On the fast track to organizer gene expression.
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ABSTRACT:
SUBMITTER: Kimelman D
PROVIDER: S-EPMC5568668 | biostudies-other | 2010 Aug
REPOSITORIES: biostudies-other
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Project description:Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequences enables not only the recombinant expression of the original antibody for further applications, but opens the road for antibody engineering towards innovative diagnostic or therapeutic applications. A time- and cost-efficient production system enabling the detailed analysis of the antibodies is an essential requirement in this context.Sequences were rescued from three hybridoma cell lines, subjected to sequence analysis, subcloned into binary expression vectors and recombinantly expressed as chimeric mAb (constant regions of human IgG1:k1) in Nicotiana benthamiana plants. The properties of the recombinant and the murine mAbs were compared using competition enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. The recognition of native PfMSP4 by the recombinant mAb was analysed by immunofluorescence staining of Pf 3D7A schizonts and by western blot analysis of merozoite extract.The rescued sequences of all three hybridoma cell lines were identical. The recombinant mAb was successfully expressed as IgG in plants at moderate levels (45 mg/kg fresh leaf weight). Preservation of the original epitope was demonstrated in a competition ELISA, using recombinant mAb and the three murine mAbs. EGF_PfMSP4-specific affinities were determined by SPR spectroscopy to 8 nM and 10 nM for the murine or recombinant mAb, respectively. Binding to parasite PfMSP4 was confirmed in an immunofluorescence assay showing a characteristic staining pattern and by western blot analysis using merozoite extract.As demonstrated by the example of an EGF_PfMSP4-specific antibody, the described combination of a simple and efficient hybridoma antibody cloning approach with the flexible, robust and cost-efficient transient expression system suitable to rapidly produce mg-amounts of functional recombinant antibodies provides an attractive method for the generation of mAbs and their derivatives as research tool, novel therapeutics or diagnostics.
| S-EPMC4323031 | biostudies-literature
Project description:The 3 day workshop "Alzheimer's Fast Track" is a unique opportunity for graduate students, postdoctoral fellows, or other early-career scientists, focused on Alzheimer's disease research, to gain new knowledge and become an expert in where this emerging scientific field is moving. In addition, it is not only about receiving a good overview, but also learning to write and defend a successful application for securing funding for Alzheimer's disease research projects.
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Project description:Considerable technological advancements have recently been made with endovascular stent grafts for the treatment of abdominal aortic aneurysm (AAA). However, there is opportunity to further improve the efficiency of endovascular aneurysm repair (EVAR), which may yield better patient outcomes and lower perioperative treatment costs.The Least Invasive Fast-Track EVAR (LIFE) registry was developed to determine the clinical utility and cost effectiveness of the Ovation® Prime stent graft when used under least invasive conditions using a defined fast-track protocol. The LIFE study is a prospective multicenter post-market registry of the ultra-low profile (14F) Ovation Prime stent graft when used in the treatment of patients with AAA using a fast-track protocol, consisting of appropriate patient selection, bilateral percutaneous access, avoidance of general anesthesia and intensive care unit admission, and next-day discharge. The primary endpoint of the study is the proportion of subjects that experience a major adverse event within 30 days of the initial procedure. Primary endpoint data will be compared to a target performance goal. A total of 250 subjects will be enrolled at up to 40 sites in the United States. The first subject in this study was enrolled in October 2014 and enrollment is anticipated to continue through mid-2016.The recent development of ultra low-profile stent grafts enables EVAR using least invasive methods. A structured fast-track EVAR protocol may yield clinical and cost benefits versus standard EVAR.ClinicalTrials.gov Identifier: NCT02224794.
