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1.
Proc Natl Acad Sci U S A ; 121(20): e2321711121, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38713624

RESUMO

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Córtex Cerebral , Receptores ErbB , Proteínas Hedgehog , Proteínas do Tecido Nervoso , Células-Tronco Neurais , Neurogênese , Fator de Transcrição 2 de Oligodendrócitos , Fator de Transcrição PAX6 , Animais , Neurogênese/fisiologia , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Camundongos , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fator de Transcrição PAX6/metabolismo , Fator de Transcrição PAX6/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Neuroglia/metabolismo , Neuroglia/citologia , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Bulbo Olfatório/metabolismo , Bulbo Olfatório/citologia , Linhagem da Célula , Humanos
2.
Proc Natl Acad Sci U S A ; 117(46): 28625-28631, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33139554

RESUMO

Evidence linking amyloid beta (Aß) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aß uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aß depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrPC) has emerged as an important mediator of Aß-induced toxicity and of neuronal Aß internalization. Here, we report that the soluble, nonfibrillizing Aß (1-30) peptide recapitulates full-length Aß stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aß (1-30) uptake is also dependent on PrPC expression. NMR-based molecular-level characterization identified the docking site on PrPC that underlies the stereoselective binding of Aß (1-30). Our findings therefore identify a specific sequence within Aß that is responsible for the recognition of the peptide by PrPC, as well as PrPC-dependent cellular uptake. Further uptake stereodifferentiation in PrPC-free cells points toward additional receptor-mediated interactions as likely contributors for Aß cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aß cellular uptake as an alternative route for future therapeutic development for Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas PrPC/metabolismo , Células HEK293 , Humanos
3.
Emerg Infect Dis ; 28(5)2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35451367

RESUMO

We describe the global molecular epidemiology of 229 carbapenemase-producing Escherichia coli in 36 countries during 2015-2017. Common carbapenemases were oxacillinase (OXA) 181 (23%), New Delhi metallo-ß-lactamase (NDM) 5 (20%), OXA-48 (17%), Klebsiella pneumoniae carbapenemase 2 (15%), and NDM-1 (10%). We identified 5 dominant sequence types (STs); 4 were global (ST410, ST131, ST167, and ST405), and 1 (ST1284) was limited to Turkey. OXA-181 was frequent in Jordan (because of the ST410-B4/H24RxC subclade) and Turkey (because of ST1284). We found nearly identical IncX3-blaOXA-181 plasmids among 11 STs from 12 countries. NDM-5 was frequent in Egypt, Thailand (linked with ST410-B4/H24RxC and ST167-B subclades), and Vietnam (because of ST448). OXA-48 was common in Turkey (linked with ST11260). Global K. pneumoniae carbapenemases were linked with ST131 C1/H30 subclade and NDM-1 with various STs. The global carbapenemase E. coli population is dominated by diverse STs with different characteristics and varied geographic distributions, requiring ongoing genomic surveillance.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Escherichia coli , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Humanos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
4.
J Antimicrob Chemother ; 76(5): 1135-1139, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33547472

RESUMO

INTRODUCTION: Escherichia coli ST131 is the most common multidrug-resistant (MDR) E. coli clone causing bloodstream infections (BSIs) in Calgary. This study describes patient characteristics and spatial distribution of ST131 subclades C1 and C2 causing BSIs in Calgary. METHODS: E. coli from blood (n = 685) obtained in Calgary, Canada, (2016) were PCR screened for ST131 and positives (n = 141) underwent whole genome sequencing. Patient characteristics were analysed using Fisher's Exact/t-tests and spatial analysis was used to identify clusters. RESULTS: Overall, 21% of E. coli was identified as ST131 and clade C dominated the population. ST131-C2 was associated with blaCTX-M-15 and significantly more MDR than ST131-C1. The spatial distribution in Calgary showed that ST131-C1 was mainly present in long-term care (LTC) residents whereas ST131-C2 clustered in a specific North East (NE) Calgary sector comprising of six neighbourhoods without LTC centres. This NE sector has high immigration and travel rates from the Indian subcontinent. CONCLUSIONS: This study showed that ST131 C subclades have different geographical distribution patterns in Calgary. We believe that recent travel to and immigration from certain high-risk regions for antimicrobial resistance are responsible for the ST131-C2 NE Calgary clustering pattern.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Antibacterianos/farmacologia , Canadá/epidemiologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Proteínas de Escherichia coli/genética , Humanos , beta-Lactamases/genética
5.
Emerg Infect Dis ; 26(12): 2907-2915, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33219650

