Support Protocol Research Articles

Generating iAstrocytes From Human Induced Pluripotent Stem Cells by Combining Low-Density Passaging of Neural Progenitor Cells and Transcription Factor NFIA Transdifferentiation.

Astrocytes are key regulators of central nervous system (CNS) homeostasis, and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from human induced pluripotent stem cells (hiPSCs) using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediate allows for facile differentiation into both purified neuronal and astrocyte cell types in parallel from the same genetic background, depending on the experimental needs. This article presents a protocol to generate NPCs from hiPSCs, which are then differentiated into hiPSC-derived astrocytes, termed iAstrocytes. The resulting iAstrocytes express key markers of astrocyte identity at transcript and protein levels by bulk RNA-Seq and immunocytochemistry, respectively. Additionally, they respond to the inflammatory stimuli poly(I:C) and generate waves of calcium activity in response to either physical activity or the addition of ATP. Our approach offers a simple and robust method to generate and characterize human astrocytes, which can be used to model human disease affecting this cell type. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of hiPSCs to NPCs Basic Protocol 2: Differentiation of NPCs into iAstrocytes Support Protocol 1: Molecular validation of iAstrocytes Support Protocol 2: Calcium imaging-based validation of iAstrocyte function Support Protocol 3: Differentiation of NPCs into neurons.

Analysis of Tandem Repeats in Short-Read Sequencing Data: From Genotyping Known Pathogenic Repeats to Discovering Novel Expansions.

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genotyping known pathogenic tandem repeat loci Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy Support Protocol 1: Installation of tools and ExpansionHunter catalog modification Basic Protocol 2: Performing genome-wide genotyping of tandem repeats Basic Protocol 3: Discovering de novo tandem repeat expansions Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles.

A Hybrid Simulation‐Based Workshop Improves Knowledge and Confidence in the Management of Hemorrhagic Conversion of Stroke Among Interventional Neurology Trainees

Background Mechanical thrombectomy is the standard of care in acute ischemic stroke with anterior large vessel occlusion presenting within 6 hours of symptom onset, but complications do exist. We assessed the feasibility of a hybrid simulation‐based workshop at a national meeting for interventional neurology trainees focused on the management of acute ischemic stroke and tissue‐plasminogen activator‐related hemorrhage. Methods In this prospective, observational, hybrid simulation‐based study at a fellows’ workshop at a national conference, participants were asked to manage a patient with acute ischemic stroke in the neurointerventional suite followed by thrombolytic‐related hemorrhage leading to cerebral herniation during mechanical thrombectomy. We evaluated the participants’ ability to complete critical actions that were developed based on best practices and relevant American Heart Association guidelines and the Neurocritical Care Society's Emergency Neurological Life Support protocols. The primary outcome was the improvement in knowledge from a precourse to postcourse test. Secondary outcomes included participant reactions. Results Sixty trainees completed the simulation session in 8 groups. The mean sum of critical actions completed by trainees was 9.75/14 (70%). There was a moderate effect of the intervention on trainees’ knowledge from pretest (mean 3.8, SD = 0.3) to posttest (mean 4.3, SD = 0.3). The simulation scenario was described as moderately realistic, very engaging, and extremely satisfactory. Following the workshop, all fellows endorsed an increase in proficiency and confidence in neurological emergency management. Conclusion Simulation‐based workshops at national conferences are feasible and a potentially useful tool for safely educating a large audience of trainees who may not have access to high‐fidelity simulation platforms at their institutions.

Green spectrophotometric approaches applied to tertiary mixture for management of common cold and COVID-19 symptoms