| S-EPMC4684921 | biostudies-literature
Project description:The present study examines whether the Fast Track (FT) intervention, a 10-year randomized controlled trial with children at risk for conduct problems, affects family formation in adulthood, as indexed by partnerships, parenthood, and family structure, and whether the intervention effect differs across participants' gender and race/ethnicity. Participants included 891 children (intervention n = 445; control n = 446; 69% male; 51% Black, 47% White) who were recruited in kindergarten and followed to age 32 or 34 (80% participation of still-living participants), when they reported on their romantic partnerships, parenthood, and family structure. Controlling for numerous covariates that are related to family formation, intervention participants were more likely than those in the control group to be married rather than single and to have a larger number of children; the intervention and control groups did not differ on cohabitation status, age at first marriage, whether they had ever been divorced, their likelihood of being a parent, the age at which they first became a parent, the spacing of births, family structure (partnered or not, with or without children), or in whether they were residentially independent of their parents and grandparents. Intervention effects were not moderated by gender, but race/ethnicity moderated the effect of the intervention on the probability of having any children and the number of children. These findings suggest that several elements of family formation may remain unchanged by an intervention that changes many other behavioral and psychological trajectories of participants. (PsycInfo Database Record (c) 2023 APA, all rights reserved).
| S-EPMC9870928 | biostudies-literature
Project description:Promoters are key components for the application of biotechnological techniques in crop plants. Reporter genes such as GUS or GFP have been used to test the activity of promoters for diverse applications. A huge number of T-DNAs carrying promoterless GUS near their right borders have been inserted into the rice genome, and 105,739 flanking sequence tags from rice lines with this T-DNA insertion have been identified, establishing potential promoter trap lines for 20,899 out of 55,986 genes in the rice genome. Anatomical meta-expression data and information on abiotic stress related to these promoter trap lines enable us to quickly identify new promoters associated with various expression patterns. In the present report, we introduce a strategy to identify new promoters in a very short period of time using a combination of meta-expression analysis and promoter trap lines.
| S-EPMC7020180 | biostudies-literature
Project description:Postoperative joint stiffness following total knee arthroplasty (TKA) may compromise the outcome and necessitate manipulation. Previous studies have not been in a fast-track setting with optimized pain treatment, early mobilization, and short length of stay (LOS), which may have influenced the prevalence of joint stiffness and subsequent manipulation. We investigated the prevalence of manipulation following fast-track TKA and identified patients at risk of needing manipulation.3,145 consecutive unselected elective primary unilateral TKA patients operated in 6 departments with well-defined fast-track settings were included in the study. Demographic data, prevalence, type and timing of manipulation, and preoperative and postoperative ROM were recorded prospectively, ensuring complete 1-year follow-up.70 manipulations were performed within 1 year (2.2%) at a mean of 4 months after index surgery. Younger age and not using walking aids preoperatively were associated with a higher risk of manipulation. LOS ? 4 days (as opposed to a longer LOS) was not associated with an increased risk of manipulation.The prevalence of manipulation was lower or comparable to that in most published studies following more conventional pathways. Inherent patient demographics were identified as risk factors for manipulation whereas LOS ? 4 days was not. Thus, fast-track TKA does not result in increased risk of manipulation-despite a shorter LOS. Optimized pain treatment and early mobilization may contribute to these favorable results that support the use of fast-track.
| S-EPMC4366662 | biostudies-literature
Project description:BackgroundRepositioning of existing drugs has been suggested as a fast track for developing new anti-malarial agents. The compound libraries of GlaxoSmithKline (GSK), Pfizer and AstraZeneca (AZ) comprising drugs that have undergone clinical studies in other therapeutic areas, but not achieved approval, and a set of US Food and Drug Administration (FDA)-approved drugs and other bio-actives were tested against Plasmodium falciparum blood stages.MethodsMolecules were tested initially against erythrocytic co-cultures of P. falciparum to measure proliferation inhibition using one of the following methods: SYBR®I dye DNA staining assay (3D7, K1 or NF54 strains); [(3)H] hypoxanthine radioisotope incorporation assay (3D7 and 3D7A strain); or 4',6-diamidino-2-phenylindole (DAPI) DNA imaging assay (3D7 and Dd2 strains). After review of the available clinical pharmacokinetic and safety data, selected compounds with low ?M activity and a suitable clinical profile were tested in vivo either in a Plasmodium berghei four-day test or in the P. falciparum Pf3D7(0087/N9) huSCID 'humanized' mouse model.ResultsOf the compounds included in the GSK and Pfizer sets, 3.8% (9/238) had relevant in vitro anti-malarial activity while 6/100 compounds from the AZ candidate drug library were active. In comparison, around 0.6% (24/3,800) of the FDA-approved drugs and other bio-actives were active. After evaluation of available clinical data, four investigational drugs, active in vitro were tested in the P. falciparum humanized mouse model: UK-112,214 (PAF-H1 inhibitor), CEP-701 (protein kinase inhibitor), CEP-1347 (protein kinase inhibitor), and PSC-833 (p-glycoprotein inhibitor). Only UK-112,214 showed significant efficacy against P. falciparum in vivo, although at high doses (ED90 131.3 mg/kg [95% CI 112.3, 156.7]), and parasitaemia was still present 96 hours after treatment commencement. Of the six actives from the AZ library, two compounds (AZ-1 and AZ-3) were marginally efficacious in vivo in a P. berghei model.ConclusionsRepositioning of existing therapeutics in malaria is an attractive proposal. Compounds active in vitro at ?M concentrations were identified. However, therapeutic concentrations may not be effectively achieved in mice or humans because of poor bio-availability and/or safety concerns. Stringent safety requirements for anti-malarial drugs, given their widespread use in children, make this a challenging area in which to reposition therapy.