RESUMO

Global expansion of antimicrobial drug-resistant Escherichia coli sequence type (ST) 131 is unrivaled among human bacteria. Understanding trends among ST131 clades will help with designing prevention strategies. We screened E. coli from blood samples (n = 1,784) obtained in Calgary, Alberta, Canada, during 2006, 2012, and 2016 by PCR for ST131 and positive samples (n = 344) underwent whole-genome sequencing. The incidence rate per 100,000 residents increased from 4.91 during 2006 to 12.35 during 2012 and 10.12 during 2016. ST131 belonged to clades A (10%), B (9%), and C (81%). Clades C1-nonM27 and B were common during 2006, and C2 containing blaCTX-M-15, C1-M27 containing blaCTX-M-27, and A were responsible for the increase of ST131 during 2012 and 2016. C2 was the most antimicrobial drug-resistant subclade and increased exponentially over time. Eradicating ST131, more specifically the C2 subclade, will lead to considerable public health benefits for persons in Calgary.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Alberta/epidemiologia , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Humanos , Dinâmica Populacional , beta-Lactamases/genética
6.
Appl Environ Microbiol ; 86(4)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31811043

RESUMO

In the ten years since its discovery, the Escherichia coli clone sequence type 131 (ST131) has become a major international health threat, with the multidrug-resistant and extended-spectrum ß-lactamase (ESBL)-producing clade C emerging as the globally dominant form. ST131 has previously been isolated from wastewater; however, most of these studies selectively screened for ESBL-producing organisms, thereby missing the majority of remaining ST131 clades. In this study, we used a high-throughput PCR-based screening strategy to comprehensively examine wastewater for the presence of ST131 over a 1-year period. Additional multiplex PCRs were used to differentiate clades and obtain an unbiased account of the total ST131 population structure within the collection. Furthermore, antimicrobial susceptibility profiles of all ST131-positive samples were tested against a range of commonly used antibiotics. From a total of over 3,762 E. coli wastewater samples, 1.86% (n = 70) tested positive for ST131, with the majority being clade A isolates. In total, 63% (n = 44) were clade A, 29% (n = 20) were clade B, 1% (n = 1) were clade C0, 6% (n = 4) were clade C1, and 1% (n = 1) were clade C2. In addition, a very high rate of resistance to commonly used antibiotics among wastewater isolates is reported, with 72.7% (n = 32) of clade A resistant to ciprofloxacin and high rates of resistance to gentamicin, sulfamethoxazole-trimethoprim, and tetracycline in clades that are typically sensitive to antibiotics.IMPORTANCE ST131 is a global pathogen. This clone causes urinary tract infections and is frequently isolated from human sources. However, little is known about ST131 from environmental sources. With the widely reported increase in antibiotic concentrations found in wastewater, there is additional selection pressure for the emergence of antibiotic-resistant ST131 in this niche. The unbiased screening approach reported herein revealed that previously antibiotic-sensitive lineages of ST131 are now resistant to commonly used antibiotics present in wastewater systems and may be capable of surviving UV sterilization. This is the most comprehensive account of ST131 in the wastewater niche to date and an important step in better understanding the ecology of this global pathogen.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Águas Residuárias/microbiologia , Alberta , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase
7.
PLoS Genet ; 13(1): e1006570, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28103245

RESUMO

Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes.


Assuntos
Escherichia coli/genética , Frequência do Gene , Taxa de Mutação , Oxigênio/metabolismo , Anaerobiose , Reparo do DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Sequências Repetitivas de Ácido Nucleico/genética
8.
Proteins ; 87(8): 699-705, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30958578

RESUMO

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Fator G para Elongação de Peptídeos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Ribossomos/química
9.
Chembiochem ; 20(8): 1023-1026, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30550626

RESUMO

Amyloidâ€…ß is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloidâ€…ß and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Estereoisomerismo
10.
Biol Lett ; 12(8)2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27555646

RESUMO

Life has existed on the Earth for approximately four billion years. The sheer depth of evolutionary time, and the diversity of extant species, makes it tempting to assume that all the key biochemical innovations underpinning life have already happened. But we are only a little over halfway through the trajectory of life on our planet. In this Opinion piece, we argue: (i) that sufficient time remains for the evolution of new processes at the heart of metabolic biochemistry and (ii) that synthetic biology is providing predictive insights into the nature of these innovations. By way of example, we focus on engineered solutions to existing inefficiencies in energy generation, and on the complex, synthetic regulatory circuits that are currently being implemented.


