Current Practice in Bicistronic IRES Reporter Use: A Systematic Review.
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ABSTRACT:
SUBMITTER: van den Akker GGH
PROVIDER: S-EPMC8156625 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bicistronic reporter assay systems have become a mainstay of molecular biology. While the assays themselves encompass a broad range of diverse and unrelated experimental protocols, the numerical data garnered from these experiments often have similar statistical properties. In general, a primary dataset measures the paired expression of two internally controlled reporter genes. The expression ratio of these two genes is then normalized to an external control reporter. The end result is a 'ratio of ratios' that is inherently sensitive to propagation of the error contributed by each of the respective numerical components. The statistical analysis of this data therefore requires careful handling in order to control for the propagation of error and its potentially misleading effects. A careful survey of the literature found no consistent method for the statistical analysis of data generated from these important and informative assay systems. In this report, we present a detailed statistical framework for the systematic analysis of data obtained from bicistronic reporter assay systems. Specifically, a dual luciferase reporter assay was employed to measure the efficiency of four programmed -1 frameshift signals. These frameshift signals originate from the L-A virus, the SARS-associated Coronavirus and computationally identified frameshift signals from two Saccharomyces cerevisiae genes. Furthermore, these statistical methods were applied to prove that the effects of anisomycin on programmed -1 frameshifting are statistically significant. A set of Microsoft Excel spreadsheets, which can be used as templates for data generated by dual reporter assay systems, and an online tutorial are available at our website (http://dinmanlab.umd.edu/statistics). These spreadsheets could be easily adapted to any bicistronic reporter assay system.
| S-EPMC534638 | biostudies-literature
Project description:BackgroundInternal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies.ResultsWe observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function.ConclusionFrom these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP).
| S-EPMC1373653 | biostudies-literature
Project description:To give a comprehensive review of current literature on robotic rectal cancer surgery.A systematic review of current literature via PubMed and Embase search engines was performed to identify relevant articles from january 2007 to november 2013. The keywords used were: "robotic surgery", "surgical robotics", "laparoscopic computer-assisted surgery", "colectomy" and "rectal resection".After the initial screen of 380 articles, 20 papers were selected for review. A total of 1062 patients (male 64.0%) with a mean age of 61.1 years and body mass index of 24.9 kg/m(2) were included in the review. Out of 1062 robotic-assisted operations, 831 (78.2%) anterior and low anterior resections, 132 (12.4%) intersphincteric resection with coloanal anastomosis, 98 (9.3%) abdominoperineal resections and 1 (0.1%) Hartmann's operation were included in the review. Robotic rectal surgery was associated with longer operative time but with comparable oncological results and anastomotic leak rate when compared with laparoscopic rectal surgery.Robotic colorectal surgery has continued to evolve to its current state with promising results; feasible surgical option with low conversion rate and comparable short-term oncological results. The challenges faced with robotic surgery are for more high quality studies to justify its cost.
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Project description:Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5-8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.
| S-EPMC6842805 | biostudies-literature
Project description:The coronavirus disease (COVID-19) outbreak </mac_aq>started in Wuhan, China, in December 2019, and evolved into a global problem in a short period. The pandemic has led to many social and health-care challenges. In this context, surgery is an area that is facing the need for many adaptations. In this systematic literature review, we analyzed different perspectives concerning this situation, aiming to provide recommendations that could guide surgeons and </mac_aq>entities toward screening, elective and emergency surgeries, decision making, and operating room management. A computerized search in PubMed, Scopus, and Scientific Electronic Library Online (SciELO) for relevant literature up to April 4, 2020, was performed. Articles were included if they were related to surgery dynamics in the context of the COVID-19 pandemic. Of the 281 articles found in our initial search and 15 articles from alternative sources, 39 were included in our review after a systematic evaluation. Concerning preoperative testing </mac_aq>for severe acute respiratory syndrome coronavirus 2 infection, 29 (74.4%) articles recommended some kind of </mac_aq>screening. Another major suggestion was postponing all (or at least selected) elective operations (29 articles, </mac_aq>74.4%). Several additional recommendations with respect to surgical practice or surgical staff were also assessed and discussed, such as performing laparoscopic surgeries and avoiding the use of electrocauterization. On the basis of the current literature, we concluded that any surgery that can be delayed should be postponed. COVID-19 screening is strongly recommended for all surgical cases. Moreover, surgical staff should be reduced to the essential members and provided with institutional psychological support.
