Support Protocol Research Articles

Blunt Trauma Neck with Thyroid Cartilage Subluxation with no External Sign

Trauma to the neck can produce catastrophic results as neck houses plethora of vital structures and is relatively an unprotected anatomical territory. Blunt trauma to the neck, excluding cervical spine injuries, represents only 5% of all neck trauma, but can be very challenging to assess since its presentation is often delayed. Penetrating injuries, on the other hand, are more common, and even when they seem to be only superficial and minor, always need thorough investigation and observation. Primary stabilization of the patient followed by an extensive evaluation needs to be done in all cases of neck trauma. CECT neck and thorax along with fibreoptic laryngoscopy remain the main modalities of diagnosis following a neck trauma. The initial approach to any kind of neck injury, whether penetrating or blunt, is performed according current Advanced Trauma Life Support (ATLS) or European Trauma Course (ETC) protocols, which both use the structured ‘ABCD’ approach. A motor vehicle accident (MVA) remains the most common cause of blunt neck injury, causing direct pressure to the anterior neck from the dashboard, steering wheel or airbag deployment. Direct pressure to the neck is transduced to the trachea and causes compression of the oesophagus against the cervical spine. Laryngotracheal trauma continues to be a rare entity and is the second most common cause of death in patients with head and neck trauma after intracranial injury. Only 0.5% of multiple trauma patients were reported to have injury to the airway at any level. Stabilize the airway first. Immediate surgical airway procedure can be necessary in less familiar circumstances and environments. If possible, define landmarks before the procedure. Defining anatomical zones is useful in penetrating injuries, although these do not guide diagnostic or therapeutic management completely. In unstable patients, elective surgical exploration is recommended instead of extensive diagnostic work-up. Unstable patients still need immediate exploration, whereas all stable patients will first be assessed with clinical examination and CT angiography and fibreoptic laryngoscopy. Thus the take home message is to consider all neck injuries an emergency and proceed with the diagnosis and management without delay.

P-396 Comparison of luteal support protocols in fresh IVF/ICSI cycles: A network meta-analysis

Abstract Study question What is the optimal LPS in fresh cycles in agonist and antagonist cycles? Summary answer Vaginal progesterone, with the addition of SCGnRH-a results in improved clinical pregnancy and live birth events in both GnRH-agonist and antagonist ovarian stimulation protocols. What is known already Despite the proven superiority of various Luteal Phase Support protocols (LPS) over placebo in view of improved pregnancy rates in fresh cycles of IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) cycles, there is ongoing controversy over specific LPS protocol selection, dosage, and duration. Study design, size, duration Network Meta-analysis. Twelve databases, namely Embase (OVID), MEDLINE (R) (OVID), GlobalHealth (Archive), GlobalHealth, Health and Psychosocial Instruments, Maternity & Infant Care Database (MIDIRS), APA PsycTests, ClinicalTrials.gov, HMIC Health Management Information Consortium, CENTRAL, Web of Science, Scopus and two prospective registers, MedRxiv, Research Square were searched from inception to Aug.1st, 2023.The study was prospectively registered under the PROSPERO database. Participants/materials, setting, methods Only RCTs were included. Primary outcomes included clinical pregnancy and live birth events, while secondary outcomes included biochemical pregnancy, miscarriage, multiple pregnancy and OHSS events. Bayesian network meta-analysis (NMA) model was employed for outcome analysis, presenting fixed effects, odds ratios (ORs) with 95% credibility intervals (CrIs). Vaginal Progesterone (VP) was considered the reference LPS for the purposes of the NMA given its’ clinical relevance. Main results and the role of chance Seventy-six RCTs, comparing 22 interventions, and including 26536 participants were included in the present NMA.Combinatorial regimens, with subcutaneous GnRH-a (SCGnRH-a) on a vaginal progesterone base and oral oestrogen (OE) appeared to overall improve clinical pregnancy events; VP+OE+SCGnRH-a [OR 1.57 (95% CrI 1.11 to 2.22)], VP+SCGnRH-a [OR 1.28 (95% CrI 1.05 to 1.55)] as well as live pregnancy events, VP+OE+SCGnRH-a [OR 8.81 (95% CrI 2.35 to 39.1)], VP+SCGnRH-a [OR 1.76 (95% CrI 1.45 to 2.15)]. Equally, the progesterone free LPS, intramuscular human chorionic gonadotrophin, [OR 9.67 (95%CrI 2.34, 73.2)] was also found to increase live birth events, however was also associated with an increased probability of ovarian hyperstimulation, [OR 1.64 (95%CrI 0.75, 3.71)]. Of all LPS protocols, VP+SC GnRH-a was found to significantly reduce miscarriage events, OR 0.54 (95% CrI 0.37 to 0.80). Subgroup analysis according to ovarian stimulation (OS) protocol revealed that the optimal LPS across both long and short OS, taking into account increase in live birth and reduction in miscarriage as well as OHSS events, was VP+SCGnRH-a, with an OR 2.89 [95%CrI 1.08, 2.96] and OR 2.84 [95%CrI 1.35, 6.26] respectively. Limitations, reasons for caution Given the anticipated diversity of measured outcomes, a Bayesian meta-synthesis approach has been adopted to account for the expected heterogeneity and to incorporate modelling flexibility by allowing for posterior distributions interpreted as SUCRA probabilities with the later enabling crisper communication of the uncertainty in the treatment effects estimates. Wider implications of the findings Overall, NMA data suggest that combinatorial treatments, with the addition of SCGnRH-a on a VP base result in improved clinical pregnancy and live birth events in both GnRH-agonist and antagonist ovarian stimulation protocols. Trial registration number Not applicable

