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CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.
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Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Expressão Gênica , Homeostase/genética , RNA Guia de Cinetoplastídeos/genética , Autoimunidade/genética , Sequência de Bases , Sequência Conservada , Regulação para Baixo/genética , Modelos Genéticos , Mutação/genética , Óperon/genética , Regiões Promotoras Genéticas/genética , Streptococcus pyogenes/genética , Estresse Fisiológico/genética , Transcrição Gênica , Ativação Transcricional/genéticaRESUMO
Regulation of bacterial transcription is a complex and multi-faceted phenomenon that is critical for growth and adaptation. Proteins in the CarD_CdnL_TRCF family are widespread, often essential, regulators of transcription of genes required for growth and metabolic homeostasis. Research in the last decade has described the mechanistic and structural bases of CarD-CdnL-mediated regulation of transcription initiation. More recently, studies in a range of bacteria have begun to elucidate the physiological roles of CarD-CdnL proteins as well as mechanisms by which these proteins, themselves, are regulated. A theme has emerged wherein regulation of CarD-CdnL proteins is central to bacterial adaptation to stress and/or changing environmental conditions.
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This study aimed to determine the prevalence of V. parahaemolyticus in oysters from the northwestern coast of Mexico and to identify the serotypes, virulence factors, and antibiotic resistance of the strains. Oyster samples were collected from 2012 to 2020 from the northwest coast of Mexico; biochemical and molecular methods were used to identify V. parahaemolyticus from oysters; antiserum reaction to determine V. parahaemolyticus serotypes, and PCR assays were performed to identify pathogenic (tdh and/or trh) or pandemic (toxRS/new, and/or orf8) strains and antibiotic resistance testing. A total of 441 oyster samples were collected and tested for V. parahaemolyticus. Forty-seven percent of oyster samples were positive for V. parahaemolyticus. Ten different O serogroups and 72 serovars were identified, predominantly serotype O1:KUT with 22.2% and OUT:KUT with 17.3%. Twenty new serotypes that had not been previously reported in our region were identified. We detected 4.3% of pathogenic clones but no pandemic strains. About 73.5% of strains were resistant to at least one antibiotic, mainly ampicillin and ciprofloxacin; 25% were multi-drug resistant. In conclusion, the pathogenic strains in oysters and antibiotic resistance are of public health concern, as the potential for outbreaks throughout northwestern Mexico is well established.
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Antibacterianos , Ostreidae , Frutos do Mar , Vibrio parahaemolyticus , Fatores de Virulência , Animais , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/isolamento & purificação , México/epidemiologia , Ostreidae/microbiologia , Fatores de Virulência/genética , Antibacterianos/farmacologia , Frutos do Mar/microbiologia , Farmacorresistência Bacteriana , Sorogrupo , Virulência/genética , Testes de Sensibilidade MicrobianaRESUMO
The problem of unaffordable prescription medications in the United States is complex and can result in poor patient adherence to therapy, worse clinical outcomes, and high costs to the healthcare system. While providers are aware of the financial burden of healthcare for patients, there is a lack of actionable price transparency at the point of prescribing. Real-time prescription benefit (RTPB) tools are new electronic clinical decision support tools that retrieve patient- and medication-specific out-of-pocket cost information and display it to clinicians at the point of prescribing. The rise in US healthcare costs has been a major driver for efforts to increase medication price transparency, and mandates from the Centers for Medicare & Medicaid Services for Medicare Part D sponsors to adopt RTPB tools may spur integration of such tools into electronic health records. Although multiple factors affect the implementation of RTPB tools, there is limited evidence on outcomes. Further research will be needed to understand the impact of RTPB tools on end results such as prescribing behavior, out-of-pocket medication costs for patients, and adherence to pharmacologic treatment. We review the terminology and concepts essential in understanding the landscape of RTPB tools, implementation considerations, barriers to adoption, and directions for future research that will be important to patients, prescribers, health systems, and insurers.
