Discovery, structure, and function of filamentous 3-methylcrotonyl-CoA carboxylase.

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent mitochondrial enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments, and determined the structures of different filament regions at 3.4, 3.9, and 7.3 Å resolution using cryoEM. α6ß6 LtMCCs assemble in a twisted-stacks architecture, manifesting as supramolecular rods up to 400 nm. Filamentous LtMCCs bind biotin non-covalently and lack coenzyme A. Filaments elongate by stacking α6ß6 LtMCCs onto the exterior α-trimer of the terminal LtMCC. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the function of polymerization in the carboxylase superfamily and beyond.

CryoEM reveals oligomeric isomers of a multienzyme complex and assembly mechanics.

Propionyl-CoA carboxylase (PCC) is a multienzyme complex consisting of up to six α-subunits and six ß-subunits. Belonging to a metabolic pathway converging on the citric acid cycle, it is present in most forms of life and irregularities in its assembly lead to serious illness in humans, known as propionic acidemia. Here, we report the cryogenic electron microscopy (cryoEM) structures and assembly of different oligomeric isomers of endogenous PCC from the parasitic protozoan Leishmania tarentolae (LtPCC). These structures and their statistical distribution reveal the mechanics of PCC assembly and disassembly at equilibrium. We show that, in solution, endogenous LtPCC ß-subunits form stable homohexamers, to which different numbers of α-subunits attach. Sorting LtPCC particles into seven classes (i.e., oligomeric formulae α0ß6, α1ß6, α2ß6, α3ß6, α4ß6, α5ß6, α6ß6) enables formulation of a model for PCC assembly. Our results suggest how multimerization regulates PCC enzymatic activity and showcase the utility of cryoEM in revealing the statistical mechanics of reaction pathways.

Structural basis of gRNA stabilization and mRNA recognition in trypanosomal RNA editing.

In Trypanosoma brucei, the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)-programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo-electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA-binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome's catalytic modality.

Social determinants of impaired functioning among Nepali widows: A mixed methods study.

Despite considerable stigmatisation of widows in Nepal, little is known about factors affecting their ability to function in society. Using mixed methods, we studied psychosocial factors associated with impaired functioning among Nepali widows. For the qualitative analysis, we analysed 3 focus groups, 25 in-depth interviews, and 12 key-informant interviews. The quantitative analysis was based on data from 204 widows. Odds ratios were calculated linking psychosocial exposures to impaired functioning using adjusted logistic regression models. Low social support (aOR = 2.35, 95% CI: 1.2, 4.6, generally; aOR = 3.3, 95% CI: 1.7, 6.42, specifically from family members), experiences of discrimination (aOR = 2.97, 95% CI: 1.43, 6.14), and low life control (aOR = 4.3, 95% CI: 1.86, 10.1) were risk factors for impaired functioning. Qualitative findings suggested how discrimination and lack of control contribute to impaired functioning. Support from the husband's family appeared to be more important to a widow's functioning, compared to from her own parents. Knowledge about risk factors for impaired functioning can help inform interventions for Nepali widows.

Traumatic abdominal wall hernias: a single-center case series of surgical management.

BACKGROUND: Traumatic abdominal wall hernias (TAWHs) are a rare clinical entity that can be difficult to diagnose and manage. There is no consensus on management of TAWH due to its low incidence and complex concomitant injury patterns. We hereby present the largest single-center case series in the USA to characterize associated injury patterns, identify optimal strategies for hernia management, and determine outcomes. METHODS: Patients who presented with a TAWH from blunt trauma requiring operative management were retrospectively identified over a 14-year period. Demographic data, Injury Severity Score (ISS), associated injuries, type of repair, durability of repair, and complications were collected, and descriptive statistics were calculated. RESULTS: Fifteen patients were identified. The average age was 31±11 years, ISS 15±9, and body mass index 33.4±7.1 kg/m2. Mechanisms included falls (13%), motor vehicle collisions (60%), motorcycle accidents (20%), and pedestrian versus motor vehicle collisions (7%). The most commonly associated injuries included colonic injuries (53%), long bone fractures (47%), pelvic fractures (40%), and small bowel injuries (33%). Nineteen hernia repairs were performed: 6 underwent primary suture repair (32%) and 13 used mesh (68%). There were four recurrences. We could not find any significant relationship between contamination and mesh use or recurrence. There was one mortality related to sepsis. DISCUSSION: TAWHs have an associated injury pattern involving fractures and abdominopelvic visceral injuries where a tailored approach is advisable. Without hollow viscous injuries and gross contamination, these hernias can be repaired safely with mesh in the acute setting. However, in patients with gross contamination or hemodynamic instability, the risk of recurrence with primary repair must be weighed against the risk of infection and prolonged surgery with mesh repair. In those cases, a delayed reconstruction in the elective setting may be optimal.

