RESUMO
Background: Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005. Methods: We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates. Results: Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3. Conclusions: The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the "perfect storm" for sustained endemicity of these multidrug-resistant organisms in Colombia.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Epidemias , Evolução Molecular , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Cidades/epidemiologia , Colômbia/epidemiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Transmissão de Doença Infecciosa , Transferência Genética Horizontal , Humanos , Sequências Repetitivas Dispersas , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/isolamento & purificação , Epidemiologia Molecular , Filogenia , Análise de Sequência de DNA , Centros de Atenção Terciária , Sequenciamento Completo do GenomaRESUMO
P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)-a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.
RESUMO
Mutations in SARS-CoV-2 variants of concern (VOCs) have expanded the viral host range beyond primates, and a limited range of other mammals, to mice, affording the opportunity to exploit genetically diverse mouse panels to model the broad range of responses to infection in patient populations. Here we surveyed responses to VOC infection in genetically diverse Collaborative Cross (CC) founder strains. Infection of wild-derived CC founder strains produced a broad range of viral burden, disease susceptibility and survival, whereas most other strains were resistant to disease despite measurable lung viral titers. In particular, CAST/EiJ, a wild-derived strain, developed high lung viral burdens, more severe lung pathology than seen in other CC strains, and a dysregulated cytokine profile resulting in morbidity and mortality. These inbred mouse strains may serve as a valuable platform to evaluate therapeutic countermeasures against severe COVID-19 and other coronavirus pandemics in the future.
RESUMO
The Escherichia coli actin homologue MreB is part of a helical cytoskeletal structure that winds around the cell between the two poles. It has been shown that MreB redistributes during the cell cycle to form circumferential ring structures that flank the cytokinetic FtsZ ring and appear to be associated with division and segregation of the helical cytoskeleton. We show here that the MreB cytoskeletal ring also contains the MreC, MreD, Pbp2 and RodA proteins. Assembly of MreB, MreC, MreD and Pbp2 into the ring structure required the FtsZ ring but no other known components of the cell division machinery, whereas assembly of RodA into the cytoskeletal ring required one or more additional septasomal components. Strikingly, MreB, MreC, MreD and RodA were each able to independently assemble into the cytoskeletal ring and coiled cytoskeletal structures in the absence of any of the other ring components. This excludes the possibility that one or more of these proteins acts as a scaffold for incorporation of the other proteins into these structures. In contrast, incorporation of Pbp2 required the presence of MreC, which may provide a docking site for Pbp2 entry.
Assuntos
Divisão Celular , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/citologia , Complexos Multiproteicos , Multimerização ProteicaRESUMO
Three of the four well-established bacterial cytoskeletal systems-the MreB, MinCDE, and FtsZ systems-undergo a variety of short-range and long-range dynamic behaviors. These include the cellular reorganization of the cytoskeletal elements, in which the proteins redistribute from a predominantly helical pole-to-pole pattern into annular structures near midcell. Despite their apparent similarity, these dramatic redistributional events in the three systems are in large part independent of each other. In addition, some of the cytoskeletal structures undergo oscillatory behavior in which the helical elements move repetitively back-and-forth between the two ends of the cell. The details and mechanisms underlying these dynamic cellular events are just now being revealed by fluorescence microscopy of intact cells, fluorescence photobleaching recovery studies, single molecule tracking techniques, and in vitro studies of the purified proteins.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Bactérias/metabolismo , Polaridade Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Membrana/metabolismo , Microscopia de FluorescênciaRESUMO
LC-MS technique described here is a new way for the separation and direct determination of UV-Vis insensitive inositol phosphates (InsP(2)-InsP(6)). This circumvents the need of radioisotopic labeling and post-column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP(6) on C(18)-column using MeOH/H(2)O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI-MS). The LC-MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP(6 )in a sequential manner leading to InsP(2 )(InsP(2) x 2Na, t(R) 4.4-4.54 min, base peak m/z 382.9) as the end product.
Assuntos
6-Fitase/química , Cromatografia Líquida/métodos , Ácido Fítico/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Aspergillus niger/enzimologia , Cromatografia em Camada Fina , FosforilaçãoRESUMO
The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.