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Medicare Part D , Medicamentos sob Prescrição , Idoso , Humanos , Estados Unidos , Prescrições , Gastos em SaúdeRESUMO
Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, ΔcdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, α-ketoglutarate, ATP, NAD+, UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that ΔcdnL cells are glutamate auxotrophs, and ΔcdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites.
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Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Homeostase , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Divisão Celular , Sequência Conservada , Metaboloma , Fatores de Transcrição/genéticaRESUMO
Staphylococcus aureus is a common pathogen causing infections in humans with various degrees of severity, with pneumonia being one of the most severe infections. In as much as staphylococcal pneumonia is a disease driven in large part by α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL), we evaluated whether active immunization with attenuated forms of Hla (HlaH35L/H48L) alone, PVL components (LukS-PVT28F/K97A/S209A and LukF-PVK102A) alone, or combination of all 3 toxoids could prevent lethal challenge in a rabbit model of necrotizing pneumonia caused by the USA300 community-associated methicillin-resistant S. aureus (MRSA). Rabbits vaccinated with Hla toxoid alone or PVL components alone were only partially protected against lethal pneumonia, whereas those vaccinated with all 3 toxoids had 100% protection against lethality. Vaccine-mediated protection correlated with induction of polyclonal antibody response that neutralized not only α-hemolysin and PVL, but also other related toxins, produced by USA300 and other epidemic MRSA clones.
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Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Leucocidinas/imunologia , Pneumonia Necrosante/prevenção & controle , Pneumonia Estafilocócica/prevenção & controle , Animais , Toxinas Bacterianas/administração & dosagem , Modelos Animais de Doenças , Exotoxinas/administração & dosagem , Proteínas Hemolisinas/administração & dosagem , Humanos , Leucocidinas/administração & dosagem , Staphylococcus aureus Resistente à Meticilina , Pneumonia Necrosante/imunologia , Pneumonia Estafilocócica/imunologia , Coelhos , VacinaçãoRESUMO
Pseudomonas aeruginosa is a challenge for clinicians due to increasing drug resistance and dwindling treatment options. We report on the activity of MEDI3902, an antibody targeting type 3 secretion protein PcrV and Psl exopolysaccharide, in rabbit bloodstream and lung infection models. MEDI3902 prophylaxis or treatment was protective in both acute models and exhibited enhanced activity when combined with a subtherapeutic dose of meropenem. These findings further support MEDI3902 for the prevention or treatment of serious P. aeruginosa infections.
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Anticorpos Biespecíficos/uso terapêutico , Pneumonia/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/patogenicidade , Animais , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Bacteriemia/terapia , Imunoterapia , Meropeném/uso terapêutico , Pneumonia/microbiologia , Pneumonia/terapia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/efeitos dos fármacos , Coelhos , Resultado do TratamentoRESUMO
Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 µmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.
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Antibacterianos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Lactoferrina/farmacologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , HumanosRESUMO
Coronary collateral growth (CCG) is impaired in metabolic syndrome. microRNA-21 (miR-21) is a proproliferative and antiapoptotic miR, which we showed to be elevated in metabolic syndrome. Here we investigate whether impaired CCG in metabolic syndrome involved miR-21-mediated aberrant apoptosis. Normal Sprague-Dawley (SD) and metabolic syndrome [J. C. Russel (JCR)] rats underwent transient, repetitive coronary artery occlusion [repetitive ischemia (RI)]. Antiapoptotic Bcl-2, phospho-Bad, and Bcl-2/Bax dimers were increased on days 6 and 9 RI, and proapoptotic Bax and Bax/Bax dimers and cytochrome-c release concurrently decreased in JCR versus SD rats. Active caspases were decreased in JCR versus SD rats (~50%). Neutrophils increased transiently on day 3 RI in the collateral-dependent zone of SD rats but remained elevated in JCR rats, paralleling miR-21 expression. miR-21 downregulation by anti-miR-21 induced neutrophil apoptosis and decreased Bcl-2 and Bcl-2/Bax dimers (~75%) while increasing Bax/Bax dimers, cytochrome-c release, and caspase activation (~70, 400, and 400%). Anti-miR-21 also improved CCG in JCR rats (~60%). Preventing neutrophil infiltration with blocking antibodies resulted in equivalent CCG recovery, confirming a major role for deregulated neutrophil apoptosis in CCG impairment. Neutrophil and miR-21-dependent CCG inhibition was in significant part mediated by increased oxidative stress. We conclude that neutrophil apoptosis is integral to normal CCG and that inappropriate prolonged miR-21-mediated survival of neutrophils plays a major role in impaired CCG, in part via oxidative stress generation.
