RESUMEN
Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.
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ARN Polimerasas Dirigidas por ADN , ADN Polimerasa Dirigida por ARN , ADN Polimerasa Dirigida por ARN/metabolismo , ADN Polimerasa Dirigida por ARN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Evolución Molecular , Mutación , ADN Complementario/genéticaRESUMEN
Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host-parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts.
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Cuscuta , MicroARNs , Parásitos , Animales , MicroARNs/genética , MicroARNs/metabolismo , Cuscuta/genética , Cuscuta/metabolismo , Parásitos/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN Polimerasa III/metabolismo , Interacciones Huésped-Parásitos , Plantas/genéticaRESUMEN
Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.
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Infecciones por VIH/virología , VIH-1/fisiología , FN-kappa B/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus , Alquinos/farmacología , Animales , Antirretrovirales/farmacología , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , Macaca mulatta , Ratones , Oligopéptidos/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
Circular RNA (circRNA) has recently gained attention for its emerging biological activities, relevance to disease, potential as biomarkers, and promising an alternative modality for RNA vaccines. Nevertheless, sequencing circRNAs has presented challenges. In this context, we introduce a novel circRNA sequencing method called Induro-RT mediated circRNA-sequencing (IMCR-seq), which relies on a group II intron reverse transcriptase with robust rolling circle reverse transcription activity. The IMCR-seq protocol eliminates the need for conventional circRNA enrichment methods such as rRNA depletion and RNaseR digestion yet achieved the highest circRNA enrichment and detected 6-1000 times more circRNAs for the benchmarked human samples compared to other methods. IMCR-seq is applicable to any organism, capable of detecting circRNAs of longer than 7000 nucleotides, and is effective on samples as small as 10 ng of total RNA. These enhancements render IMCR-seq suitable for clinical samples, including disease tissues and liquid biopsies. We demonstrated the clinical relevance of IMCR-seq by detecting cancer-specific circRNAs as potential biomarkers from IMCR-seq results on lung tumor tissues together with blood plasma samples from both a healthy individual and a lung cancer patient. In summary, IMCR-seq presents an efficient and versatile circRNA sequencing method with high potential for research and clinical applications.
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Neoplasias Pulmonares , ARN Circular , Análisis de Secuencia de ARN , ARN Circular/genética , Humanos , Análisis de Secuencia de ARN/métodos , Neoplasias Pulmonares/genética , Biomarcadores de Tumor/genética , Transcripción Reversa , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5' cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5' cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA-a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.
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Caperuzas de ARN/metabolismo , ARN/análisis , Análisis de Secuencia de ARN/métodos , Humanos , Fijación del TejidoRESUMEN
Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.
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Biblioteca de Genes , ARN Pequeño no Traducido/aislamiento & purificación , Análisis de Secuencia de ARN/métodos , Electroforesis Capilar , Femenino , Humanos , Masculino , Metilación , MicroARNs/química , MicroARNs/genética , MicroARNs/aislamiento & purificación , Hibridación de Ácido Nucleico , Oligorribonucleótidos/química , ARN Neoplásico/química , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , ARN de Planta/química , ARN de Planta/genética , ARN de Planta/aislamiento & purificación , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , ARN de Transferencia/química , ARN de Transferencia/aislamiento & purificación , Distribución Aleatoria , Sensibilidad y Especificidad , Alineación de SecuenciaRESUMEN
The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper's content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.
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Descubrimiento de Drogas , Primates , Animales , Evaluación Preclínica de Medicamentos/métodos , Industria Farmacéutica/métodos , Preparaciones FarmacéuticasRESUMEN
Group living confers many benefits while simultaneously exposing group members to intense competition. An individual's rise to prominence within a group may conflict with the overall functioning of the group. There is therefore a complex and dynamic relationship between the behavioral displays that directly benefit an individual, the consequences of these actions for the community, and how they feed back on individual-level fitness. We used a network analysis approach to study the link between behavior, social stability, and steroid hormone levels in replicate communities of the cichlid fish, Astatotilapia burtoni, which live in social groups with a dominance hierarchy. We demonstrate that individual behavior can have direct and indirect effects on the behavior of others while also affecting group characteristics. Our results show that A. burtoni males form stable social networks, where dominant individuals act as hubs for social interactions. However, there was variation in the temporal stability in these networks, and this variation in stability impacted hormone levels. Dominant males had higher testosterone levels, however, the differences in testosterone levels between dominant and subordinate males were greatest in stable communities. In sum, our analyses provide novel insights into the processes by which individual and community properties interact.