P-585 Vaginal progesterone associated with oral dydrogesterone versus vaginal progesterone for luteal support in letrozole-hcg cycles for thawed embryo transfer: a non-inferiority prospective cohort study

Abstract Study question Does micronized vaginal progesterone (MVP) associated with oral dydrogesterone (DYG) an alternative for luteal support in Letrozole-hcg cycles for thawed embryo transfer? Summary answer The association of MVP and DYG is a non-inferior alternative to only MVP for luteal support in thawed embryo transfer in Letrozole-hcg cycles. What is known already Endometrial preparation is essential for thawed embryo transfer. Several protocols involving different luteal support with progesterones are available. Moreover, the presence of corpus luteum decreases the obstetrical morbidity mainly linked to hypertensive disorders. Letrozole-hcg administration is a good alternative for frozen embryo transfer (FET). However, the literature lacks information about the comparison among the different protocols for luteal support in Letrozole-hcg cycles. Dydrogesterone is an oral progesterone with some unique characteristics which could be utilized with some advantage for luteal support, including oral administration and some immunological activity. Study design, size, duration This is a prospective non-inferiority cohort study performed at an infertility clinic including patients performing the first FET cycle during 2023. Reproductive outcomes were compared between patients receiving 600 mg (3x200 mg) of MPV or 40 mg of DYG plus 400 mg of MVP after Letrozole-hcg administration. Participants/materials, setting, methods All patients (aged 23-39 years) undergoing their first FET cycle during 2023 were included in this study (n = 169), divided into two groups: MVP+DYG (n = 54) or MVP (n = 115) for luteal support after Letrozole-hcg. Letrozole was administered for 5 days (2,5 mg/bid) and hcg was done when a dominant follicle reached 18 mm with a follicular endometrium measuring at least 7 mm. Main results and the role of chance Both groups were equivalent in terms of age, infertility aetiology, BMI and AMH (P > 0.05). Moreover, the proportion of cleavage or blastocyst stage was also similar between both groups (57% blastocyst transfer for MVP+DYG and 50% blastocyst transfer for MVP group, P > 0.05). The number of transferred embryos was (mean ±SD) 1.6±0.5 for both groups, P > 0.05. Furthermore, the number of top-quality embryos (cleavage stage or blastocyst) was also similar, P > 0.05. Moreover, clinical pregnancy (> 12 weeks) was 42% in MVP+DYG group versus 31% in the MVP group (-0.22-1.27, P = 0.156) and the early pregnancy loss per positive hcg test did not differ (18% for MVP+DGY and 22% for MVP, P = 0.687). In addition, we performed a multivariable analysis controlling possible confounders for clinical pregnancy: BMI, age, AMH, embryo quality, infertility aetiology and embryo stage during the transfer, and only age (0.86; 95%CI -0.229 to -0.064, P = 0.001) was related to clinical pregnancy. Limitations, reasons for caution The main limitation is the cohort design of our study, with an inherent risk of bias (selection bias). Moreover, we calculated this study to achieve the power for a non-inferiority comparison, not superiority comparison. Wider implications of the findings This is the first study comparing MVP+DYG to only MVP in patients submitted to FET using the Letrozole-hcg protocol. We demonstrated in this non-inferiority study the usefulness of adding DYG. Moreover, our results were promising showing a trend towards better pregnancy rates after MVP+DYG. Trial registration number not applicable