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Apoptose , Oclusão Coronária/metabolismo , Vasos Coronários/metabolismo , Síndrome Metabólica/metabolismo , MicroRNAs/metabolismo , Neutrófilos/metabolismo , Animais , Células Cultivadas , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Citocromos c/metabolismo , Masculino , MicroRNAs/genética , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Inadequate cell proliferation is considered a major causative factor for impaired coronary collateral growth (CCG). Proangiogenic growth factors (GFs) stimulate cell proliferation, but their administration does not promote CCG in patients. These GFs are increased in patients with metabolic syndrome and in animal models, where CCG is impaired. Here, we investigated whether excessive cell proliferation underlies impaired CCG in metabolic syndrome. Normal [Sprague-Dawley (SD)] and metabolic syndrome [James C. Russell (JCR)] rats underwent repetitive ischemia (RI; transient, repetitive coronary artery occlusion and myocardial ischemia). We have shown that CCG was maximal at d 9 of RI in SD rats but did not occur in JCR rats. The increase in cell proliferation (PCNA, Ki-67, cyclin A, phospho- cdc2, p21Waf, p27Kip) was transient (â¼4-fold, d 3 RI) in SD rats but greater and sustained in JCR rats (â¼8- to 6-fold, d 3-9 RI). In JCR rats, this was associated with increased and sustained miR-21 expression and accumulation of proliferating synthetic vascular smooth muscle cells in the lumen of small arterioles, which failed to undergo outward expansion. Administration of anti-miR-21 blocked RI-induced cell proliferation and significantly improved CCG in JCR rats (â¼60%). miR-21-dependent excessive cell proliferation in the later stages of collateral remodeling correlates with impaired CCG in metabolic syndrome.
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Proliferação de Células/genética , Circulação Colateral/fisiologia , Doença da Artéria Coronariana/prevenção & controle , Circulação Coronária/fisiologia , Síndrome Metabólica/fisiopatologia , MicroRNAs/metabolismo , Músculo Liso Vascular/citologia , Isquemia Miocárdica/prevenção & controle , Animais , Apoptose , Western Blotting , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Modelos Animais de Doenças , Técnicas Imunoenzimáticas , Masculino , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Neovascularização Patológica/prevenção & controle , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: We have previously shown that transient coronary artery occlusion stimulated coronary collateral growth (CCG) in healthy (Sprague Dawley) but not in metabolic syndrome (JCR:LA-cp [JCR] ) rats. Here, we sought to determine whether matrix metalloproteinases (MMPs) negatively regulate CCG in the metabolic syndrome via release of endostatin and angiostatin. APPROACH AND RESULTS: Rats underwent transient, repetitive left anterior descending occlusion and resultant myocardial ischemia (RI) for 0 to 10 days. CCG was measured in the collateral-dependent and normal zones using microspheres, MMP activation by Western blot, and endostatin and angiostatin by ELISA on days 0, 3, 6, 9, or 10 of RI. Endostatin and angiostatin were increased in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Increased endostatin and angiostatin correlated with increased MMP12 (≈ 4-fold) activation in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Inhibition of MMP12 in JCR rats nearly completely blocked endostatin (≈ 85%) and angiostatin (≈ 90%) generation and significantly improved CCG (collateral-dependent zone flow was ≈ 66% of normal zone flow versus ≈ 12% for JCR RI). CONCLUSIONS: Compromised CCG in the metabolic syndrome is, in large part, because of increased MMP12 activation and consequent increased generation of endostatin and angiostatin, which inhibits late-stage collateral remodeling.