Recently, cold and cough dosage forms have gained significant attention due to their use in the supportive protocols for managing COVID-19 symptoms. In this study, a pharmaceutical formulation containing Paracetamol (PAR), Guaifenesin (GUA), and Phenylephrine hydrochloride (PHE) was investigated for spectral resolution and quantification using advanced spectrophotometric methods. The spectra of these components were significantly overlapped and present in their combined tablet in a challenging ratio of 250:100:5 for PAR, GUA, and PHE, respectively. The established approaches were employed for the simultaneous determination of these drugs in their pharmaceutical formulation without interference from matrix excipients. The study involved various manipulation steps, allowing each component in the combination to be analyzed by more than one approach. Integrating these methods with smart mathematical techniques, the issue of spectral data overlap was resolved without the need for preliminary separation steps. The developed methods are dual wavelength, first derivative, derivative ratio, ratio difference, constant center coupled with spectrum subtraction, and constant multiplication paired with spectrum subtraction. The proposed methods were linear over the concentration range of 3.0–35.0 μg/mL for GUA and 3.0–30.0 μg/mL for PHE. While the PAR ranges for the first derivative and constant multiplication methods were 2.5–35.0 μg/mL and 2.5–25.0 μg/mL, respectively. Excellent linearity of the suggested methods was demonstrated by the high correlation coefficients (R2), ≥ 0.9998 for all the tested compounds. These methodologies were validated according to ICH guidelines. Validation results demonstrated excellent accuracy, with recovery percentages ranging from 98 to 102 %, and precision, with RSD values less than 2 %. The obtained results were statistically compared with the official ones using F-test, Student’s t-test, and one-way ANOVA, revealing no significant differences. The proposed methods are accurate, green, smart, fast, and cost-effective. Their compliance with Green Analytical Chemistry principles was evaluated and compared to a published method using various tools to enable a more holistic evaluation from different perspectives. The promising results revealed that the investigated methods are superior green alternatives for routine analysis of the cited drugs in laboratories with limited resources and without access to expensive instruments.

Digital Health Technology on Patient Recovery and Nurse Performance in Indonesia

This study investigates the impact of digital health technology, nurse competence, patient safety protocols, and organizational support on patient recovery and nurse performance within the Indonesian healthcare sector. Utilizing a quantitative approach, data were collected from 120 healthcare professionals through structured questionnaires and analyzed using Structural Equation Modeling-Partial Least Squares (SEM-PLS 3). The findings reveal that digital health technology, nurse competence, and patient safety protocols positively influence patient recovery, while organizational support and digital health technology significantly enhance nurse performance. Organizational support and patient safety protocols emerged as the strongest predictors of nurse performance and patient recovery, respectively. These results suggest that a comprehensive approach integrating technological, safety, and support factors can substantially improve healthcare outcomes. This study offers practical recommendations for healthcare administrators and policymakers to enhance healthcare quality through strategic investments in digital tools, training, safety protocols, and supportive work environments.

Optimizing CAR-NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance.

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs) Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells Support Protocol 1: Plasmid amplification Support Protocol 2: Lentivirus preparation Support Protocol 3: Lentivirus titration.

Inclusive Life Projects for people with Serious Mental Disorder and/or addictions

Abstract Project “Inclusive Life Projects” offers comprehensive and community care to people with SMD/A within projects of autonomous living, which provide a concrete and real response to their needs, with the ultimate objective of avoiding the risk of institutionalization. Currently, 26 actions have been designed and are being implemented within JA ImpleMENTAL pilot in five municipalities in the Region of Murcia: Caravaca, Cartagena, Lorca. Molina de Segura and Yecla with a participation of 50 people with SMD/A. The following elements are selected from the Belgian good practice: • Creation of local intersectoral networks promoting person-centered care process; • Personalization of care through the Individualized Care Plan, responding to the needs, and desires of people with SMI/A, activating the appropriate support; • Professional case manager. It generates the well-being of people on an empathy and trust basis and articulates coordination so that the agreed Plan can be developed as planned. A strategic and support coordination tool for action and facilitation of institutional support, the Social and Health Coordination Protocol for people with SMI/A, has been developed. The protocol facilitates the creation of coordination structures, among them the Socio-Health Coordination Base Teams stand out, teams made up of professionals from the different agents involved, multi-professional and intersectoral, (Mental Health, Primary Health Care, Social Services, Social Initiative) taking into account the needs of the person. It also serves to share information, detect training needs, influence resources, etc. PETRA, a computer platform, has been developed for sharing information between the systems involved, especially the health system and the social services system, both primary care, hospital care and specialized care. It allows the exchange of information, in real time, between professionals, the planning of actions, and the design of indicators and control and evaluation processes.

Peri-operative strategy in resuscitation of unstable injured surgical patients: a primer.

