RESUMEN
The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols.
Asunto(s)
Anticuerpos Monoclonales/química , Proteínas Recombinantes de Fusión/química , Anticuerpos Monoclonales/genética , Células Cultivadas , Cromatografía de Afinidad , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas Recombinantes de Fusión/genética , Robótica , Proteína Estafilocócica A/química , TransfecciónRESUMEN
It is estimated that around 75% of patients with Borderline Personality Disorder (BPD) are prescribed psychotropic medication during their treatment course, although this is not recommended as first line therapy. In the UK, there are no guidelines to advise which drug treatments to use in BPD, however, numerous, but mostly small scale studies, show evidence that different medications target specific core symptoms. We report a case of a 25 year old woman with BPD, who has received treatment with five different psychotropic medications. We go on to assess not only the efficacy of these treatments in this individual case, but also whether the use of these treatments is in line with best evidence according to currently available research.
Asunto(s)
Trastorno de Personalidad Limítrofe/tratamiento farmacológico , Psicotrópicos/uso terapéutico , Adulto , Trastorno de Personalidad Limítrofe/diagnóstico , Trastorno de Personalidad Limítrofe/psicología , Terapia Combinada , Sustitución de Medicamentos , Quimioterapia Combinada , Femenino , Humanos , Psicotrópicos/efectos adversos , Resultado del TratamientoRESUMEN
There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern. IMPORTANCE: The explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.
Asunto(s)
Vacuna nCoV-2019 mRNA-1273 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacuna BNT162 , Vacunas contra la COVID-19 , COVID-19 , Pruebas de Neutralización , SARS-CoV-2 , Humanos , SARS-CoV-2/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Vacuna nCoV-2019 mRNA-1273/inmunología , Vacuna BNT162/inmunología , Vacuna BNT162/administración & dosificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Estándares de Referencia , Inmunización Secundaria , Vacunación , Ad26COVS1/inmunologíaRESUMEN
Genomic sequencing of clinical samples to identify emerging variants of SARS-CoV-2 has been a key public health tool for curbing the spread of the virus. As a result, an unprecedented number of SARS-CoV-2 genomes were sequenced during the COVID-19 pandemic, which allowed for rapid identification of genetic variants, enabling the timely design and testing of therapies and deployment of new vaccine formulations to combat the new variants. However, despite the technological advances of deep sequencing, the analysis of the raw sequence data generated globally is neither standardized nor consistent, leading to vastly disparate sequences that may impact identification of variants. Here, we show that for both Illumina and Oxford Nanopore sequencing platforms, downstream bioinformatic protocols used by industry, government, and academic groups resulted in different virus sequences from same sample. These bioinformatic workflows produced consensus genomes with differences in single nucleotide polymorphisms, inclusion and exclusion of insertions, and/or deletions, despite using the same raw sequence as input datasets. Here, we compared and characterized such discrepancies and propose a specific suite of parameters and protocols that should be adopted across the field. Consistent results from bioinformatic workflows are fundamental to SARS-CoV-2 and future pathogen surveillance efforts, including pandemic preparation, to allow for a data-driven and timely public health response.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/epidemiología , Pandemias , Flujo de Trabajo , Biología ComputacionalRESUMEN
Development and validation of rapid and easy-to-perform diagnostics continue to be a high priority during the current COVID-19 pandemic. Although vaccines are now widely available, early detection and consistent transmission control provide ideal means to mitigate the spread of SARS-CoV-2. Nucleic acid-based real-time PCR tests are widely acknowledged as the gold standard for reliable diagnosis of COVID-19 infection. These tests are based on detecting viable or nonviable viral nucleic acids. SARS-CoV-2 spike protein is an alternative and ideal target for SARS-CoV-2 diagnosis in the early phase of infection, but point-of-care kits to detect the SARS-CoV-2 spike protein are limited. Here we describe a rapid and convenient method based on Lateral Flow Immunoassay (LFIA) to detect SARS-CoV-2 spike proteins, including SARS-CoV-2 variants (A.23.1, B.1.1.1, 1.617.2, B.1.1.7, B.1.351, P.1, N501Y, R.1, P681H, P3, UK, and South African) within 5 to 10 minutes. We generated highly specific monoclonal antibodies (mAbs) against rationally designed SARS-CoV-2 spike protein. Matched pair mAbs were selected by epitope mapping and employed as antigen capture reagents by spotting onto a nitrocellulose membrane and as detector reagents by conjugation with colloidal gold nanoparticles. We evaluated the performance of the LFIA using recombinant spike proteins of SARS-CoV-2 and several SARS-CoV-2 variants. The specificity of the LFIA was assessed using heat-inactivated SARS-CoV-2 and related human coronaviruses (HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63) and an FDA-approved respiratory pathogens (RP) panel. The assay exhibited 98% specificity and acceptable performance with respect to the minimum limit of detection (25 ng/test) in validation tests. This new LFIA provides improved performance for the early diagnosis of SARS-CoV-2, particularly for home monitoring and in situations with limited access to molecular methods.