Assuntos
Evolução Biológica , Planeta Terra , Vida
11.
J Biol Chem ; 287(25): 21530-40, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22549788

RESUMO

Although serum albumin has an established function as a transport protein, evidence is emerging that serum albumin may also have a role as a molecular chaperone. Using established techniques to characterize chaperone interactions, this study demonstrates that bovine serum albumin: 1) preferentially binds stressed over unstressed client proteins; 2) forms stable, soluble, high molecular weight complexes with stressed client proteins; 3) reduces the aggregation of client proteins when it is present at physiological levels; and 4) inhibits amyloid formation by both WT and L55P transthyretin. Although the antiaggregatory effect of serum albumin is maintained in the presence of physiological levels of Ca(2+) and Cu(2+), the presence of free fatty acids significantly alters this activity: stabilizing serum albumin at normal levels but diminishing chaperone-like activity at high concentrations. Moreover, here it is shown that depletion of albumin from human plasma leads to a significant increase in aggregation under physiologically relevant heat and shear stresses. This study demonstrates that serum albumin possesses chaperone-like properties and that this activity is maintained under a number of physiologically relevant conditions.


Assuntos
Amiloide/química , Chaperonas Moleculares/química , Soroalbumina Bovina/química , Amiloide/metabolismo , Animais , Bovinos , Humanos , Chaperonas Moleculares/metabolismo , Pré-Albumina/química , Pré-Albumina/metabolismo , Ligação Proteica , Estabilidade Proteica , Soroalbumina Bovina/metabolismo
12.
J Sep Sci ; 34(19): 2759-64, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21898817

RESUMO

Centrifugal precipitation chromatography and a high-speed counter-current chromatography system equipped with a spiral tubing support rotor (spHSCCC) were successfully applied for the identification and isolation of carotenoid cleavage-like enzymes from Enteromorpha compressa (L.) Nees. This is the first study separating active enzymes from a complex natural matrix by spHSCCC. The target enzymes were identified after fractionation of the proteins in an acetone Tris-buffer gradient by centrifugal precipitation chromatography. Also, an aqueous two-phase solvent system consisting of PEG 1000 and mono- and dibasic potassium phosphate was used for the isolation of the enzymes by spHSCCC. The purified fractions contained two proteins of 65 and 72 kDa, respectively. The enzymes could cleave ß-carotene and ß-apo-8'-carotenal to produce ß-ionone.


Assuntos
Cromatografia Líquida/métodos , Distribuição Contracorrente/métodos , Dioxigenases/química , Dioxigenases/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Ulva/enzimologia , Centrifugação/métodos , Precipitação Química , Cromatografia Líquida/instrumentação , Distribuição Contracorrente/instrumentação , Peso Molecular , Ulva/química
13.
Cell Rep ; 35(12): 109269, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34161768

RESUMO

Projection neuron subtype identities in the cerebral cortex are established by expressing pan-cortical and subtype-specific effector genes that execute terminal differentiation programs bestowing neurons with a glutamatergic neuron phenotype and subtype-specific morphology, physiology, and axonal projections. Whether pan-cortical glutamatergic and subtype-specific characteristics are regulated by the same genes or controlled by distinct programs remains largely unknown. Here, we show that FEZF2 functions as a transcriptional repressor, and it regulates subtype-specific identities of both corticothalamic and subcerebral neurons by selectively repressing expression of genes inappropriate for each neuronal subtype. We report that TLE4, specifically expressed in layer 6 corticothalamic neurons, is recruited by FEZF2 to inhibit layer 5 subcerebral neuronal genes. Together with previous studies, our results indicate that a cortical glutamatergic identity is specified by multiple parallel pathways active in progenitor cells, whereas projection neuron subtype-specific identity is achieved through selectively repressing genes associated with alternate identities in differentiating neurons.