Trauma remains a leading cause of death, both for individuals under 40 in North America, and globally, where it contributes to ~10% of deaths annually. Thoughtful, timely, balanced resuscitation, especially in the peri-operative period for unstable injured surgical patients, is vital for optimizing outcomes. The advanced trauma life support protocol plays a pivotal role in early evaluation and management, emphasizing hemorrhage control and resuscitation strategies. This narrative review provides a structured, evidence-based framework aimed at enhancing the educational experience of surgical trainees. It outlines key principles in peri-operative trauma resuscitation, emphasizing timely intervention, goal-directed fluid therapy, and damage control surgery (DCS) to improve patient outcomes. A comprehensive Scale for Quality Assessment of Narrative Review Articles -guideline compliant literature search was conducted using PubMed and Google Scholar for English-language articles published between January 2000 and February 2024. The search included relevant medical subject headings terms. Additional studies were identified from reference lists. Extracted data were reviewed and organized using thematic analysis, focusing on historical perspectives, evidence-based practices, and the concept of DCS. Key findings from the 55 relevant studies selected underscore the importance of balanced fluid and blood product administration, the use of permissive hypotension in hemorrhagic shock, and the application of DCS principles. This review highlights educational strategies that foster a deeper understanding of trauma resuscitation practices, offering practical insights through case studies and technological innovations. This review serves as an educational resource for surgical trainees, equipping them with a robust understanding of evidence-based trauma resuscitation. By integrating historical context, modern practices, and emerging technologies, the review aims to enhance both the theoretical knowledge and practical skills necessary for managing unstable trauma patients. Emphasis is placed on interdisciplinary teamwork, continuous education, and personalized resuscitation strategies to improve clinical outcomes.

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

De Novo Synthesis of Error-Free Long Oligos.

This protocol describes the synthesis of long oligonucleotides (up to 401-mer), their isolation from complex mixtures using the catching-by-polymerization (CBP) method, and the selection of error-free sequence via cloning followed by Sanger sequencing. Oligo synthesis is achieved under standard automated solid-phase synthesis conditions with only minor yet critical adjustments using readily available reagents. The CBP method involves tagging the full-length sequence with a polymerizable tagging phosphoramidite (PTP), co-polymerizing the sequence into a polymer, washing away failure sequences, and cleaving the full-length sequence from the polymer. Cloning and sequencing guided selection of error-free sequence overcome the problems of substitution, deletion, and addition errors that cannot be addressed using any other methods, including CBP. Long oligos are needed in many areas such as protein engineering and synthetic biology. The methods described here are particularly important for projects requiring long oligos containing long repeats or stable higher-order structures, which are difficult or impossible to produce using any other existing technologies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Long oligo synthesis Support Protocol 1: Synthesis of polymerizable tagging phosphoramidite (PTP) Support Protocol 2: Synthesis of 5'-O-Bz phosphoramidite Basic Protocol 2: Catching-by-polymerization (CBP) purification Basic Protocol 3: Error-free sequence selection via cloning and sequencing.

Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization.

A Double Lysis Method for Animal Rotavirus RNA Extraction From Stool Samples.

Globally, porcine rotavirus is a leading cause of gastroenteritis in nursing and post-weaning piglets, as well as adult pigs. Between February 2015 and June 2016, 156 fecal samples were collected from pigs in the Northeastern part of Accra, Ghana, and screened for Group A rotavirus using the ProflowTM Kit. Here, we describe different extraction methods that were employed to recover high-quality RNA for downstream analysis, with emphasis on a novel hybrid extraction method. The hybrid approach with a kit and manual extraction method led to a 10-fold greater RNA yield versus the kit-based method alone. The new extraction method gave an average purity ratio (A260/A280) of 1.8, which was also significantly higher than that obtained solely from the manual or kit-based extraction methods. Our novel hybrid approach will be useful in the extraction of rotavirus from animal fecal samples, thus improving the yield of RNA for downstream analysis. © 2024 Wiley Periodicals LLC. Basic Protocol: Hybrid 2: A double lysis method for RNA extraction from animal stool samples Support Protocol 1: The GenElute extraction method Support Protocol 2: Hybrid 1 extraction method.