Asunto(s)
COVID-19 , Nanopartículas del Metal , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/análisis , Prueba de COVID-19 , Sistemas de Atención de Punto , Pandemias , Oro , Sensibilidad y Especificidad , Inmunoensayo/métodosRESUMEN
Multiple parasite lineages with different proliferation rates or fitness may coexist within a clinical malaria isolate, resulting in complex growth interactions and variations in phenotype. To elucidate the dynamics of parasite growth in multiclonal isolates, we measured growth rates (GRs) of three Plasmodium falciparum Cambodian isolates, including IPC_3445 (MRA-1236), IPC_5202 (MRA-1240), IPC_6403 (MRA-1285), and parasite lineages previously cloned from each of these isolates by limiting dilution. Following synchronization, in vitro cultures of each parasite line were maintained over four consecutive asexual cycles (192 h), with thin smears prepared at each 48-h cycle to estimate GR and fold change in parasitemia (FCP). Cell cycle time (CCT), the duration it takes for ring-stage parasites to develop into mature schizonts, was measured by monitoring the development of 0-3-h post-invasion rings for up to 52 h post-incubation. Laboratory lines 3D7 (MRA-102) and Dd2 (MRA-150) were used as controls. Significant differences in GR, FCP, and CCT were observed between parasite isolates and clonal lineages from each isolate. The parasite lines studied here have well-defined growth phenotypes and will facilitate basic malaria research and development of novel malaria interventions. These lines are available to malaria researchers through the MR4 collection of NIAID's BEI Resources Program.
Asunto(s)
Malaria Falciparum , Malaria , Parásitos , Animales , Plasmodium falciparum/genética , Malaria Falciparum/parasitología , FenotipoRESUMEN
During the COVID-19 pandemic, SARS-CoV-2 surveillance efforts integrated genome sequencing of clinical samples to identify emergent viral variants and to support rapid experimental examination of genome-informed vaccine and therapeutic designs. Given the broad range of methods applied to generate new viral genomes, it is critical that consensus and variant calling tools yield consistent results across disparate pipelines. Here we examine the impact of sequencing technologies (Illumina and Oxford Nanopore) and 7 different downstream bioinformatic protocols on SARS-CoV-2 variant calling as part of the NIH Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Tracking Resistance and Coronavirus Evolution (TRACE) initiative, a public-private partnership established to address the COVID-19 outbreak. Our results indicate that bioinformatic workflows can yield consensus genomes with different single nucleotide polymorphisms, insertions, and/or deletions even when using the same raw sequence input datasets. We introduce the use of a specific suite of parameters and protocols that greatly improves the agreement among pipelines developed by diverse organizations. Such consistency among bioinformatic pipelines is fundamental to SARS-CoV-2 and future pathogen surveillance efforts. The application of analysis standards is necessary to more accurately document phylogenomic trends and support data-driven public health responses.
RESUMEN
Coagulase-negative staphylococci (CoNS) are the most common cause of biofilm-associated sepsis in very low birth weight infants (VLBW). Standard biofilm assays may not predict the pathogenic potential of CoNS since biofilm production is regulated by diverse environmental stimuli. Staphylococcus epidermidis isolated from blood cultures from VLBW infants were evaluated for biofilm production in response to various environmental stimuli, including intravenous solutions and skin preparations. While responses to environmental stimuli were variable for individual isolates and products, some trends were observed. Biofilm production by hospital S. epidermidis isolates (predominantly ica and biofilm-positive) was most commonly increased at 30°C and decreased in the presence of intravenous solutions and moisturisers. Commensals (mainly biofilm-negative and lacking the ica gene) were more often induced to produce biofilm than hospital isolates. These results indicate that biofilm production in S. epidermidis can vary in response to environmental stimuli encountered in the clinical setting, that standard biofilm assays are unlikely to predict clinical outcome, and that harmless skin commensals may be induced to produce biofilm by some of the products used in neonatal units.
