivo drug responses. Thus, these findings suggest that gastric cancer assembloids, which effectively replicate the in vivo tumor microenvironment, show promise for enabling more precise high-throughput drug screening and predicting the clinical outcomes of various drugs.
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mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs to contain ovalbumin mRNA and protein (EVOvaM+P) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs, natural nanoparticle carriers shed by all cells, that contain mRNA and protein Spike (S) protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.
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Cancer models play critical roles in basic cancer research and precision medicine. However, current in vitro cancer models are limited by their inability to mimic the three-dimensional architecture and heterogeneous tumor microenvironments (TME) of in vivo tumors. Here, we develop an innovative patient-specific lung cancer assembloid (LCA) model by using droplet microfluidic technology based on a microinjection strategy. This method enables precise manipulation of clinical microsamples and rapid generation of LCAs with good intra-batch consistency in size and cell composition by evenly encapsulating patient tumor-derived TME cells and lung cancer organoids inside microgels. LCAs recapitulate the inter- and intratumoral heterogeneity, TME cellular diversity, and genomic and transcriptomic landscape of their parental tumors. LCA model could reconstruct the functional heterogeneity of cancer-associated fibroblasts and reflect the influence of TME on drug responses compared to cancer organoids. Notably, LCAs accurately replicate the clinical outcomes of patients, suggesting the potential of the LCA model to predict personalized treatments. Collectively, our studies provide a valuable method for precisely fabricating cancer assembloids and a promising LCA model for cancer research and personalized medicine.
Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Microambiente Tumoral , Organoides/patologia , Medicina de Precisão/métodosRESUMO
Objective and aims: Osteopontin (OPN), an oxidant stress sensitive cytokine, plays a central role in liver fibrosis. While OPN expression can be reduced by small interfering RNA (siRNA), the challenge to deliver siRNA safely and effectively into liver remains unresolved. Exosomes are promising natural nanocarriers for drug delivery that are able to enter cells with different biological barriers efficiently. In this study, we used exosomes as a delivery vehicle to target OPN in liver fibrosis. Methods: Exosomes selectively home to fibrotic liver according to small animal imaging system. Electroporation technique was used to engineer exosomes to carry siRNA targeting OPN (ExosiRNA-OPN). Primary hepatic stellate cells (HSCs) were isolated and treated with ExosiRNA-OPN to assess the effect on activated HSCs (aHSCs). Immunofluorescence for α-SMA, an aHSCs marker, and sirius red staining were performed to assess ECM deposition. Finally, plasma OPN from patients with liver fibrosis was identified by ELISA assay. Results: Exosome-mediated siRNA delivery systems show high uptake and low toxicity. Besides, ExosiRNA-OPN suppressed HSCs activation and ECM deposition and more efficiently improved liver function when compared to naked siRNA-OPN. Moreover, ExosiRNA-OPN was assumed inhibiting TGF-ß1 signaling activation, along with other fibrotic-related genes based on a GEO datasheet of liver fibrosis samples for correlation analyzes. ExosiRNA-OPN inhibited TGF-ß1 signaling by decreasing high-mobility group box-1 (HMGB1). Plasma proteins from chronic HBV-induced fibrosis patients were identified that patients with high OPN expression correlates with more advanced fibrosis progression. Discussion: This study shows that exosome-mediated siRNA-OPN delivery may be an effective option for the treatment of liver fibrosis.
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INTRODUCTION: Adiponectin is a potent vascular protective molecule. Recent findings have suggested adiponectin resistance during early diabetes. However, the molecular mechanisms responsible remain unidentified. Here, we took an unbiased approach to identify whether hyperlipidemic plasma molecules exist that bind and inhibit adiponectin function, contributing to adiponectin resistance and diabetic vascular injury. METHODS: Adult rats were randomly assigned to receive either a normal or a high-fat diet for 8 weeks. Plasma was co-immunoprecipitated with anti-APN antibody and analyzed by mass spectrometry. The APN binding molecules and their effect upon APN biological activity were determined. RESULTS: As expected, the high-fat-diet increased plasma triglyceride, total cholesterol, and low-density lipoprotein. Importantly, the circulating APN level was significantly increased at this time point. Mass spectrometry identified 18 proteins with increased APN binding in hyperlipidemic plasma, among which four proteins critical in lipid metabolism, including apolipoprotein A1 (APOA1), APOA4, APOC1, and paraoxonase 1, were further investigated. Incubating recombinant APN with APOA1 markedly (P < 0.01), and incubating with APOC1 significantly (P < 0.05), inhibited APN activity as evidenced by the reduced AMPK activation in HUVECs. APOA4 and paraoxonase 1 incubation had no effect upon APN activity. Finally, plasma APOA1 was significantly increased (P < 0.05) in hyperlipidemic plasma compared with the control plasma. CONCLUSIONS: It was demonstrated for the first time that increased APOA1 and APOC1 in hyperlipidemic plasma binds and inhibits APN activity. This result not only identifies a novel molecular mechanism responsible for adiponectin resistance during early stage diabetes, but also provides additional new insight into the diverse/controversial (protective and harmful) functions of high-density lipoprotein.
