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Bacterial microcompartments are prokaryotic organelles comprising encapsulated enzymes within a thin protein shell. They facilitate metabolic processing including propanediol, choline, glycerol, and ethanolamine utilization, and they accelerate carbon fixation in cyanobacteria. Enzymes targeted to the inside of the microcompartment frequently possess a cargo-encapsulation peptide, but the site to which the peptide binds is unclear. We provide evidence that the encapsulation peptides bind to the hydrophobic groove formed between tessellating subunits of the shell proteins. In silico docking studies provide a compelling model of peptide binding to this prominent hydrophobic groove. This result is consistent with the now widely accepted view that the convex side of the shell oligomers faces the lumen of the microcompartment. The binding of the encapsulation peptide to the groove between tessellating shell protein tiles explains why it has been difficult to define the peptide binding site using other methods, provides a mechanism by which encapsulation-peptide bearing enzymes can promote shell assembly, and explains how the presence of cargo affects the size and shape of the bacterial microcompartment. This knowledge may be exploited in engineering microcompartments or disease prevention by hampering cargo encapsulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Internal medicine (IM) residency programs select applicants based on several metrics. Factors predicting success during residency are unclear across studies. To identify whether specific applicant or resident factors are associated with IM resident performance using ACGME milestones. We tested for associations between applicant factors available prior to the start of IM residency and resident factors measured during IM residency training, and resident performance on ACGME milestones across three consecutive years of IM training between 2015-2020. Univariable and multivariable linear regression modeling was used to test associations. Eighty-nine categorical IM residents that completed 3 consecutive years of training were included. Median age was 28 years (IQR 27-29) and 59.6% were male. Mean ACGME milestone scores increased with each post-graduate year (PGY) from 3.36 (SD 0.19) for PGY-1, to 3.80 (SD 0.15) for PGY-2, to 4.14 (SD 0.15) for PGY-3. Univariable modeling suggested referral to the clinical competency committee (CCC) for professionalism concerns was negatively associated with resident performance during each PGY. No applicant or resident factors included in the final multivariable regression models (age at starting residency, USMLE Step scores, interview score, rank list position, ITE scores) were associated with ACGME milestone scores for PGY-1 and PGY-2. Referral to the CCC for professionalism was negatively associated with resident performance during PGY-3. Residency selection factors did not predict resident milestone evaluation scores. Referral to the CCC was associated with significantly worse resident evaluation scores, suggesting professionalism may correlate with clinical performance.

Introduction: Evidence-based ventilation strategies for infants with severe bronchopulmonary dysplasia (BPD) remain unknown. Determining whether contemporary ventilation approaches cluster as specific BPD strategies may better characterize care and enhance the design of clinical trials. The objective of this study was to test the hypothesis that unsupervised, multifactorial clustering analysis of point prevalence ventilator setting data would classify a discrete number of physiology-based approaches to mechanical ventilation in a multicenter cohort of infants with severe BPD., Methods: We performed a secondary analysis of a multicenter point prevalence study of infants with severe BPD treated with invasive mechanical ventilation. We clustered the cohort by mean airway pressure (MAP), positive end expiratory pressure (PEEP), set respiratory rate, and inspiratory time (Ti) using Ward's hierarchical clustering analysis (HCA)., Results: Seventy-eight patients with severe BPD were included from 14 centers. HCA classified three discrete clusters as determined by an agglomerative coefficient of 0.97. Cluster stability was relatively strong as determined by Jaccard coefficient means of 0.79, 0.85, and 0.77 for clusters 1, 2, and 3, respectively. The median PEEP, MAP, rate, Ti, and PIP differed significantly between clusters for each comparison by Kruskall-Wallis testing (p < 0.0001)., Conclusions: In this study, unsupervised clustering analysis of ventilator setting data identified three discrete approaches to mechanical ventilation in a multicenter cohort of infants with severe BPD. Prospective trials are needed to determine whether these approaches to mechanical ventilation are associated with specific severe BPD clinical phenotypes and differentially modify respiratory outcomes., (© 2023 Wiley Periodicals LLC.)