Assuntos
Córtex Cerebral/citologia , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transcrição Gênica , Alelos , Animais , Diferenciação Celular/genética , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Camundongos Knockout , Mitose/genética , Neurônios/citologia , Ligação Proteica , Proteínas Repressoras/metabolismo
14.
Infect Genet Evol ; 81: 104265, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32112974

RESUMO

The abrupt expansion of Escherichia coli sequence type (ST) 131 is unmatched among Gram negative bacteria. In many ways, ST131 can be considered a real-world model for the complexities involved in the evolution of a multidrug resistant pathogen. While much progress has been made on our insights into the organism's population structure, pathogenicity and drug resistance profile, significant gaps in our knowledge remain. Whole genome studies have shed light on key mutations and genes that have been selected against the background of antibiotics, but in most cases such events are inferred and not supported by experimental data. Notable examples include the unknown fitness contribution made by specific plasmids, genomic islands and compensatory mutations. Furthermore, questions remain like why this organism in particular achieved such considerable success in such a short time span, compared to other more pathogenic and resistant clones. Herein, we document what is known regarding the genetics of this organism since its first description in 2008, but also highlight where work remains to be done for a truly comprehensive understanding of the biology of ST131, in order to account for its dramatic rise to prominence.


Assuntos
Escherichia coli/genética , Animais , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Genoma Bacteriano/genética , Humanos , Mutação/genética , Plasmídeos/genética
15.
Chem Commun (Camb) ; 56(87): 13359-13362, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33030162

RESUMO

A range of N-heterocyclic carbene-supported copper diphenylphosphides (NHC = IPr, 6-Dipp, SIMes and 6-Mes) were synthesised. These include the first reports of ring-expanded NHC-copper(i) phosphides. The compounds were characterised by NMR spectroscopy and X-ray crystallography. Reaction of (6-Dipp)CuPPh2 with isocyanates, isothiocyanates and carbon disulfide results in the insertion of the heterocumulene into the Cu-P bond. The NHC-copper phosphides were found to be the most selective catalysts yet reported for the hydrophosphination of isocyanates. They provide access to a broad range of phosphinocarboxamides in excellent conversion and good yield.

16.
PLoS One ; 14(12): e0219879, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31851668

RESUMO

The crisis of antimicrobial resistance is driving research into the phenomenon of collateral sensitivity. Sometimes, when a bacterium evolves resistance to one antimicrobial, it becomes sensitive to others. In this study, we have investigated the utility of Phenotype Microarray (PM) plates for identifying collateral sensitivities with unprecedented throughput. We assessed the relative resistance/sensitivity phenotypes of nine strains of Staphylococcus aureus (two laboratory strains and seven clinical isolates) towards the 72 antimicrobials contained in three PM plates. In general, the PM plates reported on resistance and sensitivity with a high degree of reproducibility. However, a rigorous comparison of PM growth phenotypes with minimum inhibitory concentration (MIC) measurements revealed a trade-off between throughput and accuracy. Small differences in PM growth phenotype did not necessarily correlate with changes in MIC. Thus, we conclude that PM plates are useful for the rapid and high-throughput assessment of large changes in collateral sensitivity phenotypes during the evolution of antimicrobial resistance, but more subtle examples of cross-resistance or collateral sensitivity cannot be identified reliably using this approach.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Antibacterianos/farmacologia , Sensibilidade Colateral a Medicamentos/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação
17.
Emerg Med Australas ; 31(6): 1115-1118, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31456338

RESUMO

Focused echocardiography may be a useful tool in cardiopulmonary resuscitation for prognostication, to identify certain reversible causes of cardiac arrest, and to guide further management and procedures. Nonetheless, many clinicians have reservations regarding its widespread adoption due to evidence that it leads to prolonged interruption of cardiac compressions. Furthermore, the lack of a clear protocol for the inclusion of focused echocardiography into the rhythm check can lead to confusion in teams not familiar with incorporating the modality, as well as safety concerns for the echosonographer during delivery of a shock. We propose the protocol COACHRED to guide the use of focused echocardiography during rhythm check in a safe and timely manner. This approach incorporates the best strategies identified to date that minimise interruptions to chest compressions. We demonstrate that, in a simulation environment, it is achievable to incorporate focused echocardiography into the rhythm check while keeping the interruption to chest compressions within the timeframe prescribed by international guidelines.