Management of blunt force bladder injuries: A practice management guideline from the Eastern Association for the Surgery of Trauma.

BACKGROUND: The diagnostic evaluation and clinical management of bladder injuries caused by blunt force trauma are variable. We aim to formulate a practice management guideline using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology. METHODS: The working group, patient, intervention, comparator, outcome (PICO), formulated four questions regarding the following topics: (1) diagnostic evaluation based on patient baseline risk of bladder injury (computed tomography cystography vs. no imaging); (2) management of intraperitoneal bladder injuries (operative versus nonoperative); (3) management of extraperitoneal bladder injuries based on complexity of injury (operative vs. nonoperative); and (4) diagnostic follow-up of bladder injuries based on complexity of repair (cystography vs. no cystography). A systematic review of the MEDLINE database for English language articles with adult patients was undertaken. RevMan 5 (Review Manager (RevMan) [Computer program]. Version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) and GRADEpro (GRADEpro GDT: GRADEpro Guideline Development Tool [Software]. McMaster University, 2015) software were used. Recommendations were voted on by working group members. Consensus was obtained for each recommendation. RESULTS: Three hundred ninety-three articles were screened, resulting in a full-text review of 64 articles. Seventeen articles were used to formulate the recommendations of this guideline. Several recommendations are made. The need for initial computed tomography cystography after trauma depends on characteristics of the trauma itself, but it is not recommended in patients without gross hematuria. In general, patients with intraperitoneal bladder ruptures should undergo operative repair. This is not routinely necessary in those with extraperitoneal ruptures unless the injury is complex. The need for follow-up cystography after bladder repair depends on the risk of urine leak. Those with low risk of urine leak do not require a follow-up study. CONCLUSION: Using the GRADE process, the panel made nine recommendations based on four PICO questions concerning the evaluation and management of blunt force bladder injuries.

Outcomes in patients with gunshot wounds to the brain.

INTRODUCTION: Gunshot wounds to the brain (GSWB) confer high lethality and uncertain recovery. It is unclear which patients benefit from aggressive resuscitation, and furthermore whether patients with GSWB undergoing cardiopulmonary resuscitation (CPR) have potential for survival or organ donation. Therefore, we sought to determine the rates of survival and organ donation, as well as identify factors associated with both outcomes in patients with GSWB undergoing CPR. METHODS: We performed a retrospective, multicenter study at 25 US trauma centers including dates between June 1, 2011 and December 31, 2017. Patients were included if they suffered isolated GSWB and required CPR at a referring hospital, in the field, or in the trauma resuscitation room. Patients were excluded for significant torso or extremity injuries, or if pregnant. Binomial regression models were used to determine predictors of survival/organ donation. RESULTS: 825 patients met study criteria; the majority were male (87.6%) with a mean age of 36.5 years. Most (67%) underwent CPR in the field and 2.1% (n=17) survived to discharge. Of the non-survivors, 17.5% (n=141) were considered eligible donors, with a donation rate of 58.9% (n=83) in this group. Regression models found several predictors of survival. Hormone replacement was predictive of both survival and organ donation. CONCLUSION: We found that GSWB requiring CPR during trauma resuscitation was associated with a 2.1% survival rate and overall organ donation rate of 10.3%. Several factors appear to be favorably associated with survival, although predictions are uncertain due to the low number of survivors in this patient population. Hormone replacement was predictive of both survival and organ donation. These results are a starting point for determining appropriate treatment algorithms for this devastating clinical condition. LEVEL OF EVIDENCE: Level II.

