RESUMO
Use of whole-genome sequencing (WGS) for routine mycobacterial species identification and drug susceptibility testing (DST) is becoming a reality. We compared the performances of WGS and standard laboratory workflows prospectively, by parallel processing at a major mycobacterial reference service over the course of 1 year, for species identification, first-line Mycobacterium tuberculosis resistance prediction, and turnaround time. Among 2,039 isolates with line probe assay results for species identification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these isolates, clinically important species were identified for 1,902 isolates, of which 1,825 (96.0%) were identified as the same species by WGS and the line probe assay. A total of 2,157 line probe test results for detection of resistance to the first-line drugs isoniazid and rifampin were available for 728 M. tuberculosis complex isolates. Excluding 216 (10.0%) cases where there were insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% confidence interval [CI], 98.9 to 99.6%), sensitivity was 97.6% (91.7 to 99.7%), and specificity was 99.5% (99.0 to 99.7%). A total of 2,982 phenotypic DST results were available for 777 M. tuberculosis complex isolates. Of these, 356 (11.9%) had no WGS comparator due to insufficient sequencing data, and in 154 (5.2%) cases the WGS prediction was indeterminate due to discovery of novel, previously uncharacterized mutations. Excluding these data, overall concordance was 99.2% (98.7 to 99.5%), sensitivity was 94.2% (88.4 to 97.6%), and specificity was 99.4% (99.0 to 99.7%). Median processing times for the routine laboratory tests versus WGS were similar overall, i.e., 20 days (interquartile range [IQR], 15 to 31 days) and 21 days (15 to 29 days), respectively (P = 0.41). In conclusion, WGS predicts species and drug susceptibility with great accuracy, but work is needed to increase the proportion of predictions made.
Assuntos
Farmacorresistência Bacteriana/genética , Genoma Bacteriano/genética , Tipagem Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/microbiologia , Antituberculosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Estudos Prospectivos , Rifampina/farmacologia , Sensibilidade e Especificidade , Fatores de Tempo , Tuberculose/diagnósticoRESUMO
Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/química , Proteínas de Membrana/biossíntese , Pichia/genética , Plasmídeos/química , Saccharomyces cerevisiae/genética , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Bases de Dados Factuais , Expressão Gênica , Vetores Genéticos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Pichia/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/metabolismo , SolubilidadeRESUMO
BACKGROUND: In July 2013, a rotavirus vaccination program for 2- to 3-month-olds was introduced in the United Kingdom. We present an initial impact analysis of this new vaccine program using national syndromic surveillance systems. METHODS: General practitioner (GP) in-hours, GP out-of-hours, and emergency department (ED) syndromic surveillance systems were used to monitor GP consultations and ED visits for gastroenteritis, diarrhea, and vomiting. Data were stratified by age group and compared between pre- and postvaccine-year rotavirus seasons. Incidence rate ratios (IRRs) and percentage ratios were calculated for GP in-hours consultations and GP out-of-hours and ED data, respectively. RESULTS: There was a significant reduction in gastroenteritis, diarrhea, and vomiting GP in-hours consultations in children aged 0-4 years when comparing the rotavirus season in the pre- and postvaccine years (P < .001 for all indicators). IRRs illustrated a 26%-33% and 23%-31% decrease in gastroenteritis incidence in the <1 and 1-4 years age groups, respectively, across the syndromic surveillance systems. There was also an 8% decrease recorded in the 5-14 years age group in the GP in-hours and ED systems. CONCLUSIONS: Syndromic surveillance revealed a marked decline in gastroenteritis, coinciding with the introduction of the new rotavirus vaccine program in England. The largest reduction in disease was observed in infants, although some impact was also demonstrated in children aged 1-4 and 5-14 years, suggesting possible herd protection in older age groups. This study was limited to the first postvaccine year, and further analysis is required to assess the longer-term impact of the vaccine.
Assuntos
Gastroenterite/epidemiologia , Gastroenterite/prevenção & controle , Programas de Imunização , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/administração & dosagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Diarreia/epidemiologia , Diarreia/prevenção & controle , Inglaterra/epidemiologia , Monitoramento Epidemiológico , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-ß-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
Assuntos
Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Linhagem Celular , Células HEK293 , Humanos , Maltose/análogos & derivados , Maltose/química , Pichia/química , Pichia/genética , Pichia/metabolismo , Estabilidade Proteica , Receptor A2A de Adenosina/biossíntese , Receptor A2A de Adenosina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
During March and early April 2014 there was widespread poor air quality across the United Kingdom. Public Health England used existing syndromic surveillance systems to monitor community health during the period. Short lived statistically significant rises in a variety of respiratory conditions, including asthma and wheeze, were detected. This incident has demonstrated the value of real-time syndromic surveillance systems, during an air pollution episode, for helping to explore the impact of poor air quality on community health in real-time.
Assuntos
Poluição do Ar , Vigilância da População , Inglaterra/epidemiologia , HumanosRESUMO
BACKGROUND: Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A(2a) adenosine receptor (hA(2a)R), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). RESULTS: Functional hA(2a)R was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA(2a)R and GFP were still produced in the pre-induction phases. Both hA(2a)R and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake-flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. CONCLUSIONS: The production of recombinant hA(2a)R, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.
Assuntos
Reatores Biológicos/microbiologia , Expressão Gênica , Microbiologia Industrial/métodos , Pichia/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores Purinérgicos P1/metabolismo , Biomassa , Glucose/metabolismo , Glicerol/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microbiologia Industrial/instrumentação , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Receptores Purinérgicos P1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.
Assuntos
Proteínas Recombinantes/metabolismo , Leveduras/metabolismo , Proteínas Recombinantes/genética , Leveduras/genéticaRESUMO
A common method for inducing the production of recombinant proteins in Pichia pastoris is through the use of methanol. However, the by-products of methanol metabolism are toxic to yeast cells and therefore its addition to recombinant cultures must be controlled and monitored throughout the process in order to maximise recombinant protein yields. Described here are online and off-line methods to monitor and control methanol addition to bench-top-scale bioreactors.
Assuntos
Reatores Biológicos/microbiologia , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Fermentação , Metanol/efeitos adversos , Metanol/metabolismo , Pichia/efeitos dos fármacos , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.