Assuntos
Reanimação Cardiopulmonar , Protocolos Clínicos , Ecocardiografia , Parada Cardíaca/diagnóstico por imagem , Parada Cardíaca/terapia , Humanos , Prognóstico
18.
ACS Chem Neurosci ; 10(8): 3880-3887, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31319029

RESUMO

Amyloid ß (Aß) 42 is an aggregation-prone peptide and the believed seminal etiological agent of Alzheimer's disease (AD). Intermediates of Aß42 aggregation, commonly referred to as diffusible oligomers, are considered to be among the most toxic forms of the peptide. Here, we studied the effect of the age-related epimerization of Ser26 (i.e., S26s chiral edit) in Aß42 and discovered that this subtle molecular change led to reduced fibril formation propensity. Surprisingly, the resultant soluble aggregates were nontoxic. To gain insight into the structural changes that occurred in the peptide upon S26s substitution, the system was probed using an array of biophysical and biochemical methods. These experiments consistently pointed to the stabilization of aggregation intermediates in the Aß42-S26s system. To better understand the changes arising as a consequence of the S26s substitution, molecular level structural studies were performed. Using a combined nuclear magnetic resonance (NMR)- and density functional theory (DFT)-computational approach, we found that the S26s chiral edit induced only local structural changes in the Gly25-Ser26-Asn27 region. Interestingly, these subtle changes enabled the formation of an intramolecular Ser26-Asn27 H-bond, which disrupted the ability of Asn27 to engage in the fibrillogenic side chain-to-side chain H-bonding pattern. This reveals that intermolecular stabilizing interactions between Asn27 side chains are a key element controlling Aß42 aggregation and toxicity.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Ratos
19.
PeerJ ; 5: e3244, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28480139

RESUMO

BACKGROUND: Many bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe, Escherichia coli, adapts to anaerobic environments. METHODS: Twenty-one populations of E. coli REL4536, an aerobically evolved 10,000th generation descendent of the E. coli B strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations. Mutations were identified relative to the ancestral genome, and based on the extent of parallelism, traits that were likely to have contributed towards adaptation were inferred. RESULTS: There were increases in fitness relative to the ancestor among anaerobically evolved lineages when tested in the anaerobic environment, but no increases were found in the aerobic environment. For lineages that had evolved under the fluctuating regime, relative fitness increased significantly in the anaerobic environment, but did not increase in the aerobic environment. The aerobically-evolved lineages did not increase in fitness when tested in either the aerobic or anaerobic environments. The strictly anaerobic lineages adapted more rapidly to the anaerobic environment than did the fluctuating lineages. Two main strategies appeared to predominate during adaptation to the anaerobic environment: modification of energy generation pathways, and inactivation of non-essential functions. Fermentation pathways appeared to alter through selection for mutations in genes such as nadR, adhE, dcuS/R, and pflB. Mutations were frequently identified in genes for presumably dispensable functions such as toxin-antitoxin systems, prophages, virulence and amino acid transport. Adaptation of the fluctuating lineages to the anaerobic environments involved mutations affecting traits similar to those observed in the anaerobically evolved lineages. DISCUSSION: There appeared to be strong selective pressure for activities that conferred cell yield advantages during anaerobic growth, which include restoring activities that had previously been inactivated under long-term continuous aerobic evolution of the ancestor.

20.
J Chromatogr A ; 1508: 7-15, 2017 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-28619589

RESUMO

Spiral countercurrent-chromatography has great potential for improving the capacity and efficiency of purification of secondary metabolites, and here we describe applications useful for the isolation of flavonoids from the widely used South African medicinal plant, Sutherlandia frutescens (L.) R. Br. In the spiral tubing support rotor, STS-4 for high-speed counter-current chromatography, several polar butanol aqueous solvent systems were selected using a logK plot, and the novel flavonol glycosides (sutherlandins A-D) were well separated by the optimized solvent system (ethyl acetate:n-butanol:acetic acid:water; 5:1:0.3:6 by vol.). The yield of purified flavonoids from 0.9g extract varied from 8.6mg to 54mg of the sutherlandins for a total of 85.3mg. The same extract was fractionated in the new STS-12 rotor of the same outside dimensions but with more radial channels forming 12 loops of the tubing instead of 4. The rotor holds more layers and increased length of tubing. From 0.9g extract the STS-12 rotor yielded more recovery of 110.4mg total with amounts varying from 11.2mg to 64mg of the sutherlandins and apparent increased separation efficiency as noted by less volume of each fraction peak. Thus from 1-g amounts of extract, good recovery of the flavonoids was achieved in the butanol aqueous solvent system.


Assuntos
Distribuição Contracorrente/métodos , Fabaceae/química , Extratos Vegetais/isolamento & purificação , Distribuição Contracorrente/instrumentação , Flavonoides/análise , Flavonoides/isolamento & purificação , Extratos Vegetais/análise , Plantas Medicinais/química
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