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Angiostatinas/metabolismo , Circulação Colateral/fisiologia , Oclusão Coronária/metabolismo , Endostatinas/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Síndrome Metabólica/metabolismo , Angiostatinas/análise , Animais , Western Blotting , Circulação Coronária/fisiologia , Oclusão Coronária/fisiopatologia , Modelos Animais de Doenças , Endostatinas/análise , Ensaio de Imunoadsorção Enzimática , Síndrome Metabólica/fisiopatologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
OBJECTIVE: Transient, repetitive occlusion stimulates coronary collateral growth (CCG) in normal animals. Vascular smooth muscle cells (VSMCs) switch to synthetic phenotype early in CCG, then return to contractile phenotype. CCG is impaired in the metabolic syndrome. We determined whether impaired CCG was attributable to aberrant VSMC phenotypic modulation by miR-145-mediated mechanisms, and whether restoration of physiological miR-145 levels in metabolic syndrome (JCR rat) improved CCG. APPROACH AND RESULTS: CCG was stimulated by transient, repetitive left anterior descending artery occlusion and evaluated after 9 days by coronary blood flow measurements (microspheres). miR-145 was delivered to JCR VSMCs via adenoviral vector (miR-145-Adv). In JCR rats, miR-145 was decreased late in CCG (≈ 2-fold day 6; ≈ 4-fold day 9 versus SD), which correlated with decreased expression of smooth muscle-specific contractile proteins (≈ 5-fold day 6; ≈ 10-fold day 9 versus SD), indicative of VSMCs' failure to return to the contractile phenotype late in CCG. miR-145 expression in JCR rats (miR-145-Adv) on days 6 to 9 of CCG completely restored VSMCs contractile phenotype and CCG (collateral/normal zone flow ratio was 0.93 ± 0.09 JCR+miR-145-Adv versus 0.12 ± 0.02 JCR versus 0.87 ± 0.02 SD). CONCLUSIONS: Restoration of VSMC contractile phenotype through miR-145 delivery is a highly promising intervention for restoration of CCG in the metabolic syndrome.
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Circulação Colateral , Circulação Coronária , Terapia Genética , Síndrome Metabólica/terapia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição , Adenoviridae/genética , Animais , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Síndrome Metabólica/fisiopatologia , Proteínas Musculares/biossíntese , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Fenótipo , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Ratos Zucker , Fatores de TempoRESUMO
OBJECTIVE: Chronic blood transfusion therapy reduces clinical events in children with sickle cell anemia but increases risk for an iron-related liver injury. Liver biopsy is the gold standard technique for quantifying liver iron content (LIC) and evaluating liver pathology. Ferritin, liver enzymes, and R2* magnetic resonance imaging of the liver are obtained as surrogate markers. In this study we compared surrogate markers with the gold standard, liver biopsy, in assessing liver histology. METHODS: We conducted a retrospective review of 259 liver biopsies in 109 children with sickle cell anemia on chronic transfusion therapy and chelation therapy during a 9-year period at a single center. Liver pathology was compared with LIC, ferritin, and alanine aminotransferase. RESULTS: Ferritin correlates with LIC (râ=â0.74, Pâ<â0.001), although there is a broad range of ferritin values for a given LIC. Furthermore, patients with a high LIC (≥7 mg Fe/g dry weight) demonstrated significantly higher ferritin as compared to the patients with lower LIC <7 (Pâ<â0.001). Periportal/portal inflammation also showed a significant relation. There was no significance when comparing ferritin and lobular inflammation or ferritin and alanine aminotransferase. When evaluating LIC in relation to fibrosis, the present study revealed that there was only a significant correlation with severe fibrosis (Fâ=â36, Pâ<â0.001). CONCLUSIONS: The results suggest that although correlations exist among ferritin and LIC and severe fibrosis and LIC, caution should be taken when they are used in isolation. Liver biopsy provides important pathologic information that cannot be obtained through surrogate markers.