Objective: To evaluate use of a standardized, 3-tiered, seizure burden-based protocol for treatment of all electroencephalography (EEG)-confirmed seizures in a level IV neonatal intensive care unit (NICU)., Study Design: All infants admitted to the NICU with EEG-confirmed seizures over a 25-month period were enrolled in the study. We compared short-term outcomes before and after implementation of a standardized, 3-tiered protocol., Results: A total of 107 infants were enrolled in the study. Use of midazolam infusions was reduced by 53.7% ( p   = 0.02). Midazolam infusion duration increased from 4 to 7.5 days ( p = 0.003); however, when excluding 3 outliers, there was no significant difference between groups (- p   = 0.67). Duration of EEG monitoring decreased from 5 to 3 days ( p   = 0.005). Hospital length of stay was unchanged., Conclusion: Implementation of a standardized, 3-tiered protocol for treatment of neonatal seizures improved short-term outcomes. Although not measured directly, reductions in EEG duration and midazolam use are promising indicators of overall seizure burden. More research is needed to evaluate impact on long-term neurodevelopmental outcomes.

Despite the improving understanding of how lung mechanics and tidal volume requirements evolve during the evolution of bronchopulmonary dysplasia (BPD), clinical management continues to be heterogeneous and inconsistent at many institutions. Recent reports have examined the use of high tidal-volume low respiratory rate strategies in these patients once disease has been well established to help facilitate their eventual extubation and improve their long-term neurodevelopmental outcomes. In this retrospective observational research study, we describe how intentional adjustment of ventilator settings based on patient lung mechanics by an interdisciplinary BPD team improved the care of the at-risk population of infants, reduced the need for tracheostomies, as well as length of stay over a period of over 3 years. The team aimed to establish consistency in the management of these children using a high tidal volume, low-rate approach, and titrating PEEP to address the autoPEEP and bronchomalacia that is frequently observed in this patient population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Sammour, Conlon, Bauer, Montgomery, Cristea and Rose.)

Background: Bronchopulmonary dysplasia (BPD) remains the most common late morbidity of preterm birth. Clinical care and research have largely focused on the pathogenesis and prevention of BPD. Preterm infants who develop BPD have significant medical needs that persist throughout their hospital course and continue after discharge, including those associated with growth and nutrition. The objective of this study was to review the available literature on nutrition and growth in infants with established BPD and to identify the knowledge and research gaps to provide direction for future studies., Methods: We conducted a literature search in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines using Ovid MEDLINE, CINAHL, and Embase. Titles, abstracts, and full texts were independently reviewed by the authors and selected based on predetermined inclusion/exclusion criteria. Results were summarized qualitatively., Results: Excluding duplicates, 2315 articles were identified. Thirty articles were selected for inclusion. We identified the following key components of nutrition support and clinical care: energy expenditure, growth, and metabolism; body composition; enteral nutrition; supplements; parenteral nutrition; and respiratory outcomes., Conclusions: Despite a large body of literature describing the role of growth and nutrition in the prevention of BPD, research is lacking with respect to interventions and management in the population with established BPD. Thus, organized approaches for clinical interventions and trials with respect to growth and nutrition in infants and young children with established BPD are needed. These studies should include multiple centers because of the small numbers of patients with BPD at each site., (© 2022 American Society for Parenteral and Enteral Nutrition.)

Objective: To measure the effects of a quality improvement intervention on length of stay and benzodiazepine use among patients admitted for alcohol use disorder., Methods: This retrospective cohort study was performed at the Salt Lake City Veterans Affairs Medical Center. Patients 18 years and older admitted to a general medical ward with a diagnosis of alcohol related disorders who were treated for alcohol withdrawal were included. The baseline cohort included patients admitted over 12 months. The post-intervention cohort included patients admitted over 12 months. Primary outcomes were total benzodiazepine dose and length of stay. Secondary outcomes included episodes of delirium tremens and seizures., Results: Total benzodiazepine dose decreased significantly over the intervention period. Length of stay also decreased. No episodes of delirium tremens or seizures were observed., Conclusions: A quality improvement intervention directed at general medicine inpatients admitted for alcohol withdrawal was associated with reductions in total benzodiazepine administration and length of stay.