Asunto(s)
Bacteriemia/microbiología , Biopelículas/crecimiento & desarrollo , Recién Nacido de Bajo Peso , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus epidermidis/aislamiento & purificación , Sangre/microbiología , Humanos , Recién Nacido , TemperaturaRESUMEN
SARS-CoV-2 pathogenesis, vaccine, and therapeutic studies rely on the use of animals challenged with highly pathogenic virus stocks produced in cell cultures. Ideally, these virus stocks should be genetically and functionally similar to the original clinical isolate, retaining wild-type properties to be reliably used in animal model studies. It is well-established that SARS-CoV-2 isolates serially passaged on Vero cell lines accumulate mutations and deletions in the furin cleavage site; however, these can be eliminated when passaged on Calu-3 lung epithelial cell lines, as presented in this study. As numerous stocks of SARS-CoV-2 variants of concern are being grown in cell cultures with the intent for use in animal models, it is essential that propagation methods generate virus stocks that are pathogenic in vivo. Here, we found that the propagation of a B.1.351 SARS-CoV-2 stock on Calu-3 cells eliminated viruses that previously accumulated mutations in the furin cleavage site. Notably, there were alternative variants that accumulated at the same nucleotide positions in virus populations grown on Calu-3 cells at multiple independent facilities. When a Calu-3-derived B.1.351 virus stock was used to infect hamsters, the virus remained pathogenic and the Calu-3-specific variants persisted in the population. These results suggest that Calu-3-derived virus stocks are pathogenic but care should still be taken to evaluate virus stocks for newly arising mutations during propagation.
Asunto(s)
SARS-CoV-2/crecimiento & desarrollo , Pase Seriado/métodos , Glicoproteína de la Espiga del Coronavirus/genética , Animales , COVID-19/virología , Línea Celular Tumoral , Chlorocebus aethiops , Cricetinae , Furina/metabolismo , Humanos , Mutación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Células VeroRESUMEN
An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.
RESUMEN
Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.
Asunto(s)
Técnicas Bacteriológicas , Infecciones por Mycobacterium/microbiología , Mycobacterium/crecimiento & desarrollo , Animales , Armadillos , Técnicas Bacteriológicas/instrumentación , Reactores Biológicos , Modelos Animales de Enfermedad , Humanos , Ratones Desnudos , Viabilidad Microbiana , Mycobacterium/aislamiento & purificación , Mycobacterium leprae/crecimiento & desarrollo , Mycobacterium leprae/aislamiento & purificación , Factores de TiempoRESUMEN
The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong. The genome sequence shows a 27 nt in-frame deletion (Δ27,264-27,290) within ORF6, predicted to result in a 9 aa deletion ( ΔFKVSIWNLD ) from the central portion of p6. This deletion is predicted to result in a dramatic alteration in the three-dimensional structure of the resultant protein (p6 Δ22-30 ), possibly with significant functional implications. Analysis of the original clinical sample indicates that the deletion was not present, while sequencing of subsequent passages of the strain identifies the deletion as a majority variant. This suggests that the deletion originated ab initio during passaging and subsequently propagated into the majority, possibly due to the removal of selective pressure through the IFN-deficient Vero E6 cell line. The specific function of the SARS-CoV-2 p6 N-terminus, if any, has not yet been determined. However, this deletion is predicted to cause a shift from N-endo to N-ecto in the transmembrane localization of the SARS-CoV-2 p6 Δ22-30 N-terminus, possibly leading to the ablation of its native function.
RESUMEN
There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.