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Adiponectina , Arildialquilfosfatase , Hiperlipidemias , Adiponectina/sangue , Animais , Arildialquilfosfatase/sangue , Arildialquilfosfatase/metabolismo , Dieta Hiperlipídica , Hiperlipidemias/sangue , Metabolismo dos Lipídeos , Distribuição Aleatória , RatosRESUMO
Mycobacteria include a large number of pathogens. Identification to species level is important for diagnoses and treatments. Here, we report the development of a Web-accessible database of the hsp65 locus sequences (http://msis.mycobacteria.info) from 149 out of 150 Mycobacterium species/subspecies. This database can serve as a reference for identifying Mycobacterium species.
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Proteínas de Bactérias/genética , Chaperonina 60/genética , Bases de Dados de Ácidos Nucleicos , Internet , Mycobacterium/classificação , Mycobacterium/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
To identify loci useful for species identification and to enhance our understanding of the population structure and genetic variability of the genus Mycobacterium, we conducted a multiple-genome comparison of a total of 27 sequenced genomes in the suborder of Corynebacterineae (18 from the Mycobacterium genus, 7 from the Corynebacterium genus, 1 each from the Nocardia and Rhodococcus genera). Our study revealed 26 informative loci for species identification in Mycobacterium. The sequences from these loci were used in a phylogenetic analysis to infer the evolutionary relations of the 18 mycobacterial genomes. Among the loci that we identified, rpoBC, dnaK, and hsp65 were amplified from 29 ATCC reference strains and 17 clinical isolates and sequenced. The phylogenetic trees generated from these loci show similar topologies. The newly identified dnaK locus is more discriminatory and more robust than the widely used hsp65 locus. The length-variable rpoBC locus is the first intergenic locus between two protein-encoding genes being used for mycobacterial species identification. A multilocus sequence analysis system including the rpoBC, dnaK, and hsp65 loci is a robust tool for accurate identification of Mycobacterium species.
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Técnicas Bacteriológicas/métodos , Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Mycobacterium/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Biologia Computacional , Corynebacterium/classificação , Corynebacterium/genética , RNA Polimerases Dirigidas por DNA/genética , Genoma Bacteriano , Proteínas de Choque Térmico HSP70/genética , Humanos , Dados de Sequência Molecular , Nocardia/classificação , Nocardia/genética , Filogenia , Rhodococcus/classificação , Rhodococcus/genética , Análise de Sequência de DNARESUMO
During Drosophila metamorphosis, dorsal and ventral wing surfaces adhere, separate, and reappose in a paradoxical process involving cell-matrix adhesion, matrix production and degradation, and long cellular projections. The identity of the intervening matrix, the logic behind the adhesion-reapposition cycle, and the role of projections are unknown. We find that laminin matrix spots devoid of other main basement membrane components mediate wing adhesion. Through live imaging, we show that long microtubule-actin cables grow from those adhesion spots because of hydrostatic pressure that pushes wing surfaces apart. Formation of cables resistant to pressure requires spectraplakin, Patronin, septins, and Sdb, a SAXO1/2 microtubule stabilizer expressed under control of wing intervein-selector SRF. Silkworms and dead-leaf butterflies display similar dorso-ventral projections and expression of Sdb in intervein SRF-like patterns. Our study supports the morphogenetic importance of atypical basement-membrane-related matrices and dissects matrix-cytoskeleton coordination in a process of great evolutionary significance.