We performed a point prevalence study on infants with severe bronchopulmonary dysplasia (BPD), collecting data on type and settings of ventilatory support; 187 infants, 51% of whom were on invasive positive-pressure ventilation (IPPV), from 15 centers were included. We found a significant center-specific variation in ventilator modes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: To compare three bronchopulmonary dysplasia (BPD) definitions against hospital outcomes in a referral-based population., Study Design: Data from the Children's Hospitals Neonatal Consortium were classified by 2018 NICHD, 2019 NRN, and Canadian Neonatal Network (CNN) BPD definitions. Multivariable models evaluated the associations between BPD severity and death, tracheostomy, or length of stay, relative to No BPD references., Results: Mortality was highest in 2019 NRN Grade 3 infants (aOR 225), followed by 2018 NICHD Grade 3 (aOR 145). Infants with lower BPD grades rarely died (<1%), but Grade 2 infants had aOR 7-21-fold higher for death and 23-56-fold higher for tracheostomy., Conclusions: Definitions with 3 BPD grades had better discrimination and Grade 3 2019 NRN had the strongest association with outcomes. No/Grade 1 infants rarely had severe outcomes, but Grade 2 infants were at risk. These data may be useful for counseling families and determining therapies for infants with BPD., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Premature birth affects millions of neonates each year, placing them at risk for respiratory disease due to prematurity. Bronchopulmonary dysplasia is the most common chronic lung disease of infancy, but recent data suggest that even premature infants who do not meet the strict definition of bronchopulmonary dysplasia can develop adverse pulmonary outcomes later in life. This post-prematurity respiratory disease (PPRD) manifests as chronic respiratory symptoms, including cough, recurrent wheezing, exercise limitation, and reduced pulmonary function. This document provides an evidence-based clinical practice guideline on the outpatient management of infants, children, and adolescents with PPRD. Methods: A multidisciplinary panel of experts posed questions regarding the outpatient management of PPRD. We conducted a systematic review of the relevant literature. The Grading of Recommendations, Assessment, Development, and Evaluation approach was used to rate the quality of evidence and the strength of the clinical recommendations. Results: The panel members considered the strength of each recommendation and evaluated the benefits and risks of applying the intervention. In formulating the recommendations, the panel considered patient and caregiver values, the cost of care, and feasibility. Recommendations were developed for or against three common medical therapies and four diagnostic evaluations in the context of the outpatient management of PPRD. Conclusions: The panel developed recommendations for the outpatient management of patients with PPRD on the basis of limited evidence and expert opinion. Important areas for future research were identified.

Background: Non-obstructive azoospermia (NOA) with history of cryptorchidism and idiopathic NOA are the most common forms of NOA without genetic aetiology. Of all patients with one of these two types of NOA, only a few will have a positive TEsticular Sperm Extraction (TESE). Of those with positive extraction followed by sperm freezing, not all will have a child after TESE-ICSI. What are the ways and probabilities of taking home a baby for patients with NOA and a history of cryptorchidism compared with patients with idiopathic NOA?, Results: Patients with idiopathic NOA or NOA and a history of cryptorchidism who underwent their first TESE were included. The patients were divided into two groups: Group 1 was composed of 125 patients with idiopathic NOA and Group 2 of 55 patients with NOA and a history of surgically treated cryptorchidism. Our results showed that more than half of the NOA patients succeeded in becoming parents. The main way to fulfil their plans for parenthood is to use sperm or embryo donation (72%) for men with idiopathic NOA, whereas the majority of men with NOA and a history of cryptorchidism had a child after TESE-ICSI (58.8%)., Conclusions: In our centre, before considering TESE for a patient with NOA, we explain systematically TESE-ICSI alternatives (sperm donation, embryo donation or adoption). As a result, the couple can consider each solution to become parents., (© 2021. The Author(s).)