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Anemia Falciforme/terapia , Ferritinas/metabolismo , Ferro/metabolismo , Cirrose Hepática/etiologia , Fígado/patologia , Reação Transfusional , Adolescente , Adulto , Alanina Transaminase/sangue , Anemia Falciforme/sangue , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Biópsia , Terapia por Quelação , Criança , Pré-Escolar , Humanos , Inflamação/metabolismo , Fígado/metabolismo , Cirrose Hepática/sangue , Cirrose Hepática/metabolismo , Estudos Retrospectivos , Índice de Gravidade de Doença , Adulto JovemRESUMO
BACKGROUND: PALB2 has emerged as a breast cancer susceptibility gene. Mutations in PALB2 have been identified in almost all breast cancer populations studied to date, but the rarity of these mutations and lack of information regarding their penetrance makes genetic counseling for these families challenging. We studied BRCA1/2 -negative breast and/or ovarian cancer families to a) assess the contribution of PALB2 mutations in this series and b) identify clinical, pathological and family history characteristics that might make PALB2 screening more efficient. METHODS: The coding region of the PALB2 gene was analyzed in 175 probands with family histories of breast and/or ovarian cancer ascertained from a single Canadian institution in Eastern Ontario. RESULTS: We identified 2 probands with PALB2 mutations that are known or strongly considered to be pathogenic and 3 probands with missense mutations that are possibly pathogenic. One of the identified truncating mutations [c.3113G > A (p.Gly1000_Trp1038del - major product)], has been previously described while the other four mutations [c.3507_3508delTC (p.H1170Ffs*19), c.1846G > C (p.D616H), c.3418 T > G (p.W1140G), c.3287A > G (p.N1096S)] have not been previously reported. Loss of heterozygosity was detected in two breast tumors from one c.3507_3508delTC mutation carrier but not in other available tumors from that family or in tumors from carriers of other mutations. CONCLUSIONS: PALB2 mutation screening identifies a small, but significant number of mutations in BRCA1/2 -negative breast and/or ovarian cancer families. We show that mutations are more likely to be found in families with three or more breast cancers as well as other BRCA2-related cancers. In our cohort, both clearly pathogenic mutations were identified in premenopausal breast cancer cases (2/77, 2.6%). Testing should be preferentially offered to affected women from such families.
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Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium Caulobacter crescentus. Unexpectedly, depletion of OpgH or attempted complementation with a catalytically dead OpgH variant results in striking asymmetric bulging and cell lysis. These shape defects are accompanied by reduced cell wall synthesis and mislocalization of morphogenetic complexes. Interestingly, overactivation of the CenKR two-component system that has been implicated in cell envelope stress homeostasis in α-proteobacteria phenocopies the morphogenetic defects associated with OpgH depletion. Each of these perturbations leads to an increase in the levels of the elongasome protein, MreB, and decreases in the levels of divisome proteins FtsZ and MipZ as well as OpgH, itself. Constitutive production of OpgH during CenKR overactivation prevents cell bulging, but cells still exhibit morphogenetic defects. We propose that OPG depletion activates CenKR, leading to changes in the expression of cell envelope-related genes, but that OPGs also exert CenKR-independent effects on morphogenesis. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining cell morphology in Caulobacter.IMPORTANCEBacteria must synthesize and fortify the cell envelope in a tightly regulated manner to orchestrate growth and adaptation. Osmoregulated periplasmic glucans (OPGs) are important, but poorly understood, constituents of Gram-negative cell envelopes that contribute to envelope integrity and protect against osmotic stress. Here, we determined that the OPG synthase OpgH plays a surprising, essential role in morphogenesis in Caulobacter crescentus. Loss of OpgH causes asymmetric cell bulging and lysis via misregulation of the localization and activity of morphogenetic complexes. Overactivation of the CenKR two-component system involved in envelope homeostasis phenocopies OpgH depletion, suggesting that depletion of OpgH activates CenKR. Because cell envelope integrity is critical for bacterial survival, understanding how OpgH activity contributes to morphogenesis and maintenance of envelope integrity could aid in the development of antibiotic therapies.