Fertility Treatment and Preservation Options for Transgender and Gender Diverse People.

Years of growing research demonstrate that transgender and gender diverse (TGD) people desire fertility counseling and family building, however social and medical factors can impact future fertility options. Fortunately, TGD individuals have many viable options for family building using their own gametes and/or reproductive organs. However, the nuanced ways in which different gender affirming treatments affect reproduction, the interplay with non-treatment related infertility factors, and mitigation of likely dysphoria triggers are all critical to actual utilization. This review focuses on fertility treatment and preservation options for TGD patients and highlights these influential social and medical factors. Fertility treatments may be associated with worsening gender dysphoria in TGD people, and an affirming clinical environment and conscientious provider approach is paramount to treatment success. However, reducing gender dysphoria can also require specific changes to medically assisted reproduction and sperm collection protocols, some which carry the potential for diminished outcomes or unknown effects. Adolescents undergoing fertility preservation treatments may need more support or additional protocol modifications, and outcomes may be poorer in this age group compared with adults. Testicular and ovarian tissue cryopreservation may present a fertility preservation option for prepubertal TGD children, however in-vitro gamete maturation remains experimental in this situation.

Going in Blind: A Common Scenario in an Uncommon Situation.

Medical students, interns, junior resident physicians, senior resident physicians. Power outages have been increasing in frequency in the past few years, therefore becoming an increased threat to healthcare delivery.1 While most studies related to the effects of power outages are focused on outpatient care, such as acute exacerbations of chronic lung conditions and the lack of chargeable equipment, with the increasing number of power outages, hospitals must be prepared for this situation as well.2,3 Although agencies such as the Federal Emergency Management Agency (FEMA) and the US Department of Health and Human Services (HHS) have provided guidelines for the response of hospitals to temporary loss of power,12,13 hospitals generally rely on institutional policies in response to the event of a power outage. Given the relative rarity but increasing frequency of power outages in hospital settings, this medical simulation was created to present a common occurrence in the emergency department (eg, cardiac arrest) in an uncommon setting of a power outage. Simulation has been shown to improve learner self-efficacy, confidence, and leadership skills among resuscitation teams.4,5 The role of simulation also helps learners identify latent safety threats, in this case a power outage.6 The goal of this simulation is to improve the skills of healthcare professionals with regards to managing cardiac arrest and to encourage these practitioners to consider their own hospital guidelines in response to a power outage. By the end of this simulation, learners will be able to (1) evaluate and treat a patient experiencing myocardial infarction and subsequent cardiac arrest during a power outage, (2) describe the local protocols for managing patient care during a power outage, (3) demonstrate the ability to coordinate a medical team during a simulated power outage in an emergency department with limited resources, (4) manage a cardiac arrest patient by following Advanced Cardiac Life Support (ACLS) protocols for bradycardia and ventricular fibrillation, and (5) justify the urgency of transfer to a certified ST segment elevation myocardial infarction center/cardiac intensive care unit, referencing the recommended 120-minute door-to-balloon time. This simulation was conducted with a high-fidelity mannequin. A total of six residents of various post-graduate year (PGY) levels participated in the simulated patient encounter as part of the simulation competition at the Western Regional meeting of the Society for Academic Emergency Medicine. This case was assessed for educational content and piloted by emergency medicine attendings from several institutions prior to running the case for the Western Regional meeting. The efficacy of the content was assessed by oral feedback. The case was well-received by both the attending physicians who evaluated the case prior to running the scenario at the Western Regional meeting and the emergency medicine residents who participated in the case at the Western Regional meeting. Overall, this simulation was well received by both the learners and the debriefers. General feedback was positive, with the perception of increased confidence among learners and reflection upon individual hospital policy in the event of a power outage. Simulation, acute myocardial infarction, cardiac arrest, power outage.

IntAct Database for Accessing IMEx's Contextual Metadata of Molecular Interactions.