P-418 What is the optimal midluteal phase circulating progesterone level during a fresh IVF cycle? The answer differs from what one expects in a natural cycle

Abstract Study question Do midluteal phase circulating progesterone (P4) levels relate to the fresh embryo transfer clinical outcome and is rescue P4 useful in such cases? Summary answer Live birth rate (LBR) was significantly lower when P4 levels were below 20ng/ml and did not seem to benefit from rescue intramuscular P4. What is known already Preceding literature on the predictive value of midluteal P4 for LBR has shown conflicting results and frequently included small sample sets and heterogeneous ovarian stimulation protocols. The findings reported ranged from the lack of an association between P4 serum levels and clinical outcome to worse success rates seen either in the groups with the highest or the lowest P4 in midluteal phase. Furthermore, while rescue P4 has been empirically proposed, it has lacked formal assessment thus far. Study design, size, duration In total, 652 patients who underwent a fresh single blastocyst transfer in an IVF cycle in our center between 2014 and 2021 were included in this retrospective cohort study. All were GnRH-antagonist cycles using hCG trigger and luteal support with micronized vaginal progesterone 200mg twice daily. Rescue intramuscular progesterone was systematically administered whenever low midluteal P4 (below 1 standard deviation of the center’s mean) levels were detected. Participants/materials, setting, methods The cycles were subdivided into three groups according to the midluteal P4 levels: <10ng/ml, 10-20ng/ml and >20ng/ml. The primary outcome assessed was LBR, with secondary outcomes including clinical pregnancy (CPR) and pregnancy loss rates. Maternal age and body mass index (BMI), year of treatment, ovarian response, endometrial thickness, cause of infertility and embryo quality were controlled for using multivariable logistic regression. Main results and the role of chance The mean age ± standard deviation of patients was 36.8±3.8 years, while mean weight and BMI were 63.2±11.5kg and 23.3±3.9kg/m2, respectively. LBR in the group with P4>20ng/ml (46.5%) was significantly higher than in the groups with low and intermediate P4 serum levels (23.1% and 28.1%, respectively, p < 0.001). The same was true for CPR (54.5%, 30.8% and 37.9%, respectively, p < 0.001). Conversely, the rate of pregnancy loss in the group with P4>20ng/ml (23.8%) was significantly lower than in the groups with low and intermediate P4 levels (42.6% and 37.5%, respectively, p = 0.018). The multivariable logistic regression confirmed that, after controlling for the confounding variables described above, P4>20ng/ml remained significantly associated with a higher LBR (OR: 2.285; 95% CI: 1.506-3.469) and a lower pregnancy loss rate (OR: 0.488; 95% CI: 0.273-0.873), irrespective of the use of rescue IM P4. Finally, a multivariable linear regression analysis of the patients baseline characteristics suggested that the only parameter predictive of midluteal P4 levels was the female patients’ weight, with an inverse relationship (adjusted regression coefficient: -0,012; 95% CI: -0,016/-0,009). Limitations, reasons for caution As the pituitary suppression, ovulation trigger and luteal support protocols used in the cycles included in the study were homogeneous (in order to maximize the internal validity of the results), this limits the extrapolibility of the results to other stimulation protocols. Furthermore, we caution that this is a retrospective study. Wider implications of the findings The usefulness of both midluteal P4 serum measurements and rescue P4 should continue to be assessed. The reasons behind the need for higher levels of P4 in a fresh cycle, when compared to natural cycles, must also be investigated. Luteal support may require personalization according to female body weight. Trial registration number Not Applicable

Enrichment of Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Strictly Anaerobic Sulfate-Reducing Cultures from Contaminated Soil and Sediment.