| S-EPMC7213672 | biostudies-literature
Project description:BackgroundThere is growing consensus on the importance of identifying age-related inequities in the receipt of public health and healthcare interventions, but concerns regarding conceptual and methodological rigour in this area of research. Establishing age inequity in receipt requires evidence of a difference that is not an artefact of poor measurement of need or receipt; is not warranted on the grounds of patient preference or clinical safety; and is judged to be unfair.MethodA systematic, thematic literature review was undertaken with the objective of characterising recent research approaches. Studies were eligible if the population was in a country within the Organisation for Economic Co-operation and Development and analyses included an explicit focus on age-related patterns of healthcare receipt including those 60 years or older. A structured extraction template was applied. Extracted material was synthesised in thematic memos. A set of categorical codes were then defined and applied to produce summary counts across key dimensions. This process was iterative to allow reconciliation of discrepancies and ensure reliability.ResultsForty nine studies met the eligibility criteria. A wide variety of concepts, terms and methodologies were used across these studies. Thirty five studies employed multivariable techniques to produce adjusted receipt-need ratios, though few clearly articulated their rationale, indicating the need for great conceptual clarity. Eighteen studies made reference to patient preference as a relevant consideration, but just one incorporated any kind of adjustment for this factor. Twenty five studies discussed effectiveness among older adults, with fourteen raising the possibility of differential effectiveness, and one differential cost-effectiveness, by age. Just three studies made explicit reference to the ethical nature of healthcare resource allocation by age. While many authors presented suitably cautious conclusions, some appeared to over-stretch their findings concluding that observed differences were 'inequitable'. Limitations include possible biases in the retrieved material due to inconsistent database indexing and a focus on OECD country populations and studies with English titles.ConclusionsCaution is needed among clinicians and other evidence-users in accepting claims of healthcare 'ageism' in some published papers. Principles for improved research practice are proposed.
| S-EPMC5505033 | biostudies-literature
Project description:Many healthcare professionals use smartphones and tablets to inform patient care. Contemporary research suggests that handheld computers may support aspects of clinical diagnosis and management. This systematic review was designed to synthesise high quality evidence to answer the question; Does healthcare professionals' use of handheld computers improve their access to information and support clinical decision making at the point of care?A detailed search was conducted using Cochrane, MEDLINE, EMBASE, PsycINFO, Science and Social Science Citation Indices since 2001. Interventions promoting healthcare professionals seeking information or making clinical decisions using handheld computers were included. Classroom learning and the use of laptop computers were excluded. Two authors independently selected studies, assessed quality using the Cochrane Risk of Bias tool and extracted data. High levels of data heterogeneity negated statistical synthesis. Instead, evidence for effectiveness was summarised narratively, according to each study's aim for assessing the impact of handheld computer use.We included seven randomised trials investigating medical or nursing staffs' use of Personal Digital Assistants. Effectiveness was demonstrated across three distinct functions that emerged from the data: accessing information for clinical knowledge, adherence to guidelines and diagnostic decision making. When healthcare professionals used handheld computers to access clinical information, their knowledge improved significantly more than peers who used paper resources. When clinical guideline recommendations were presented on handheld computers, clinicians made significantly safer prescribing decisions and adhered more closely to recommendations than peers using paper resources. Finally, healthcare professionals made significantly more appropriate diagnostic decisions using clinical decision making tools on handheld computers compared to colleagues who did not have access to these tools. For these clinical decisions, the numbers need to test/screen were all less than 11.Healthcare professionals' use of handheld computers may improve their information seeking, adherence to guidelines and clinical decision making. Handheld computers can provide real time access to and analysis of clinical information. The integration of clinical decision support systems within handheld computers offers clinicians the highest level of synthesised evidence at the point of care. Future research is needed to replicate these early results and to identify beneficial clinical outcomes.
