Helical remodeling augments 5-lipoxygenase activity in the synthesis of proinflammatory mediators.

The synthesis of proinflammatory leukotrienes implicated in asthma, allergic rhinitis, and atherosclerosis is initiated by the enzyme 5-lipoxygenase (5-LOX). The crystal structure of human Stable-5-LOX revealed a conformation where the catalytic iron was inaccessible to bulk solvent as two aromatic residues on a conserved helix-α2 (Hα2) plugged the substrate access portal. Whether 5-LOX can also adopt a more open conformation has not been resolved. Here, we present a new conformation of 5-LOX where Hα2 adopts an elongated conformation equivalent to that described in other animal lipoxygenase structures. Our observation of the sigmoidal kinetic behavior of 5-LOX, which is indicative of positive cooperativity, is consistent with a substrate-induced conformational change that shifts the ensemble of enzyme populations to favor the catalytically competent state. Strategic point mutations along Hα2 designed to unlock the closed conformation and elongate Hα2 resulted in improved kinetic parameters, altered limited proteolysis data, and a drastic reduction in the length of the lag phase yielding the most active Stable-5-LOX to date. Structural predictions by AlphaFold2 of these variants statistically favor an elongated Hα2 and reinforce a model in which improved kinetic parameters correlate with a more readily adopted open conformation. Taken together, these data provide valuable insights into the synthesis of leukotrienes.

Electronic Health Record Recording of Patient Pain: Challenges and Discrepancies.

PURPOSE OF REVIEW: In the present review, various categories of pain, clinician-observed pain scales, and patient-reported pain scales are evaluated to better understand factors that impact patient pain perceptions. Additionally, the expansion of areas that require further research to determine the optimal way to evaluate pain scale data for treatment and management are discussed. RECENT FINDINGS: Electronic health record (EHR) data provides a starting point for evaluating whether patient predictors influence postoperative pain. There are several ways to assess pain and choosing the most effective form of pain treatment. Identifying individuals at high risk for severe postoperative pain enables more effective pain treatment. However, there are discrepancies in patient pain reporting dependent on instruments used to measure pain and their storage in the EHR. Additionally, whether administered by a physician or another healthcare practitioner, differences in patient pain perception occur. While each scale has distinct advantages and limitations, pain scale data is a valuable therapeutic tool for assisting clinicians in providing patients with optimal pain control. Accurate assessment of patient pain perceptions by data extraction from electronic health records provides a potential for pain alleviation improvement. Predicting high-risk postoperative pain syndromes is a difficult clinical challenge. Numerous studies have been conducted on factors that impact pain prediction. Postoperative pain is significantly predicted by the kind of operation, the existence of prior discomfort, patient anxiety, and age.

Does the use of double hormone replacement therapy for trauma patient organ donors improve organ recovery for transplant.

BACKGROUND: With an ongoing demand for transplantable organs, optimization of donor management protocols, specifically in trauma populations, is important for obtaining a high yield of viable organs per patient. Endocrine management of brain-dead potential organ donors (BPODs) is controversial, leading to heterogeneous clinical management approaches. Previous studies have shown that when levothyroxine was combined with other treatments, including steroids, vasopressin, and insulin, BPODs had better organ recovery and survival outcomes were increased for transplant recipients. AIM: To determine if levothyroxine use in combination with steroids in BPODs increased the number of organs donated in trauma patients. METHODS: A retrospective review of adult BPODs from a single level 1 trauma center over ten years was performed. Exclusion criteria included patients who were not solid organ donors, patients who were not declared brain dead (donation after circulatory death), and patients who did not receive steroids in their hospital course. Levothyroxine and steroid administration, the number of organs donated, the types of organs donated, and demographic information were recorded. Univariate analyses were performed with P < 0.05 considered to be statistically significant. RESULTS: A total of 88 patients met inclusion criteria, 69 (78%) of whom received levothyroxine and steroids (ST/LT group) vs 19 (22%) receiving steroids without levothyroxine (ST group). No differences were observed between the groups for gender, race, pertinent injury factors, age, or other hormone therapies used (P > 0.05). In the ST/LT group, 68.1% (n = 47) donated a high yield (3-5) of organ types per donor compared to 42.1% (n = 8) in the ST group (P = 0.038). There was no difference in the total number of organ types donated between the groups (P = 0.068). CONCLUSION: This study suggests that combining levothyroxine and steroid administration increases high-yield organ donation per donor in BPODs in the trauma patient population. Limitations to this study include the retrospective design and the relatively small number of organ donors who met inclusion criteria. This study is unique in that it mitigates steroid administration as a confounding variable and focuses specifically on the adjunctive use of levothyroxine.

Traumatic Abdominal Wall Hernia: Management Using a Standardized Approach.

Traumatic abdominal wall hernias are a rare complication of high energy blunt trauma. There exist several studies evaluating and outlining potential management options but still no generalized consensus on management. This series was meant to evaluate the diagnosis and management of traumatic abdominal wall hernias. A prospectively maintained database was used to identify patients with TAWH from 2021 to 2022. The primary outcome was operative management. Secondary outcomes included: time to diagnosis and post-operative outcomes. Of the 19 patients in this case series, 100% (n = 19/19) were secondary to blunt trauma with a mean ISS of 21. Exploratory laparotomy was performed in 17 cases. 14 cases had concomitant traumatic injuries to visceral structures. Complications were found in nearly half of the patients with 3 experiencing wound dehiscence. Future studies should be aimed at standardizing management approach taking into account nature of the mechanism and concomitant injuries.

Quantitative Analysis of Gene Expression in RNAscope-processed Brain Tissue.

Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope. Graphical abstract.