Sulfate-reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH-degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH-degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH-degrading SRB cultures. In this set of protocols, we describe step-by-step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate-reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow-growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography-mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium-throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH-degrading sulfate-reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Obtaining PAH-degrading strictly anaerobic sulfate-reducing enrichment cultures from contaminated soil and sediment Support Protocol 1: Operation and maintenance of an anaerobic workstation Support Protocol 2: Setup of gas purging systems for preparing anoxic solutions Support Protocol 3: Verification of viability in slow-growing SRB enrichment cultures Support Protocol 4: Extraction of genomic DNA from low-biomass cultures Basic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC-MS Basic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB cultures Basic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue method Alternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

Update: Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Injection of Naïve CD4+ T cells (T Cell Transfer Model of Colitis).

The intestinal inflammation induced by injection of naïve CD4+ T cells into lymphocyte-deficient hosts (more commonly known as the T cell transfer model of colitis) shares many features of idiopathic inflammatory bowel disease (IBD) in humans, such as epithelial cell hyperplasia, crypt abscess formation, and dense lamina propria lymphocyte infiltration. As such, it provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. In the IBD model described here, colitis is induced in Rag (recombination-activating gene)-deficient mice by reconstitution of these mice with naïve CD4+CD45RBhi T cells through adoptive T cell transfer. Although different recipient hosts of cell transfer can be used, Rag-deficient mice are the best characterized and support studies that are both flexible and reproduceable. As described in the Basic Protocol, in most studies the transferred cells consist of naïve CD4+ T cells (CD45RBhi T cells) derived by fluorescence-activated cell sorting from total CD4+ T cells previously purified using immunomagnetic negative selection beads. In a Support Protocol, methods to characterize colonic disease progression are described, including the monitoring of weight loss and diarrhea and the histological assessment of colon pathology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Induction of IBD in Rag-deficient mice by the transfer of naïve CD4+CD45RBhi T cells Support Protocol: Monitoring development of colitis.

Describing and Sharing Molecular Visualizations Using the MolViewSpec Toolkit.

With the ever-expanding toolkit of molecular viewers, the ability to visualize macromolecular structures has never been more accessible. Yet, the idiosyncratic technical intricacies across tools and the integration complexities associated with handling structure annotation data present significant barriers to seamless interoperability and steep learning curves for many users. The necessity for reproducible data visualizations is at the forefront of the current challenges. Recently, we introduced MolViewSpec (homepage: https://molstar.org/mol-view-spec/, GitHub project: https://github.com/molstar/mol-view-spec), a specification approach that defines molecular visualizations, decoupling them from the varying implementation details of different molecular viewers. Through the protocols presented herein, we demonstrate how to use MolViewSpec and its 3D view-building Python library for creating sophisticated, customized 3D views covering all standard molecular visualizations. MolViewSpec supports representations like cartoon and ball-and-stick with coloring, labeling, and applying complex transformations such as superposition to any macromolecular structure file in mmCIF, BinaryCIF, and PDB formats. These examples showcase progress towards reusability and interoperability of molecular 3D visualization in an era when handling molecular structures at scale is a timely and pressing matter in structural bioinformatics as well as research and education across the life sciences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Creating a MolViewSpec view using the MolViewSpec Python package Basic Protocol 2: Creating a MolViewSpec view with reference to MolViewSpec annotation files Basic Protocol 3: Creating a MolViewSpec view with labels and other advanced features Support Protocol 1: Computing rotation and translation vectors Support Protocol 2: Creating a MolViewSpec annotation file.

Orsay Virus Infection in Caenorhabditis elegans.

Orsay virus infection in the nematode Caenorhabditis elegans presents an opportunity to study host-virus interactions in an easily culturable, whole-animal host. Previously, a major limitation of C. elegans as a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen, C. elegans has emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability of C. elegans enables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection of C. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: Synchronize C. elegans development by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT-PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

Generation and Characterization of hiPSC-Derived Vascularized-, Perfusable Cardiac Microtissues-on-Chip.