The International Molecular Exchange Consortium (IMEx) has evolved into a vital partnership of open resources dedicated to curating molecular interaction data from the scientific literature. This consortium, which includes IntAct, MINT, MatrixDB, and DIP, is a collaborative effort with a central mission of aggregating detailed molecular interaction experimental evidence in a machine-readable format, supported by controlled vocabularies and standard ontologies. The IntAct molecular interaction database (www.ebi.ac.uk/intact), as an IMEx partner, serves as a valuable portal for accessing IMEx data through user-friendly search options and an array of interactive filters. The resource currently hosts an extensive repository of 1,293,508 binary interactions meticulously captured from 75,098 experiments documented in 23,366 publications (as of the February 2024 release), with this corpora being added to by regular data releases. IMEx curation policy has consistently prioritized a fine-grained data and curation model, with a focus on capturing the relevant experimental details essential for interpreting molecular interaction data effectively. Our curation process is designed to support the generation of interactomes tailored to contexts such as disease-specific or tissue-/cell-type-specific interactomes. These interactions are ranked according to a scoring system based on the Proteomics Standard Initiative Molecular Interaction (PSI MI) standards. This scoring system allows users to assess the degree of confidence in binary interactions, enhancing the value of the data. The resource provides insights into the nature of relationships among interacting partners as defined by the experimental setup and the associated biological context. Interactive filters enable users to navigate these rich, multilayered data, promoting a deeper understanding of biological complexity. Additionally, the IntAct website fosters the creation of networks for collaborative analyses by the scientific community. The recent transformation of the IntAct website, supported by a graph-type database, empowers users to execute custom queries tailored to their specific research interests. This article illustrates the diverse levels of annotations available for interactions and the multiple search options at users' disposal to access data of interest. © 2024 European Molecular Biology Laboratory, European Bioinformatics Institute. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Using Quick Search, network visualization, and filters Support Protocol: Accessing fine annotations from intact: Unlocking the molecular details Alternate Protocol: Using batch search: Querying multiple interactors Basic Protocol 2: Using advanced search: Precision and customization.

TIBIAL DIAPHYSEAL FRACTURE, PANORAMIC REVISION

Introduction: Tibial fractures are quite common injuries. Since the tibia has a subcutaneous location, it is more likely to show exposed fractures. Exposed fractures of the tibial diaphysis are serious injuries that can result in significant long-term disability if not managed correctly. Objective: to detail current information related to tibial diaphysis fractures, etiology, epidemiology, anamnesis, physical examination, assessment, treatment, differential diagnosis, prognosis and complications. Methodology: a total of 29 articles were analyzed in this review, including review and original articles, as well as clinical cases, of which 17 bibliographies were used because the other articles were not relevant for this study. The sources of information were PubMed, Google Scholar and Cochrane; the terms used to search for information in Spanish, Portuguese and English were: tibia fractures, osteosynthesis, tibia fracture, intramedullary nails, leg trauma, exposed fractures. Results: tibial diaphysis fractures present an incidence of 16.9 per 100,000 individuals per year, being more common in the male sex, with 21.5 per 100,000 individuals per year, compared to 12.3 per 100,000 in the female sex. The most common triggers in males of tibial diaphysis fractures are motor vehicle accident injuries and sports. Conclusions: It is important to recognize the management of tibial fractures since they have a relatively high incidence. It is not uncommon to encounter an exposed tibial diaphysis fracture, so it is essential to identify, as well as perform damage control and appropriate management. The individual should undergo a complete examination and it may be necessary to implement the trauma life support protocol. In this type of fracture it is crucial to assess the neurovascular situation. In addition, complementary examinations should be requested to allow the best diagnosis, classification and treatment of the injuries. The treatment of tibia diaphysis fractures will depend on the specific situation and the patient's condition; however, external fixators are usually used for damage control and later intramedullary nails are used. Tibial diaphysis fractures may be accompanied by compartment syndrome and other complications. KEY WORDS: trauma, fractures, tibia, treatment.

Concussion Incidence, Mechanism, and Perspectives Among Australian Elite Surfers: Implications for Medical Support and Safety Protocols.