Asunto(s)
Bancos de Muestras Biológicas , Investigación Biomédica/métodos , Biología Computacional/métodos , Bases de Datos Genéticas , Infecciones por Mycobacterium/microbiología , Mycobacterium/genética , Mycobacterium/patogenicidad , Humanos , Reproducibilidad de los ResultadosRESUMEN
Pseudomonas aeruginosa and Candida albicans (are opportunistic pathogens that cause systemic infections in immune-suppressed patients. They show important bacterial-fungal interactions including quorum sensing. This involves cell signaling to communicate between the cells of their own colony and the cells of rival microbes or the host. It is thought that this phenomenon is vital in the potential competition and virulence of the organisms. We report a case of a previously healthy 2-year-old boy, where an accidental injury had been sustained resulting in a closed fracture of femur. He subsequently developed sepsis related to co-infection by C. albicans and P. aeruginosa. Trauma may result in a transient immune-suppression and predispose to sepsis caused by opportunistic microorganisms. They can engage in bacterial-fungal interaction. Clinicians should consider invasive co-infection when initial cultures show evidence for only 1 pathogen.
Asunto(s)
Candidemia , Coinfección/microbiología , Fracturas del Fémur/complicaciones , Infecciones por Pseudomonas , Sepsis/microbiología , Candida albicans , Preescolar , Humanos , Masculino , Interacciones Microbianas , Infecciones Oportunistas , Pseudomonas aeruginosa , Percepción de QuorumRESUMEN
PURPOSE: The chick embryo is a powerful model system for the study of retinal development. However, analysis of gene expression in the chick retina has lagged behind biological studies. The purpose of this study was to identity and characterize genes expressed in the chick embryo retina as candidate molecules involved in the development and function of photoreceptors and other retinal cell types. METHODS: RNA from embryonic day (ED) 18 White Leghorn chick embryo retinae was used to generate an oligo dT-primed cDNA library. Bacterial colonies representing five thousand individual clones were arrayed onto nylon membranes using a microarray robot. Replicate membranes were hybridized with cDNA probes synthesized from ED 18 retina, brain and liver. Clones that appeared preferentially expressed in retina were identified by homology searches, and their spatial and temporal expression patterns were analyzed by in situ hybridization. RESULTS: Two hundred and seventy-two clones were identified. Approximately forty percent of the clones represented potential novel genes, including ESTs, hypothetical proteins and clones with no assigned identities. Furthermore, many genes were identified that are the putative chick orthologues of genes cloned from other species. We determined the expression pattern of several clones for which sequence homologies suggested possible roles in transcriptional regulation, apoptosis or intercellular signaling. Their corresponding mRNAs were expressed in the embryonic retina in topographically specific, developmentally regulated patterns. CONCLUSIONS: We identified and characterized genes in the chick embryo retina using a combination of microarray analysis and in situ hybridization. Analysis of the expression patterns suggests involvement of several of these genes in key events during embryogenesis.
Asunto(s)
Embrión de Pollo , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Retina/metabolismo , Animales , ADN Complementario/análisis , Bases de Datos Factuales , Proteínas del Ojo/metabolismo , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Retina/embriología , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
Blocking the action of inhibitory molecules at sites of central nervous system injury has been proposed as a strategy to promote axonal regeneration and functional recovery. We have previously shown that genetic deletion or competitive antagonism of EphA4 receptor activity promotes axonal regeneration and functional recovery in a mouse model of lateral hemisection spinal cord injury. Here we have assessed the effect of blocking EphA4 activation using the competitive antagonist EphA4-Fc in a rat model of thoracic contusive spinal cord injury. Using a ledged tapered balance beam and open-field testing, we observed significant improvements in recovery of locomotor function after EphA4-Fc treatment. Consistent with functional improvement, using high-resolution ex vivo magnetic resonance imaging at 16.4T, we found that rats treated with EphA4-Fc had a significantly increased cross-sectional area of the dorsal funiculus caudal to the injury epicenter compared with controls. Our findings indicate that EphA4-Fc promotes functional recovery following contusive spinal cord injury and provides further support for the therapeutic benefit of treatment with the competitive antagonist in acute cases of spinal cord injury.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/farmacología , Receptor EphA4/antagonistas & inhibidores , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Western Blotting , Encéfalo/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Imagen por Resonancia Magnética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/farmacología , Traumatismos de la Médula Espinal/patología , TransfecciónRESUMEN
Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR, and real-time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5-8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14-16) for others. Treatment of retinal cell cultures with activin, staurosporine, or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific "marker" as a proxy to identify photoreceptor cell fate is problematic.