Objective: To evaluate the association between the time of first systemic corticosteroid initiation and bronchopulmonary dysplasia (BPD) in preterm infants., Study Design: A multi-center retrospective cohort study from January 2010 to December 2016 using the Children's Hospitals Neonatal Database and Pediatric Health Information System database was conducted. The study population included preterm infants <32 weeks' gestation treated with systemic corticosteroids after 7 days of age and before 34 weeks' postmenstrual age. Stepwise multivariable logistic regression was used to assess the association between timing of corticosteroid initiation and the development of Grade 2 or 3 BPD as defined by the 2019 Neonatal Research Network criteria., Results: We identified 598 corticosteroid-treated infants (median gestational age 25 weeks, median birth weight 760 g). Of these, 47% (280 of 598) were first treated at 8-21 days, 25% (148 of 598) were first treated at 22-35 days, 14% (86 of 598) were first treated at 36-49 days, and 14% (84 of 598) were first treated at >50 days. Infants first treated at 36-49 days (aOR 2.0, 95% CI 1.1-3.7) and >50 days (aOR 1.9, 95% CI 1.04-3.3) had higher independent odds of developing Grade 2 or 3 BPD when compared to infants treated at 8-21 days after adjusting for birth characteristics, admission characteristics, center, and co-morbidities., Conclusions: Among preterm infants treated with systemic corticosteroids in routine clinical practice, later initiation of treatment was associated with a higher likelihood to develop Grade 2 or 3 BPD when compared to earlier treatment., (© 2021 Wiley Periodicals LLC.)

The design and assembly of peptide-based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a "disulfide pin" between two elements of secondary structure that drive self-assembly. Specifically, homodimerizing and homotrimerizing de novo coiled-coil α-helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required., (© 2021 The Authors. Small published by Wiley-VCH GmbH.)

The environmental accumulation of plastics worldwide is a consequence of the durability of the material. Alternative polymers, marketed as biodegradable, present a potential solution to mitigate their ecological damage. However, understanding of biodegradability has been hindered by a lack of reproducible testing methods. We developed a novel method to evaluate the biodegradability of plastic samples based on the monitoring of bacterial respiration in aqueous media via the quantification of CO 2 produced, where the only carbon source available is from the polymer. Rhodococcus rhodochrous and Alcanivorax borkumensis were used as model organisms for soil and marine systems, respectively. Our results demonstrate that this approach is reproducible and can be used with a variety of plastics, allowing comparison of the relative biodegradability of the different materials. In the case of low-density polyethylene, the study demonstrated a clear correlation between the molecular weight of the sample and CO 2 released, taken as a measure of biodegradability., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Infants born between 34 weeks 0 days and 36 weeks 6 days of gestation are termed late preterm. This group accounts for the majority of premature births in the United States, with rates increasing in each of the last 3 years. This increase is significant given their large number: nearly 280,000 in 2016 alone. Late preterm infants place a significant burden on the health care and education systems because of their increased risk of morbidities and mortality compared with more mature infants. This increased risk persists past the newborn period, leading to the need for continued health monitoring throughout life., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cellular prion protein (PrP C ) can act as a cell-surface receptor for β-amyloid (Aβ) peptide; however, a role for PrP C in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aβ isoforms and PrP C in the Drosophila brain. We found that co-expression of Aβ and PrP C significantly reduces the lifespan, disrupts circadian rhythms, and increases Aβ deposition in the fly brain. In contrast, under the same conditions, expression of Aβ or PrP C individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrP C trap Aβ as oligomeric assemblies and fragment-preformed Aβ fibers. The ability of membrane-anchored PrP C to trap Aβ as cytotoxic oligomers at the membrane surface and fragment inert Aβ fibers suggests a mechanism by which PrP C exacerbates Aβ deposition and pathogenic phenotypes in the fly, supporting a role for PrP C in AD. This study provides a second animal model linking PrP C expression with Aβ toxicity and supports a role for PrP C in AD pathogenesis. Blocking the interaction of Aβ and PrP C represents a potential therapeutic strategy., (© 2018 Younan et al.)