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Cíclidos , Agresión , Animales , Jerarquia Social , Hormonas , Humanos , Masculino , Conducta Social , Predominio Social , Red SocialRESUMEN
Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.
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ADN Complementario/química , ADN Viral/química , Virus de la Leucemia Murina de Moloney/enzimología , ARN Viral/química , ADN Polimerasa Dirigida por ARN/química , Transcripción Reversa , ADN Complementario/biosíntesis , ADN Viral/biosíntesis , Motivos de Nucleótidos , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
OBJECTIVE: Concerns about the long-term durability of endovascular aortic aneurysm repair and the requirement for explantation of stents in the case of infection demonstrate the continued need for open abdominal aortic aneurysm (AAA) repair. However, with the increased complexity and decreasing volume of open cases performed, maintenance of skills and training of younger surgeons are challenging. The aim of this review was to identify and to examine studies pertaining to open AAA simulation, with focus on methods and outcomes. METHODS: We performed a systematic review of the literature to identify primary research pertaining to open AAA repair through the use of simulators. The primary outcome was to identify predominant modes of simulator design and validated assessment tools that could demonstrate improvement in trainee skills. Secondary outcomes included identifying participant numbers needed to power studies and whether tools not validated externally contributed to the studies. RESULTS: There were 309 unique papers identified, from which five papers met the inclusion criteria. The selected papers used a combination of synthetic (commercial and homemade) and cadaveric simulators. A variety of validated and nonvalidated assessment metrics were used, including Objective Structured Assessment of Technical Skills, global rating scales, and realism surveys. Three of the five papers used blinding as part of their assessments. Mean participant numbers were 30.8 ± 25.7 and with the exception of one paper consisted entirely of surgical trainees in dedicated general or vascular surgery training programs. CONCLUSIONS: Several options are currently available for open AAA simulation, all of which demonstrate improved scoring metrics after simulator use. Validated scoring systems, the Objective Structured Assessment of Technical Skills in particular, were most frequently used to deliver objective results. Whereas junior trainees derive the most benefit, senior trainees also showed significant improvements, demonstrating that simulation benefits all levels of surgical trainees. Low numbers of participants were sufficient to achieve statistical benefit within individual studies.
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Aneurisma de la Aorta Abdominal/cirugía , Entrenamiento Simulado , Procedimientos Quirúrgicos Vasculares/educación , Competencia Clínica , Humanos , StentsRESUMEN
GlaxoSmithKline has recently made significant organizational changes to its nonclinical safety, drug metabolism and pharmacokinetic, and laboratory animal science/veterinary functions, with the goal to increase our focus on scientific partnership with the discovery part of the organization. One specific change was bringing together pathologists and comparative medicine veterinarians and scientists into a single functional unit. We describe our early activities (assessing our capabilities and gaps, external benchmarking, listening to our discovery partners, redesigning some of our working practices) aimed at implementing these changes. In addition, early on we held a Discovery Engagement Workshop attended by all pathologists and comparative medicine veterinarians and scientists, as well as selected discovery scientists. The purpose of this workshop was to share learnings from the above activities and devise plans aimed at achieving our overall goal of functional integration: driving pathobiology expertise into drug discovery and increasing the human (translational) relevance of experimental data. This review describes the new organizational structure, the workshop activities, and implementation plans; updates our progress; and considers the opportunity for a pan-industry network of discovery-focused pathologists and comparative medicine veterinarians and scientists.
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Descubrimiento de Drogas/métodos , Industria Farmacéutica/organización & administración , Personal de Laboratorio , Patólogos , Animales , Evaluación Preclínica de Medicamentos/métodos , Humanos , Patología , VeterinariosRESUMEN
The recent Scientific Committee on Health, Environmental and Emerging Risks Final Opinion on "The need for nonhuman primates in biomedical research, production and testing of products and devices" (2017 SCHEER) highlights approaches that could significantly contribute to the replacement, reduction, and refinement of nonhuman primate (NHP) studies. Initiatives that have the potential to affect NHP welfare and/or their use are expected to be appropriate, fair, and objective and publicly disseminated information focused on NHPs in biomedical research, which includes toxicologic and pathologic research and testing, should be objectively evaluated by stakeholder scientists, researchers, and veterinarians. Thus, IQ Consortium member companies convened to develop an informed and objective response, focusing on identifying areas of agreement, potential gaps, or missing information in 2017 SCHEER. Overall, the authors agree that many positions in the 2017 SCHEER Opinion generally align with industry views on the use of NHPs in research and testing, including the ongoing need of NHPs in many areas of research. From the perspective of the IQ Consortium, there are several topics in the 2017 SCHEER that merit additional comment, attention, or research, as well as consideration in future opinions.