The primary objective was to investigate the incidence of concussion and the associated mechanisms of injury in elite Australian surfers. The secondary objective was to investigate the current perspectives and behaviors related to experiencing concussion in surfing. A cross-sectional, retrospective, descriptive survey. Surfing Australia high-performance program. Forty nationally identified elite Australian surfing athletes. A retrospective survey collected information pertaining to participant demographics, concussion history, potential concussive symptoms, such as headache, neck pain, dizziness, or unusual fatigue, following a wipeout, and participants' perspectives on concussion. Investigating concussion incidence among elite Australian surfers compared with potential undiagnosed concussive episodes. Surfers with a history of diagnosed surfing-related concussion (DC) were compared with those with no history of a diagnosed surfing-related concussion (NDC). A total of 13 of 40 surfers had a DC, with "contact versus the water surface" identified as the primary mechanism. Both DC and NDC groups had a high frequency of concussive symptoms after a surfing wipeout with a total of 447 and 573 concussive symptom occurrences reported in the DC and NDC groups, respectively. Concern regarding the long-term impacts of concussion were reported in 25 of 40 surfers. The number of concussive symptoms reported by surfers who had not previously been diagnosed with concussion suggests that concussion may be underreported and underdiagnosed in elite surfing. This underscores the need for increased medical support, education, and improved safety protocols.

Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats.

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.

A Cost-Effective Approach for Single-Stranded DNA Amplification Using Primer-Blocked Asymmetric PCR.

In vitro amplification of single-stranded oligonucleotide libraries presents a significant challenge due to the potential for excessive byproduct formation. This phenomenon largely affects the quality of the ssDNAs created using the most commonly used methods, e.g., asymmetric PCR, biotin-streptavidin separation, or lambda exonuclease digestion of dsDNA. Here, we describe an improved protocol that combines primer-blocked asymmetric PCR (PBA-PCR) with emulsion PCR and a cost-effective downstream process that altogether alleviates byproduct formation without distorting the sequence space of the ssDNA library. In PBA-PCR, the reaction mixture is complemented with a 3'-phosphate-blocked limiting primer that decreases mispriming, thus reducing polymerization of DNA byproducts. The downstream process includes mixing of the PBA-PCR product with excess reverse complement of the 3'-phosphate-blocked limiting primer and removal of dsDNA strands via biotin-streptavidin separation, yielding purified ssDNAs. In conclusion, we have devised a universally applicable approach for simple and cost-effective production of ssDNA libraries and unique ssDNA sequences with on-demand labeling. Our protocol could be beneficial for a variety of uses, such as generating aptamer libraries for SELEX, creating unique molecular identifiers for a wide range of sequencing applications, providing donor DNA for CRISPR-Cas9 systems, developing scaffold nanostructures, and enabling DNA-based data storage. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Amplification of ssDNA libraries using PBA-PCR Alternate Protocol 1: Amplification of ssDNA libraries using emulsion PBA-PCR with a simplified extraction of PBA-PCR products Basic Protocol 2: Purification of PBA-PCR products to remove dsDNA and conversion of 3'-blocked primer to double-stranded complexes Alternate Protocol 2: Purification of PBA-PCR products to remove both dsDNA and blocking primers from the reaction mixture Support Protocol: Analysis of PBA-PCR products by gel electrophoresis.

Management of Sideline Medical Emergencies.

Sideline medical care is typically provided by musculoskeletal specialists and orthopaedic surgeons with varying levels of training and experience. While the most common sports injuries are often benign, the potential for catastrophic injury is omnipresent. Prompt recognition of sideline emergencies and expeditious medical management are necessary to minimize the risk of calamitous events. Paramount to successful sideline coverage are both preseason and game-day preparations. Because the skillset needed for the sideline physician may involve management of injuries not commonly seen in everyday clinical practice, sideline providers should review basic life support protocols, spine boarding, and equipment removal related to their sport(s) before the season begins. Before every game, the medical bag should be adequately stocked, location of the automatic external defibrillator/emergency medical services identified, and introductions to the trainers, coaches, and referees made. In addition to musculoskeletal injuries, the sideline orthopaedic surgeon must also be acquainted with the full spectrum of nonmusculoskeletal emergencies spanning the cardiopulmonary, central nervous, and integumentary systems. Familiarity with anaphylaxis as well as abdominal and neck trauma is also critical. Prompt identification of potential life-threatening conditions, carefully orchestrated treatment, and the athlete's subsequent disposition are essential for the team physician to provide quality care.