Objective To determine the impact of Centering Pregnancy © -based group prenatal care for Hispanic gravid diabetics on pregnancy outcomes and postpartum follow-up care compared to those receiving traditional prenatal care. Methods A cohort study was performed including 460 women diagnosed with gestational diabetes mellitus (GDM) who received traditional or Centering Pregnancy © prenatal care. The primary outcome measured was completion of postpartum glucose tolerance testing. Secondary outcomes included postpartum visit attendance, birth outcomes, breastfeeding, and initiation of a family planning method. Results 203 women received Centering Pregnancy © group prenatal care and 257 received traditional individual prenatal care. Women receiving Centering Pregnancy © prenatal care were more likely to complete postpartum glucose tolerance testing than those receiving traditional prenatal care, (83.6 vs. 60.7 %, respectively; p < 0.001), had a higher rate of breastfeeding initiation (91.0 vs. 69.4 %; p < 0.001), had higher rates of strictly breastfeeding at their postpartum visit (63.1 vs. 46.3 %; p = 0.04), were less likely to need medical drug therapy compared to traditional prenatal care (30.2 vs. 42.1 %; p = 0.009), and were less likely to undergo inductions of labor (34.5 vs. 46.2 %; p = 0.014). When only Hispanic women were compared, women in the Centering group continued to have higher rates of breastfeeding and completion of postpartum diabetes screening. Conclusion for Practice Hispanic women with GDM who participate in Centering Pregnancy © group prenatal care may have improved outcomes.

We aimed to decrease practice variation in treatment of neonatal status epilepticus by implementing a standardized protocol. Our primary goal was to achieve 80% adherence to the algorithm within 12 months. Secondary outcome measures included serum phenobarbital concentrations, number of patients progressing from seizures to status epilepticus, and length of hospital stay. Data collection occurred for 6 months prior and 12 months following protocol implementation. Adherence of 80% within 12 months was partially achieved in patients diagnosed in our hospital; in pretreated patients, adherence was not achieved. Maximum phenobarbital concentrations were decreased (56.8 vs 41.0 µg/mL), fewer patients progressed from seizures to status epilepticus (46% vs 36%), and hospital length of stay decreased by 9.7 days in survivors. In conclusion, standardized, protocol-driven treatment of neonatal status epilepticus improves consistency and short-term outcome., (© The Author(s) 2016.)

Target-specific oral anticoagulants have been rapidly adopted into clinical practice for stroke prophylaxis and venous thromboembolism treatment, raising concerns about off-label prescribing practices. We conducted a retrospective review of consecutive patients prescribed dabigatran, rivaroxaban or apixaban prior to inpatient hospitalization over an 18-month period to examine the off-label prescribing frequency, contraindications and related complications. Chart review included baseline demographics, hospital admitting service, outpatient prescribing service, renal function, therapeutic indication, echocardiographic findings, contraindications, major bleeding events and vital status. We identified 160 patients who received a target-specific oral anticoagulant prior to hospitalization. Over half (53.1%) of the patients received rivaroxaban, 43.7% received dabigatran and 3.1% received apixaban. Atrial fibrillation (68.1%) and venous thromboembolism treatment (25.6%) were the most common indications. Ninety percent of patients had a U.S. Foods and Drugs Administration (FDA)-approved indication for therapy. Major bleeding events occurred in 4.4% of patients. Cardiology was the most common prescribing and admitting service (43.8 and 31.3%), and more frequently adhered to FDA-approved indications (97 vs. 84%, P = 0.01). There were no significant differences between prescribing services regarding major contraindications (P = 0.14) and major bleeding events (P = 0.77). Off-label prescription rates for target-specific oral anticoagulants were infrequent and not associated with increased adverse events.

The post-translational modifiers ubiquitin and small ubiquitin-related modifier (SUMO) regulate numerous critical signaling pathways and are key to controlling the cellular fate of proteins in eukaryotes. The attachment of ubiquitin and SUMO involves distinct, but related, machinery. However, it is now apparent that many substrates can be modified by both ubiquitin and SUMO and that some regulatory interaction takes place between the respective attachment machinery. Here, we demonstrate that the Saccharomyces cerevisiae ubiquitin ligase Rsp5p, a member of the highly conserved Nedd4 family of ubiquitin ligases, is SUMOylated in vivo. We further show that Rsp5p SUMOylation is mediated by the SUMO ligases Siz1p and Siz2p, members of the conserved family of PIAS SUMO ligases that are, in turn, substrates for Rsp5p-mediated ubiquitylation. Our experiments show that SUMOylated Rsp5p has reduced ubiquitin ligase activity, and similarly, ubiquitylated Siz1p demonstrates reduced SUMO ligase activity leading to respective changes in both ubiquitin-mediated sorting of the manganese transporter Smf1p and polySUMO chain formation. This reciprocal regulation of these highly conserved ligases represents an exciting and previously unidentified system of cross talk between the ubiquitin and SUMO systems.