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In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation in Caulobacter crescentus, SpoT synthesizes the secondary messengers guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate (collectively known as (p)ppGpp), which affect transcription by binding RNA polymerase (RNAP) to down-regulate anabolic genes. (p)ppGpp also impacts the expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. In Caulobacter, a major regulator of anabolic genes is the transcription factor CdnL. If and how CdnL is controlled during the SR and why that might be functionally important are unclear. In this study, we show that CdnL is down-regulated posttranslationally during starvation in a manner dependent on SpoT and the ClpXP protease. Artificial stabilization of CdnL during starvation causes misregulation of ribosomal and metabolic genes. Functionally, we demonstrate that the combined action of SR transcriptional regulators and CdnL clearance allows for rapid adaptation to nutrient repletion. Moreover, cells that are unable to clear CdnL during starvation are outcompeted by wild-type cells when subjected to nutrient fluctuations. We hypothesize that clearance of CdnL during the SR, in conjunction with direct binding of (p)ppGpp and DksA to RNAP, is critical for altering the transcriptome in order to permit cell survival during nutrient stress.
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Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon assembling at mid-cell, FtsZ exhibits treadmilling motion driven by GTP binding and hydrolysis. Treadmilling is proposed to play roles in Z-ring condensation and in distribution and regulation of peptidoglycan (PG) cell wall enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We addressed these gaps in knowledge by evaluating the contribution of GTPase activity to FtsZ's function in vitro and in Caulobacter crescentus cells. We observed that a lethal mutation that abrogates FtsZ GTP hydrolysis impacts FtsZ dynamics and Z-ring positioning, but not constriction. Aberrant Z-ring positioning was due to insensitivity to the FtsZ regulator MipZ when GTPase activity is reduced. Z-ring mislocalization resulted in DNA damage, likely due to constriction over the nucleoid. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell in Caulobacter. Proper Z-ring localization is required for effective coordination with chromosome segregation to prevent DNA damage and ensure successful cell division.
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Proteínas de Bactérias , Caulobacter crescentus , Divisão Celular , Proteínas do Citoesqueleto , GTP Fosfo-Hidrolases , Guanosina Trifosfato , Caulobacter crescentus/metabolismo , Caulobacter crescentus/genética , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Divisão Celular/fisiologia , Hidrólise , MutaçãoRESUMO
OBJECTIVES: Transitions of care are pivotal, vulnerable times as patients are discharged from the hospital. Telephonic care coordination is standard care, but labor intensive. We implemented a patient postdischarge digital engagement (PDDE) program to scale coordination. We hypothesized that PDDE could reduce readmissions for low-risk patients and supplement care coordination for medium- and high-risk patients. STUDY DESIGN: Pragmatic, stepped-wedge cluster randomization trial with 5 implementation waves based upon primary care clinic region. METHODS: All inpatient hospital discharges between March 2020 and November 2020 were stratified by readmission risk. Low-risk patients were offered access to PDDE, and moderate-risk and high-risk patients were offered access to PDDE and care coordination. Readmission was defined as an unplanned inpatient admission within 30 days from discharge. An intention-to-treat primary analysis was conducted using mixed-effects logistic regression clustering for wave; a treatment-on-the-treated analysis was also conducted to assess the impact among program users. RESULTS: A total of 5490 patient discharges were examined (2735 control; 2755 intervention); 1949 patients were high risk, 2032 were medium risk, and 1509 were low risk. PDDE intervention did not significantly affect readmission among low-risk (95% CI, -0.23 to 0.90; P = .23), medium-risk (95% CI, -0.14 to 0.60; P = .21), and high-risk (95% CI, -0.32 to 0.64; P = .48) groups after adjustment for time and patient factors. In a treatment-on-the-treated analysis, among patients who activated the PDDE program, readmission was also similar among the low-, medium-, and high-risk cohorts. CONCLUSIONS: Our study expanded resource-limited care coordination by offering low-risk patients a service they were unable to receive previously while having no impact on readmission. PDDE efficiently provided additional touch points between patients and providers.