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Alternativas al Uso de Animales/tendencias , Investigación Biomédica/métodos , Evaluación Preclínica de Medicamentos/tendencias , Primates , Alternativas al Uso de Animales/ética , Alternativas al Uso de Animales/legislación & jurisprudencia , Bienestar del Animal , Animales , Bioética , Investigación Biomédica/ética , Investigación Biomédica/legislación & jurisprudencia , Evaluación Preclínica de Medicamentos/ética , Evaluación Preclínica de Medicamentos/métodos , Unión Europea , Regulación GubernamentalRESUMEN
Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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Secuencia Conservada/genética , Genoma/genética , Pez Cebra/genética , Animales , Cromosomas/genética , Evolución Molecular , Femenino , Genes/genética , Genoma Humano/genética , Genómica , Humanos , Masculino , Meiosis/genética , Anotación de Secuencia Molecular , Seudogenes/genética , Estándares de Referencia , Procesos de Determinación del Sexo/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The melanocortin system is a neuroendocrine system that regulates a range of physiological and behavioural processes. We examined the extent to which the melanocortin system simultaneously regulates colour and behaviour in the cichlid fish Astatotilapia burtoni We found that yellow males are more aggressive than blue males, in line with previous studies. We then found that exogenous α-melanocyte-stimulating hormone (α-MSH) increases yellowness of the body and dispersal of xanthophore pigments in both morphs. However, α-MSH had a morph-specific effect on aggression, with only blue males showing an increase in the rate of aggression. Exogenous agouti signalling peptide (ASIP), a melanocortin antagonist, did not affect coloration but reduced the rate of aggression in both colour morphs. Blue males had higher cortisol levels than yellow males. Neural gene expression of melanocortin receptors (mcr) and ligands was not differentially regulated between colour morphs. In the skin, however, mc1r and pro-opiomelanocortin (pomc) ß were upregulated in blue males, while asip 1 was upregulated in yellow males. The effects of α-MSH on behaviour and body coloration, combined with morph-specific regulation of the stress response and the melanocortin system, suggest that the melanocortin system contributes to the polymorphism in behaviour and coloration in A. burtoni.
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Conducta Animal , Cíclidos/fisiología , Melanocortinas/fisiología , Pigmentación , Receptores de Melanocortina/fisiología , Agresión , Animales , Color , Proteínas de Peces/fisiología , Masculino , Proopiomelanocortina/fisiología , Conducta SocialRESUMEN
Extended phenotypes offer a unique opportunity to experimentally manipulate and identify sources of selection acting on traits under natural conditions. The social cichlid fish Neolamprologus multifasciatus builds nests by digging up aquatic snail shells, creating an extended sexual phenotype that is highly amenable to experimental manipulation through addition of extra shells. Here, we find sources of both positive sexual selection and opposing natural selection acting on this trait; augmenting shell nests increases access to mates, but also increases social aggression and predation risk. Increasing the attractiveness of one male also changed social interactions throughout the social network and altered the entire community structure. Manipulated males produced and received more displays from neighbouring females, who also joined augmented male territories at higher rates than unmanipulated groups. However, males in more attractive territories received more aggression from neighbouring males, potentially as a form of social policing. We also detected a significant ecological cost of the 'over-extended' phenotype; heterospecific predators usurped augmented nests at higher rates, using them as breeding sites and displacing residents. Using these natural experiments, we find that both social and ecological interactions generate clear sources of selection mediating the expression of an extended phenotype in the wild.