Thyroidectomy remains an effective treatment option for Graves' disease.

BACKGROUND: Recent experience with thyroidectomy for Graves' disease is limited. We report our current experience with thyroidectomy for Graves' disease at a tertiary hospital. METHODS: A prospective database showed 48 patients who underwent surgery for Graves' disease from April 1993 to June 2005. RESULTS: All patients had typical symptoms of Graves' disease. Twenty-three patients had ophthalmopathy. Indications for surgery were failed medical therapy (n = 24), presence of a dominant nodule (n = 12), or refusal of radioiodine (n = 12). Surgery included total thyroidectomy (n = 46) or subtotal thyroidectomy (n = 2). The incidence of cancer was 17%. Long-term follow-up data were available for 44 patients. No patients had recurrence of hyperthyroidism or cancer. Follow-up evaluation of 20 patients with ophthalmopathy showed the condition had either stabilized or resolved. CONCLUSIONS: Total thyroidectomy for Graves' disease offers rapid and durable control of hyperthyroidism, provides appropriate treatment for patients with coexisting cancer, and can stabilize or reverse ophthalmopathy.

Intraoperative PTH monitoring in parathyroid hyperplasia requires stricter criteria for success.

BACKGROUND: Intraoperative parathyroid hormone (IOPTH) monitoring in parathyroidectomy for multigland disease is less clear than for single-gland disease. This study assesses the role of IOPTH for hyperplasia. METHODS: A prospective database revealed 45 patients with hyperplasia undergoing parathyroidectomy utilizing IOPTH from February 1999 to August 2003. RESULTS: Twenty-six females and 19 males had a mean age of 55 years. Twenty-two patients underwent total parathyroidectomy. Twenty-three patients underwent subtotal parathyroidectomy. Twenty-seven patients (60%) had a drop of IOPTH greater than 50% at 10 minutes after removal of all presumably abnormal parathyroid tissue. Nine additional patients (20%) had an IOPTH drop greater than 50%, but continued exploration revealed more abnormal tissue. Nine patients failed to decrease greater than 50%, and exploration was continued. A final IOPTH less than 35 pg/mL or a greater than 90% decrease from baseline was predictive of a successful operation in 40 patients. The 5 patients who did not meet this criteria remained hyperparathyroid. CONCLUSIONS: IOPTH identifies sporadic hyperplasia and guides completeness of resection for patients with known hyperplasia. However, more rigid criteria are required than for adenomas. Failure to achieve appropriate decreases in IOPTH should prompt further neck exploration or a search for a mediastinal gland.

Optical tissue window: a novel model for optimizing engraftment of intestinal stem cell organoids.

BACKGROUND: Intestinal malabsorption disorders and short bowel syndrome lead to significant morbidity. We recently demonstrated that grafting of intestinal organoids can grow a bioengineered intestinal neomucosa and cure bile acid malabsorption in rats. Now we have developed a novel system that permits direct observation of intestinal organoids in vivo to optimize conditions for engraftment. METHODS: Optical Windows were created in C57BL/6J mice by externalizing an omental pedicle into a dorsal skin flap chamber. Following creation of windows, 5000 intestinal organoids from green-fluorescent protein transgene (GFP)+ donor mice were seeded directly either on omentum or on polyglycolic acid (PGA) disks that had been placed on omentum at 1 or 5 days. Engraftment of green fluorescent cells was evaluated on postseeding days 1, 3, 5, 7, 10, 12, and 21 using fluorescence microscopy. RESULTS: An initial group had seeding onto omentum (n = 5) or biopolymer disks (n = 5) on postoperative day 1. After 7 days, there was mucosal cell engraftment onto omental tissue and biopolymers. GFP+ organoids engrafted significantly better when seeded onto biopolymers compared to omentum (P < 0.05). In a second study with increased sample size (n = 24) up to day 12, all four groups demonstrated adherence and growth. However, GFP+ organoids seeded onto delayed PGA biopolymer demonstrated significantly better engraftment (P < 0.05). CONCLUSIONS: This novel system allows continuous in vivo observation of engrafted cells that are seeded on externalized omentum. The use of PGA mesh biopolymer may improve engraftment of intestinal organoids.