Assuntos
6-Fitase/biossíntese , Aspergillus niger/crescimento & desenvolvimento , Aspergillus niger/metabolismo , Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/métodos , Aspergillus niger/classificação , Aspergillus niger/citologia , Tamanho Celular , Estudos de Viabilidade , Concentração de Íons de Hidrogênio , Consumo de Oxigênio/fisiologia , Projetos Piloto , Especificidade da EspécieRESUMO
Phytase from Aspergillus niger van Teighem efficiently hydrolyses phytate phosphorus present in various commercial live stock feeds and was not inactivated by various formulations and antibiotics present. The enzyme retained 90-95% phytase activity at 55 degrees C, pH 2.5 after 72 h of incubation with all the commercial feeds tested, thus indicating its suitability in feed application. The phytase hydrolysis increased with the increase in temperature and a significant release of 41 nmols P(i)/ml in phytase-treated feed over control sample was observed at 55 degrees C after 48 h. Besides this, the enzyme was maximally effective when used under acidic condition, releasing 21 and 42 nmols P(i)/ml at pH 1.5 and 2.5, respectively. As the pH shifted towards 5.5, significant decline in phosphorus release was observed. However, the enzyme was able to retain almost complete phytase activity in the presence of feed constituent even after 48 h over various pH tested. Thus it can be a potential candidate in animal nutrition where the ability of present phytase to retain activity over period of time in the presence of feed constituent is desired.
Assuntos
6-Fitase/química , Ração Animal , Aspergillus niger/classificação , Aspergillus niger/enzimologia , Ácido Fítico/química , Animais , Animais Domésticos , Concentração de Íons de Hidrogênio , Hidrólise , Fosforilação , Especificidade da EspécieRESUMO
The bacterial actin homolog MreB exists as a single-copy helical cytoskeletal structure that extends between the two poles of rod-shaped bacteria. In this study, we show that equipartition of the MreB cytoskeleton into daughter cells is accomplished by division and segregation of the helical MreB array into two equivalent structures located in opposite halves of the predivisional cell. This process ensures that each daughter cell inherits one copy of the MreB cytoskeleton. The process is triggered by the membrane association of the FtsZ cell division protein. The cytoskeletal division and segregation events occur before and independently of cytokinesis and involve specialized MreB structures that appear to be intermediates in this process.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Escherichia coli/citologia , Ciclo Celular , Divisão Celular , Citoesqueleto/ultraestrutura , Proteínas de Escherichia coli/química , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Modelos Moleculares , Conformação ProteicaRESUMO
Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)-1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52-55 degrees C. The enzyme retained 97% activity after a 24-h incubation at 55 degrees C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1-5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5-8 M), significantly affected phytase activity. The maximum hydrolysis rate (Vmax) and apparent Michaelis-Menten constant (Km) were 1,074 IU/mL and 606 microM, respectively, with a catalytic turnover number of 3x10(5) s-1 and catalytic efficiency of 3.69x10(8) M-1 s-1.
Assuntos
6-Fitase/química , 6-Fitase/metabolismo , Aspergillus niger/enzimologia , 6-Fitase/isolamento & purificação , Aspergillus niger/crescimento & desenvolvimento , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Cinética , Ácido Fítico/metabolismo , Temperatura , Regulação para CimaRESUMO
HGT1 encodes a high-affinity glutathione transporter in the yeast Saccharomyces cerevisiae that is induced under sulphur limitation. The present work demonstrates that repression by organic sulphur sources is under the control of the classic sulphur regulatory network, as seen by the absence of expression in a met4delta background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4delta strains (deficient in cysteine biosynthesis) that could be repressed by elevated levels of cysteine, but not by methionine or glutathione. Investigations into cis-regulatory elements revealed that the previously described motif, a 9-bp cis element, CCGCCACAC, located at the -356 to -364 region of the promoter could in fact be refined to a 7-bp CGCCACA motif that is also repeated at -333 to -340. The second copy of this motif was essential for activity, since mutations in the core region of the second copy completely abolished activity and regulation by sulphur sources. Activity, but not regulation, could be restored by reintroducing an additional copy upstream of the first copy. A third region, GCCGTCTGCAAGGCA, conserved in the HGT1 promoters of the different Saccharomyces spp, was observed at -300 to -285 but, while mutations in this region did not lead to any loss in repression, the basal and induced levels were significantly increased. In contrast to a previous report, no evidence was found for regulation by the VDE endonuclease. The strong repression at the transport level by glutathione seen in strains overexpressing HGT1 was due to a glutathione-dependent toxicity in these cells.