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Cíclidos/fisiología , Preferencia en el Apareamiento Animal , Agresión , Animales , Cíclidos/anatomía & histología , Cíclidos/genética , Femenino , Masculino , Comportamiento de Nidificación , Fenotipo , Dinámica Poblacional , Conducta Sexual Animal , Conducta SocialRESUMEN
OBJECTIVE: A meta-analysis to determine if patients with varicose veins are at an increased risk of venous thromboembolism (VTE) when undergoing major lower limb arthroplasty. METHODS: Medline, Embase, and Cochrane Library databases were searched using appropriate terms for studies that reported post-operative VTE in patients who had lower limb arthroplasty with any history of varicose veins. Methodological quality of included studies was quantified using the Risk of Bias (ROB) assessment tools. Findings were reported using the meta-analysis of observational studies in epidemiology (MOOSE) checklist. RESULTS: A total of 129 studies were identified with 11 observational studies being eligible for inclusion. This consisted of 265,194 patients who underwent lower limb arthroplasty, 2188 of which had pre-existing varicose veins. Overall, VTE occurred in 1845 patients, and 122 cases had varicose veins present at time of arthroplasty. Meta-analysis indicates that patients undergoing lower limb arthroplasty with varicose veins are at increased risk of having a VTE, OR 2.37, 95% CI 1.54-3.63, (p < 0.001). One study evaluated if previous varicose veins surgery influenced the risk of VTE in arthroplasty patients, OR 0.96 (95% CI 0.7-1.28), p = 0.429. CONCLUSIONS: Varicose veins and lower limb arthroplasty are known independent risk factors for VTE. There is a paucity of data regarding the risk of VTE in patients undergoing lower limb arthroplasty who have co-existing varicose veins. This meta-analysis shows that patients with varicose veins are at an increased risk of VTE when undergoing major lower limb arthroplasty. Further studies are required in order to determine if such patients should undergo varicose vein surgery before undertaking major lower limb joint replacement.
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Várices , Tromboembolia Venosa , Humanos , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/etiología , Várices/cirugía , Várices/complicaciones , Factores de Riesgo , Artroplastia/efectos adversos , Medición de RiesgoRESUMEN
OBJECTIVES: The aim of this research was to ascertain the highest need areas for vascular simulation, in order to tailor training for the highest impact. DESIGN, PARTICIPANTS AND SETTING: A needs assessment was conducted according to best practices using the Delphi method. All consultant vascular surgeons/trainers in the training jurisdiction (n=33) were approached through an independent intermediary to contribute and generate a prioritized list of procedures for training. The research team were blinded to participant identities. Three rounds were conducted according to the Delphi process and scored according to the Copenhagen Needs Assessment Formula (CAMES-NAF). RESULTS: A final list of 34 vascular procedures was selected and prioritized by surgical trainers. Principles of arterial repair and endarterectomy/patching were considered the highest priority. Complex major interventions such as open abdominal aortic aneurysm (AAA) repair, carotid endarterectomy, and endovascular aortic repair (EVAR) consistently ranked higher than rarer, such as first rib resection and more basic procedures, such as foam injection for varicose veins. Major lower limb amputations typically ranked lower overall compared to arterial interventions. Surgical trainers generally agreed with the ranking according to the CAMES-NAF. There was some disagreement for a select few procedures such as iliac stenting (which rose 13 places) and open radial artery exposure (which fell 6 places) on subsequent iterations. CONCLUSIONS: Core operative principles and common major operative cases should remain the priority for vascular technical skills training. Other procedures which may be less invasive, but have the potential for major complications should also not be overlooked. In designing simulators the main focus should center on specific skill acquisition for commonly performed major procedures and management of the recognized potential complications. Lower limb amputations are considered adequately taught in clinical practice, or are too challenging to simulate in simulator models apart from cadaveric models.
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Aneurisma de la Aorta Abdominal , Procedimientos Endovasculares , Entrenamiento Simulado , Humanos , Evaluación de Necesidades , Educación de Postgrado en Medicina/métodos , Procedimientos Quirúrgicos Vasculares/educación , Aneurisma de la Aorta Abdominal/cirugía , Procedimientos Endovasculares/educaciónRESUMEN
Small RNAs (sRNAs) are an important group of non-coding RNAs that have great potential as diagnostic and prognostic biomarkers for treatment of a wide variety of diseases. The portability and affordability of nanopore sequencing technology makes it ideal for point of care and low resource settings. Currently sRNAs can't be reliably sequenced on the nanopore platform due to the short size of sRNAs and high error rate of the nanopore sequencer. Here, we developed a highly efficient nanopore-based sequencing strategy for sRNAs (SR-Cat-Seq) in which sRNAs are ligated to an adapter, circularized, and undergo rolling circle reverse transcription to generate concatemeric cDNA. After sequencing, the resulting tandem repeat sequences within the individual cDNA can be aligned to generate highly accurate consensus sequences. We compared our sequencing strategy with other sRNA sequencing methods on a short-read sequencing platform and demonstrated that SR-Cat-Seq can obtain low bias and highly accurate sRNA transcriptomes. Therefore, our method could enable nanopore sequencing for sRNA-based diagnostics and other applications.