| S-EPMC4099138 | biostudies-literature
Project description:Crohn's disease is an incurable and frequently progressive entity with major impact on affected patients. Up to half of patients require surgery in the first 10 years after diagnosis and over 75% of operated patients require at least one further surgery within lifetime. In order to minimize surgical risk, modifiable risk factors such as nutritional status need to be optimized. This systematic review on preoperative nutritional support in adult Crohn's patients between 1997 and 2017 aimed to provide an overview on target populations, screening modalities, routes of administration, and expected benefits. Pertinent study characteristics (prospective vs. retrospective, sample size, control group, limitations) were defined a priori. Twenty-nine studies were retained, of which 14 original studies (9 retrospective, 4 prospective, and 1 randomized controlled trial) and 15 reviews. Study heterogeneity was high regarding nutritional regimens and outcome, and meta-analysis could not be performed. Most studies were conducted without matched control group and thus provide modest level of evidence. Consistently, malnutrition was found to be a major risk factor for postoperative complications, and both enteral and parenteral routes were efficient in decreasing postoperative morbidity. Current guidelines for nutrition in general surgery apply also to Crohn's patients. The route of administration should be chosen according to disease presentation and patients' condition. Further studies are needed to strengthen the evidence.
| S-EPMC5490541 | biostudies-literature
Project description:Internal ribosomal entry sites (IRESs) are RNA secondary structures that mediate translation independent from the m7G RNA cap. The dicistronic luciferase assay is the most frequently used method to measure IRES-mediated translation. While this assay is quantitative, it requires numerous controls and can be time-consuming. Circular RNAs generated by splinted ligation have been shown to also accurately report on IRES-mediated translation, however suffer from low yield and other challenges. More recently, cellular sequences were shown to facilitate RNA circle formation through backsplicing. Here, we used a previously published backsplicing circular RNA split GFP reporter to create a highly sensitive and quantitative split nanoluciferase (NanoLuc) reporter. We show that NanoLuc expression requires backsplicing and correct orientation of a bona fide IRES. In response to cell stress, IRES-directed NanoLuc expression remained stable or increased while a capped control reporter decreased in translation. In addition, we detected NanoLuc expression from putative cellular IRESs and the Zika virus 5' untranslated region that is proposed to harbor IRES function. These data together show that our IRES reporter construct can be used to verify, identify and quantify the ability of sequences to mediate IRES-translation within a circular RNA.
| S-EPMC8871761 | biostudies-literature
Project description:BACKGROUND:Precision health calls for collecting and analyzing large amounts of data to capture an individual's unique behavior, lifestyle, genetics, and environmental context. The diffusion of digital tools has led to a significant growth of patient generated health data (PGHD), defined as health-related data created, gathered or inferred by or from patients and for which the patient controls data collection and data sharing. PURPOSE:We assessed the current evidence of the impact of PGHD use in clinical practice and provide recommendations for the formal integration of PGHD in clinical care. METHODS:We searched PubMed, Ovid, Embase, CINAHL, Web of Science, and Scopus up to May 2018. Inclusion criteria were applied and four reviewers screened titles and abstracts and consequently full articles. FINDINGS:Our systematic literature review identified 21 studies that examined the use of PGHD in clinical settings. Integration of PGHD into electronic records was extremely limited, and decision support capabilities were for the most part basic. DISCUSSION:PGHD and other types of patient-reported data will be part of the health care system narrative and we must continue efforts to understand its impact on health outcomes, costs, and patient satisfaction. Nursing scientists need to lead the process of defining the role of PGHD in the era of precision health.
| S-EPMC6697140 | biostudies-literature