| S-EPMC4021201 | biostudies-literature
Project description:Background In pediatric cardiac surgery, perioperative management has evolved from slow weaning of mechanical ventilation in the intensive care unit to "ultra-fast-track" anesthesia with early extubation (EE) in theater to promote a faster recovery. The strategy of EE has not been assessed in adults with congenital heart disease, a growing population of patients who often require surgery. Methods And Results Data were collected retrospectively on all patients >16 years of age who underwent adult congenital heart surgery in our tertiary center between December 2012 and January 2020. Coarsened exact matching was performed for relevant baseline variables. Overall, 711 procedures were performed: 133 (18.7%) patients underwent EE and 578 (81.3%) patients received conventional extubation. After matching, patients who received EE required less inotropic or vasopressor support in the early postoperative period (median Vasoactive-inotropic score 0.5 [0.0-2.0] versus 2.0 [0.0-3.5]; P<0.0001) and had a lower total net fluid balance than patients after conventional extubation (1168±723 versus 847±733 mL; P=0.0002). The overall reintubation rate was low at 0.3%. EE was associated with a significantly shorter postoperative length of stay in higher dependency care units before a "step-down" to ward-based care (48 [45-50] versus 50 [47-69] hours; P=0.004). Lower combined intensive care unit and high dependency unit costs were incurred by patients who received EE compared with patients who received conventional extubation (£3949 [3430-4222] versus £4166 [3893-5603]; P<0.0001). Conclusions In adult patients undergoing surgery for congenital heart disease, EE is associated with a reduced need for postoperative hemodynamic support, a shorter intensive care unit stay, and lower health-care-related costs.
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Project description:To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.
| S-EPMC6852550 | biostudies-literature
Project description:Prader-Willi syndrome (PWS) is a rare genetic neurodevelopmental disorder characterized by an insatiable appetite, leading to chronic overeating and obesity. Additional features include short stature, intellectual disability, behavioral problems and incomplete sexual development. Although significant progress has been made in understanding the genetic basis of PWS, the mechanisms underlying the pathogenesis of the disorder remain poorly understood. Treatment for PWS consists mainly of palliative therapies; curative therapies are sorely needed. Zebrafish, Danio rerio, represent a promising way forward for elucidating physiological problems such as obesity and identifying new pharmacotherapeutic options for PWS. Over the last decade, an increased appreciation for the highly conserved biology among vertebrates and the ability to perform high-throughput drug screening has seen an explosion in the use of zebrafish for disease modeling and drug discovery. Here, we review recent advances in developing zebrafish models of human disease. Aspects of zebrafish genetics and physiology that are relevant to PWS will be discussed, and the advantages and disadvantages of zebrafish models will be contrasted with current animal models for this syndrome. Finally, we will present a paradigm for drug screening in zebrafish that is potentially the fastest route for identifying and delivering curative pharmacotherapies to PWS patients.
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