In the heart in vivo, vasculature forms a semi-permeable endothelial barrier for selective nutrient and (immune) cell delivery to the myocardium and removal of waste products. Crosstalk between the vasculature and the heart cells regulates homeostasis in health and disease. To model heart development and disease in vitro it is important that essential features of this crosstalk are captured. Cardiac organoid and microtissue models often integrate endothelial cells (ECs) to form microvascular networks inside the 3D structure. However, in static culture without perfusion, these networks may fail to show essential functionality. Here, we describe a protocol to generate an in vitro model of human induced pluripotent stem cell (hiPSC)-derived vascularized cardiac microtissues on a microfluidic organ-on-chip platform (VMToC) in which the blood vessels are perfusable. First, prevascularized cardiac microtissues (MT) are formed by combining hiPSC-derived cardiomyocytes, ECs, and cardiac fibroblasts in a pre-defined ratio. Next, these prevascularized MTs are integrated in the chips in a fibrin hydrogel containing additional vascular cells, which self-organize into tubular structures. The MTs become vascularized through anastomosis between the pre-existing microvasculature in the MT and the external vascular network. The VMToCs are then ready for downstream structural and functional assays and basic characterization. Using this protocol, cardiac MTs can be efficiently and robustly vascularized and perfused within 7 days. In vitro vascularized organoid and MT models have the potential to transition current 3D cardiac models to more physiologically relevant organ models that allow the role of the endothelial barrier in drug and inflammatory response to be investigated. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Generation of VMToC Support Protocol 1: Functional Characterization of VMToC Support Protocol 2: Structural Characterization of VMToC.

Comparison of luteal support protocols in fresh IVF/ICSI cycles: a network meta-analysis

Despite the proven superiority of various luteal phase support protocols (LPS) over placebo in view of improved pregnancy rates in fresh cycles of IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) cycles, there is ongoing controversy over specific LPS protocol selection, dosage, and duration. The aim of the present study was to identify the optimal LPS under six core aspects of ART success, clinical pregnancy, live birth as primary outcomes and biochemical pregnancy, miscarriage, multiple pregnancy, ovarian hyperstimulation syndrome (OHSS) events as secondary outcomes. Twelve databases, namely Embase (OVID), MEDLINE (R) (OVID), GlobalHealth (Archive), GlobalHealth, Health and Psychosocial Instruments, Maternity & Infant Care Database (MIDIRS), APA PsycTests, ClinicalTrials.gov, HMIC Health Management Information Consortium, CENTRAL, Web of Science, Scopus and two prospective registers, MedRxiv, Research Square were searched from inception to Aug.1st, 2023, (PROSPERO Registration: CRD42022358986). Only Randomised Controlled Trials (RCTs) were included. Bayesian network meta-analysis (NMA) model was employed for outcome analysis, presenting fixed effects, odds ratios (ORs) with 95% credibility intervals (CrIs). Vaginal Progesterone (VP) was considered the reference LPS given its’ clinical relevance. Seventy-six RCTs, comparing 22 interventions, and including 26,536 participants were included in the present NMA. Overall CiNeMa risk of bias was deemed moderate, and network inconsistency per outcome was deemed low (Multiple pregnancy χ2: 0.11, OHSS χ2: 0.26), moderate (Clinical Pregnancy: χ2: 7.02, Live birth χ2: 10.95, Biochemical pregnancy: χ2: 6.60, Miscarriage: χ2: 11.305). Combinatorial regimens, with subcutaneous GnRH-a (SCGnRH-a) on a vaginal progesterone base and oral oestrogen (OE) appeared to overall improve clinical pregnancy events; VP + OE + SCGnRH-a [OR 1.57 (95% CrI 1.11 to 2.22)], VP + SCGnRH-a [OR 1.28 (95% CrI 1.05 to 1.55)] as well as live pregnancy events, VP + OE + SCGnRH-a [OR 8.81 (95% CrI 2.35 to 39.1)], VP + SCGnRH-a [OR 1.76 (95% CrI 1.45 to 2.15)]. Equally, the progesterone free LPS, intramuscular human chorionic gonadotrophin, [OR 9.67 (95% CrI 2.34, 73.2)] was also found to increase live birth events, however was also associated with an increased probability of ovarian hyperstimulation, [OR 1.64 (95% CrI 0.75, 3.71)]. The combination of intramuscular and vaginal progesterone was associated with higher multiple pregnancy events, [OR 7.09 (95% CrI 2.49, 31.)]. Of all LPS protocols, VP + SC GnRH-a was found to significantly reduce miscarriage events, OR 0.54 (95% CrI 0.37 to 0.80). Subgroup analysis according to ovarian stimulation (OS) protocol revealed that the optimal LPS across both long and short OS, taking into account increase in live birth and reduction in miscarriage as well as OHSS events, was VP + SCGnRH-a, with an OR 2.89 [95% CrI 1.08, 2.96] and OR 2.84 [95% CrI 1.35, 6.26] respectively. Overall, NMA data suggest that combinatorial treatments, with the addition of SCGnRH-a on a VP base result in improved clinical pregnancy and live birth events in both GnRH-agonist and antagonist ovarian stimulation protocols.