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Actinas/metabolismo , Laminina/metabolismo , Microtúbulos/metabolismo , Asas de Animais/metabolismo , Animais , Membrana Basal/metabolismo , Borboletas/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Metamorfose Biológica/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologiaRESUMO
The silent information regulator protein (Sir2) and its homologs are NAD(+)-dependent deacetylase enzymes that play important roles in a variety of physiological processes. However, the functions of the Sir2 family in plants are poorly understood. Here, we report that Arabidopsis AtSRT2, a homolog of yeast Sir2, negatively regulates plant basal defense against the pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). In response to PstDC3000 infection, the expression of AtSRT2 was down-regulated in a salicylic acid (SA)-independent manner. In addition, knock-out of AtSRT2 (srt2) enhanced resistance against PstDC3000 and increased expression of pathogenesis-related gene 1 (PR1). Conversely, overexpression of AtSRT2 resulted in hypersusceptibility to PstDC3000 and impaired PR1 induction. Consistent with this phenotype, expression of PAD4, EDS5 and SID2, three essential genes in the SA biosynthesis pathway, were increased in the srt2 mutant and decreased in AtSRT2-overexpressing plants. Taken together, these results demonstrate that AtSRT2 is a negative regulator of basal defense, possibly by suppressing SA biosynthesis.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Histona Desacetilases do Grupo III/metabolismo , Sirtuínas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Histona Desacetilases do Grupo III/genética , Imunidade Inata , Mutagênese Insercional , Pseudomonas syringae/patogenicidade , RNA de Plantas/genética , Ácido Salicílico/metabolismo , Sirtuínas/genéticaRESUMO
Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.
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Aquicultura , Doenças dos Peixes/microbiologia , Tilápia/microbiologia , Vibrioses/veterinária , Vibrio vulnificus/classificação , Vibrio vulnificus/genética , Animais , Técnicas de Tipagem Bacteriana , Bangladesh , Análise por Conglomerados , Impressões Digitais de DNA , Genótipo , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Vibrioses/microbiologia , Vibrio vulnificus/isolamento & purificaçãoRESUMO
BACKGROUND: Pandemic Vibrio parahaemolyticus has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In Vibrio cholerae and Vibrio vulnificus, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic V. parahaemolyticus would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic V. parahaemolyticus O3:K6 by gene deletion using a chitin based transformation strategy. RESULTS: We generated different deletion mutants of putative polysaccharide genes and examined the mutants by immuno-blots with O and K specific antisera. Our results showed that O- and K-antigen genes are separated in V. parahaemolyticus O3:K6; the region encoding both O-antigen and capsule biosynthesis in other vibrios, i.e. genes between gmhD and rjg, determines the K6-antigen but not the O3-antigen in V. parahaemolyticus. The previously identified "capsule genes" on the smaller chromosome were related to exopolysaccharide synthesis, not K-antigen. CONCLUSION: Understanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic V. parahaemolyticus. This report confirms the genetic location of K-antigen synthesis in V. parahaemolyticus O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.
Assuntos
Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Pandemias , Polissacarídeos Bacterianos/genética , Vibrioses/microbiologia , Vibrio parahaemolyticus/genética , Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Deleção de Genes , Humanos , Índia/epidemiologia , Dados de Sequência Molecular , Antígenos O/genética , Antígenos O/imunologia , Polissacarídeos Bacterianos/imunologia , Vibrioses/epidemiologia , Vibrio parahaemolyticus/imunologia , Vibrio parahaemolyticus/isolamento & purificaçãoRESUMO
The outbreak the SARS-CoV-2 (CoV-2) virus has resulted in over 6.5 million cases of COVID19, greatly stressing global healthcare infrastructure. Lacking medical prophylactic measures to combat disease spread, many nations have adopted social distancing policies in order to mitigate transmission of CoV-2. While mathematical models have suggested the efficacy of social distancing to curb the spread of CoV-2, there is a lack of systematic studies to quantify the real-world efficacy of these approaches. Here, we first demonstrate that implementation of social distancing policies in US states corresponded with a reduction in COVID19 spread rates, and that the reduction in spread rate is proportional to the average change in mobility. We validate this observation on a worldwide scale by analyzing COVID19 spread rate in 134 nations with varying social distancing policies. Globally, we find that social distancing policies significantly reduced the COVID19 spread rate, with resulting in an estimated 65% reduction (95% CI = 39-80%) in new COVID19 cases over a two week time period. These data suggest that social distancing policies may be a powerful tool to prevent spread of COVID19 in real-world scenarios.