Type I signal peptidases (SPases) cleave signal peptides from proteins during translocation across biological membranes and hence play a vital role in cellular physiology. SPase activity is also of fundamental importance to the pathogenesis of infection for many bacteria, including Pseudomonas aeruginosa, which utilizes a variety of secreted virulence factors, such as proteases and toxins. P. aeruginosa possesses two noncontiguous SPase homologues, LepB (PA0768) and PA1303, which share 43% amino acid identity. Reverse transcription (RT)-PCR showed that both proteases were expressed, while a FRET-based assay using a peptide based on the signal sequence cleavage region of the secreted LasB elastase showed that recombinant LepB and PA1303 enzymes were both active. LepB is positioned within a genetic locus that resembles the locus containing the extensively characterized SPase of E. coli and is of similar size and topology. It was also shown to be essential for viability and to have high sequence identity with SPases from other pseudomonads (≥ 78%). In contrast, PA1303, which is small for a Gram-negative SPase (20 kDa), was found to be dispensable. Mutation of PA1303 resulted in an altered protein secretion profile and increased N-butanoyl homoserine lactone production and influenced several quorum-sensing-controlled phenotypic traits, including swarming motility and the production of rhamnolipid and elastinolytic activity. The data indicate different cellular roles for these P. aeruginosa SPase paralogues; the role of PA1303 is integrated with the quorum-sensing cascade and includes the suppression of virulence factor secretion and virulence-associated phenotypes, while LepB is the primary SPase.

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.

Epidemiological studies have shown an association between statin use and a decreased risk of dementia. However, the mechanism by which this beneficial effect is brought about is unclear. In the context of Alzheimer's disease, at least three possibilities have been studied; reduction in amyloid beta peptide (Abeta) production, the promotion of alpha-secretase cleavage and positive effects on neurite outgrowth. By investigating the effects of mevalonate pathway blockade on neurite outgrowth using real-time imaging, we found that rather than promote the production of neurite extensions, inhibition rapidly induced cell rounding. Crucially, neurite-like structures were generated through the persistence of cell-cell and cell-substrate adhesions and not through a mechanism of positive outgrowth. This effect can be strikingly enhanced by the over-expression of human amyloid precursor protein and is isoprenoid rather than cholesterol dependent.

Placement of the distal interlocking screw is the most difficult part in all intramedullary nail interlocking screw systems and the Surgical Implant Generation Network (SIGN) interlocking system is not an exception. SIGN nails are interlocking implants designed with a precision instrumentation set for use in treatment of long bone fractures without an image intensifier. Locating the distal slots of SIGN nails could be challenging for young SIGN surgeons when treating very complex comminuted fractures and in obese patients. This study was stimulated by a patient who presented one year after surgery with knee pain due to a migrating nail because of missed distal screws. A total of 48 patients divided into two groups of 24 were studied retrospectively and prospectively. The retrospective studies revealed that failure to locate distal locking slots in ten antegrade nailing procedures was due to wrong entry point and comminution of the fracture. The challenges encountered led us to innovating methods to overcome the difficulties of placement of distal screws in a prospective study. Application of methods A and B made location of the distal slots easier in the prospective study even though there were more complex comminuted fractures. The methods also reduced the antegrade operation time by 1 hour 11 minutes. We concluded that SIGN nailing could be challenging and frustrating at the early learning stage. Application of the two innovative methods will make distal slot location easier. They will also make SIGN interlocking nailing less difficult for young SIGN surgeons as they journey through the learning curves.

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

In Bacillus megaterium, the synthesis of vitamin B(12) (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD(+) as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 A (1 A = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.