Assuntos
Alta do Paciente , Readmissão do Paciente , Humanos , Assistência ao Convalescente , Hospitalização , Pacientes InternadosRESUMO
Mutations in the cyclin-dependent kinase inhibitor-2A (CDKN2A) gene have been associated with a number of malignancies, most notably cutaneous malignant melanoma (CMM). Mutations in this gene have also been associated with pancreatic cancer and breast cancer, as well as astrocytomas and other nervous system tumors (NST). Among NST, rare solitary internal neurofibromas have been reported, but multiple cutaneous neurofibromas have only been described in two families. In the first family, the affected individuals all carried a heterozygous G>C mutation at the splice acceptor site of intron 1 resulting in skipping of CDKN2A exon 2, while the affected individuals in the second family had a deletion that encompassed the whole CDKN2A/CDKN2B/ANRIL locus. We now report on a proposita presenting with multiple biopsy-proven cutaneous neurofibromas and a solitary spinal neurofibroma found to have a deletion of 14 nucleotides in exon 2 of CDKN2A, providing further evidence that p14, p16, and/or ANRIL are specifically involved in the pathogenesis of neurofibromas as a feature of the familial atypical multiple malignant melanoma spectrum.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Melanoma/genética , Neurofibromatoses/genética , Neoplasias Cutâneas/genética , Neoplasias da Coluna Vertebral/genética , Adulto , Sequência de Bases , Diagnóstico Diferencial , Feminino , Humanos , Melanoma/diagnóstico , Neurofibromatoses/diagnóstico , Linhagem , Deleção de Sequência , Neoplasias Cutâneas/diagnóstico , Neoplasias da Coluna Vertebral/diagnóstico , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Holoprosencephaly (HPE), the most common malformation of the human forebrain, may result from mutations in over 12 genes. Sonic Hedgehog (SHH) was the first such gene discovered; mutations in SHH remain the most common cause of non-chromosomal HPE. The severity spectrum is wide, ranging from incompatibility with extrauterine life to isolated midline facial differences. OBJECTIVE: To characterise genetic and clinical findings in individuals with SHH mutations. METHODS: Through the National Institutes of Health and collaborating centres, DNA from approximately 2000 individuals with HPE spectrum disorders were analysed for SHH variations. Clinical details were examined and combined with published cases. RESULTS: This study describes 396 individuals, representing 157 unrelated kindreds, with SHH mutations; 141 (36%) have not been previously reported. SHH mutations more commonly resulted in non-HPE (64%) than frank HPE (36%), and non-HPE was significantly more common in patients with SHH than in those with mutations in the other common HPE related genes (p<0.0001 compared to ZIC2 or SIX3). Individuals with truncating mutations were significantly more likely to have frank HPE than those with non-truncating mutations (49% vs 35%, respectively; p=0.012). While mutations were significantly more common in the N-terminus than in the C-terminus (including accounting for the relative size of the coding regions, p=0.00010), no specific genotype-phenotype correlations could be established regarding mutation location. CONCLUSIONS: SHH mutations overall result in milder disease than mutations in other common HPE related genes. HPE is more frequent in individuals with truncating mutations, but clinical predictions at the individual level remain elusive.