Improving the nutritional care of oncology patients - Validation of a multidisciplinary protocol in the Spanish clinical setting

malnutrition is a very frequent problem in oncology patients and can have serious repercussions. Adequate nutritional management is cost-effective in terms of health and survival in this population, but it requires multidisciplinary coordination, specific training, and continuous follow-up. to validate the applicability and efficacy of a multidisciplinary nutritional support protocol in oncology patients. a multidisciplinary nutritional protocol was developed for oncology patients, with guidelines for screening and assessment of malnutrition, treatment, re-evaluation, and management of side effects, as well as guidance on supplementation and eating patterns. The protocol would be implemented in various clinical centers, collecting data through a structured questionnaire, registering variables before and after implementation. the protocol and its impact were implemented and evaluated in 39 centers. An improvement in nutritional care was observed, evidenced by an earlier initiation of nutritional assessment and an increase in the number of patients receiving adequate care following the protocol implementation. Problems related to inadequate malnutrition coding in the centers, limited resources, and the need for greater interdepartmental collaboration were identified. the conduct of this study provides insights into how the implementation of a multidisciplinary nutritional support protocol can improve the nutritional care received by patients and informs about the main obstacles to adequate implementation.

Knowledge, Attidue and Practice of Advanced Trauma Life Suppoort (ATLS) Protocol among House-Officers in Khartoum State Hospitals, Sudan, 2023

BackgroundAdvanced Trauma Life Support was originated mainly to train doctors who don’t manage trauma on a regular basis, including junior doctors as it prepares them more efficiently and effectively for treating and managing trauma patients. This study was conducted to study knowledge, attitude and practice of advanced trauma life support protocol among house-officers in Khartoum state hospitals, Sudan 2023.MethodsThis is a cross-sectional descriptive health facility based study conducted in Bahri Teaching Hospital, Omdurman Teaching Hospital and Ibrahim Malik teaching hospital, Khartoum state, Sudan. Data of 151 House-officers of all nationality working in Khartoum state hospitals was collected using a simple random technique, filling questionnaire that was designed especially for this study. Comparison between different variables by Chi-square test and statistical significance difference at P value < 0.05 was done.ResultA total of 151 house officers were included in the study. 49% aged between 20 and 25 years, females were the majority 56.3%. About 41.1% have took ATLS course before. 55.21% of the study participants didn’t take the course because it was not available, while 35.42% because it was expensive and 29.17% referred it to their busy lifestyle. 91% of the study population think that ATLS course should be compulsory and 85% think that the ATLS protocol should be recommended to both junior and senior doctors. 77% of the study participants stated that their seniors teaching skills affect how they apply ATLS.ConclusionOverall junior doctors at Khartoum state hospitals demonstrated a positive attitude towards ATLS, but they showed poor knowledge regarding the topic. It’s advised that an earlier training program is introduced by incorporating ATLS course to be part of all final year medical school’s curriculum.

Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Using Fluorescent GAP Indicators to Monitor ER Ca2+

AbstractThe endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP‐Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+‐binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5‐8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescenceBasic Protocol 2: Monitoring ER Ca2+Basic Protocol 3: Monitoring ER‐ and cytosolic‐Ca2+Support Protocol: Generation of a stable cell line expressing erGAP3

Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System.

U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.

Optimization of Fluorescence In Situ Hybridization Protocols in the Era of Precision Medicine.

Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.

Preparation and Compositional Analysis of Lignocellulosic Plant Biomass as a Precursor for Food Production During Food Crises.

In the event of a sunlight-blocking, temperature-lowering global catastrophe, such as a global nuclear war, super-volcano eruption or large asteroid strike, normal agricultural practices would be severely disrupted with a devastating impact on the global food supply. Despite the improbability of such an occurrence, it is prudent to consider how to sustain the surviving population following a global catastrophe until normal weather and climate patterns resume. Additionally, the ongoing challenges posed by climate change, droughts, flooding, soil salinization, and famine highlight the importance of developing food systems with resilient inputs such as lignocellulosic biomass. With its high proportion of cellulose, the abundant lignocellulosic biomass found across the Earth's land surfaces could be a source of energy and nutrition, but it would first need to be converted into foods. To understand the potential of lignocellulosic biomass to provide energy and nutrition to humans in post-catastrophic and other food crisis scenarios, compositional analyses should be completed to gauge the amount of energy (soluble sugars) and other macronutrients (protein and lipids) that might be available and the level of difficulty in extracting them. Suitable preparation of the lignocellulosic biomass is critical to achieve consistent and comparable results from these analyses. Here we describe a compilation of protocols to prepare lignocellulosic biomass and analyze its composition to understand its potential as a precursor to produce post-catastrophic foods which are those that could be foraged, grown, or produced under the new climate conditions to supplement reduced availability of traditional foods. These foods have sometimes been referred to in the literature as emergency, alternate, or resilient foods. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Convection oven drying (1 to 2 days) Alternate Protocol 1: Air-drying (2 to 3 days) Alternate Protocol 2: Lyophilization (1 to 4 days) Support Protocol 1: Milling plant biomass Support Protocol 2: Measuring moisture content Basic Protocol 2: Cellulose determination Basic Protocol 3: Lignin determination Basic Protocol 4: Crude protein content by total nitrogen Basic Protocol 5: Crude fat determination via soxtec extraction system Basic Protocol 6: Sugars by HPLC Basic Protocol 7: Ash content.

Ascaris Mouse Model Protocols: Advancing Research on Larval Ascariasis Biology.

Ascariasis, caused by both Ascaris lumbricoides and Ascaris suum, is the most prevalent parasitic disease worldwide, affecting both human and porcine populations. However, due to the difficulties of assessing the early events of infection in humans, most studies of human ascariasis have been restricted to the chronic intestinal phase. Therefore, the Ascaris mouse model has become a fundamental tool for investigating the immunobiology and pathogenesis of the early infection stage referred to as larval ascariasis because of the model's practicality and ability to replicate the natural processes involved. The Ascaris mouse model has been widely used to explore factors such as infection resistance/susceptibility, liver inflammation, lung immune-mediated pathology, and co-infections and, notably, as a pivotal element in preclinical vaccine trials. Exploring the immunobiology of larval ascariasis may offer new insights into disease development and provide a substantial understanding of key components that trigger a protective immune response. This article focuses on creating a comprehensive guide for conducting Ascaris experimental infections in the laboratory as a foundation for future research efforts. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Acquisition and embryonation of Ascaris suum eggs from adult females Alternate Protocol: Cleaning and purification of Ascaris suum from female A. suum uteri Basic Protocol 2: Preparation of Ascaris suum eggs and murine infection Basic Protocol 3: Measurement of larval burden and Ascaris-larva-induced pathogenesis Basic Protocol 4: In vitro hatching and purification of Ascaris L3 larvae Support Protocol: Preparation of crude antigen from Ascaris infectious stages Basic Protocol 5: Ultrastructure-expansion microscopy (U-ExM) of Ascaris suum larval stages.