Search

Acetabular wedge augmentation is a useful technique and is recommended in situations where cup revision is likely to be excessively difficult. Numerous augmentation designs that improve the stability of the hip are now available in the market. Occasionally, however, these devices are not readily available, particularly if problems such as instability and dislocation are not anticipated preoperatively. We report our experience in treating such a case, using an augment cut from a second cup with a follow-up for 30 months. Though there is no substitute for proper preoperative planning, awareness of this procedure is helpful as unexpected problems do arise in revision acetabular surgery.

Metal-on-metal hip resurfacing arthroplasty is increasingly popular for younger patients with advanced hip disease. Intra-operative or immediate postoperative femoral neck fracture after metal-on-metal hip resurfacing is a well-described technical complication, ranging from 0% to 1.5%. We report 2 cases of late femoral neck fracture occurring 8 and 15 months following the index operation, with a review of the literature. We recommend that patient selection should be of prime importance before embarking on metal-on-metal surface hip replacement to avoid such complications.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.

Breast cancer-related lymphedema (BCRL) is characterized by skin changes, swelling, fibrosis, and recurrent skin infections. Clinical studies have suggested that lymphedema results in skin barrier defects; however, the underlying cellular mechanisms and the effects of bacterial contamination on skin barrier function remain unknown. In matched biopsies from patients with unilateral BCRL, we observed decreased expression of FLG and the tight junction protein ZO-1 in skin affected by moderate lymphedema or by subclinical lymphedema in which dermal backflow of lymph was identified by indocyanine green lymphography, relative to those in the controls (areas without backflow and from the unaffected arm). In vitro stimulation of keratinocytes with lymph fluid obtained from patients undergoing lymphedema surgery led to the same changes as well as increased expression of keratin 14, a marker of immature keratinocytes. Finally, using mouse models of lymphedema, we showed that similar to the clinical scenario, the expression of skin barrier proteins was decreased relative to that in normal skin and that colonization with Staphylococcus epidermidis bacteria amplified this effect as well as lymphedema severity. Taken together, our findings suggest that lymphatic fluid stasis contributes to skin barrier dysfunction in lymphedema., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Obesity and internalising disorders, including depression and anxiety, often co-occur. There is evidence that familial confounding contributes to the co-occurrence of internalising disorders and obesity in adults. However, its impact on this association among young people is unclear. Our study investigated the extent to which familial factors confound the association between internalising disorders and obesity in adolescents and young adults., Subjects/methods: We used a matched co-twin design to investigate the impact of confounding by familial factors on associations between internalising symptoms and obesity in a sample of 4018 twins aged 16 to 27 years., Results: High levels of internalising symptoms compared to low levels increased the odds of obesity for the whole cohort (adjusted odds ratio [AOR] = 3.1, 95% confidence interval [CI]: 1.5, 6.8), and in females (AOR = 4.1, 95% CI 1.5, 11.1), but not in males (AOR = 2.8 95% CI 0.8, 10.0). We found evidence that internalising symptoms were associated with an increased between-pair odds of obesity (AOR 6.2, 95% CI 1.7, 22.8), using the paired analysis but not using a within-pair association, which controls for familial confounding. Sex-stratified analyses indicated high internalising symptoms were associated with increased between-pair odds of obesity for females (AOR 12.9, 95% CI 2.2, 76.8), but this attenuated to the null using within-pair analysis. We found no evidence of between or within-pair associations for males and weak evidence that sex modified the association between internalising symptoms and obesity (likelihood ratio test p = 0.051)., Conclusions: Some familial factors shared by twins confound the association between internalising symptoms and obesity in adolescent and young adult females. Internalising symptoms and obesity were not associated for adolescent and young adult males. Therefore, prevention and treatment efforts should especially address familial shared determinants of obesity, particularly targeted at female adolescents and young adults with internalising symptoms and those with a family history of these disorders., (© 2024. The Author(s).)

Young breast and bowel cancers (e.g., those diagnosed before age 40 or 50 years) have far greater morbidity and mortality in terms of years of life lost, and are increasing in incidence, but have been less studied. For breast and bowel cancers, the familial relative risks, and therefore the familial variances in age-specific log(incidence), are much greater at younger ages, but little of these familial variances has been explained. Studies of families and twins can address questions not easily answered by studies of unrelated individuals alone. We describe existing and emerging family and twin data that can provide special opportunities for discovery. We present designs and statistical analyses, including novel ideas such as the VALID (Variance in Age-specific Log Incidence Decomposition) model for causes of variation in risk, the DEPTH (DEPendency of association on the number of Top Hits) and other approaches to analyse genome-wide association study data, and the within-pair, ICE FALCON (Inference about Causation from Examining FAmiliaL CONfounding) and ICE CRISTAL (Inference about Causation from Examining Changes in Regression coefficients and Innovative STatistical AnaLysis) approaches to causation and familial confounding. Example applications to breast and colorectal cancer are presented. Motivated by the availability of the resources of the Breast and Colon Cancer Family Registries, we also present some ideas for future studies that could be applied to, and compared with, cancers diagnosed at older ages and address the challenges posed by young breast and bowel cancers., (© 2024 The Authors. Genetic Epidemiology published by Wiley Periodicals LLC.)

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), a receptor tyrosine kinase encoded by the FLT4 gene, plays a significant role in the morphogenesis and maintenance of lymphatic vessels. Under both normal and pathologic conditions, VEGF-C and VEGF-D bind VEGFR3 on the surface of lymphatic endothelial cells (LECs) and induce lymphatic proliferation, migration, and survival by activating intracellular PI3K-Akt and MAPK-ERK signaling pathways. Impaired lymphatic function and VEGFR3 signaling has been linked with a myriad of commonly encountered clinical conditions. This review provides a brief overview of intracellular VEGFR3 signaling in LECs and explores examples of dysregulated VEGFR3 signaling in various disease states, including (1) lymphedema, (2) tumor growth and metastasis, (3) obesity and metabolic syndrome, (4) organ transplant rejection, and (5) autoimmune disorders. A more complete understanding of the molecular mechanisms underlying the lymphatic pathology of each disease will allow for the development of novel strategies to treat these chronic and often debilitating illnesses.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC 50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

Introduction: Socially excluded populations, defined by homelessness, substance use disorder, sex work or criminal justice system contact, experience profound health inequity compared with the general population. Cumulative exposure to adverse childhood experiences (ACEs), including neglect, abuse and household dysfunction before age 18, has been found to be independently associated with both an increased risk of social exclusion and adverse health and mortality outcomes in adulthood.Despite this, the impact of ACEs on health and mortality within socially excluded populations is poorly understood., Methods and Analysis: We will search MEDLINE, Cumulative Index of Nursing and Allied Health Literature, Educational Resources Information Center, PsycINFO, Applied Social Science Index and Abstracts and Criminal Justice Database for peer-reviewed studies measuring ACEs and their impact on health and mortality in socially excluded populations.Three review questions will guide our data extraction and analysis. First, what is the prevalence of ACEs among people experiencing social exclusion in included studies? Second, what is the relationship between ACEs and health and mortality outcomes among people experiencing social exclusion? Does resilience modify the strength of association between ACEs and health outcomes among people experiencing social exclusion?We will meta-analyse the relationship between ACE exposure and health outcomes classified into six a prior categories: (1) substance use disorders; (2) sexual and reproductive health; (3) communicable diseases; (4) mental illness; (5) non-communicable diseases and (6) violence victimisation, perpetration and injury. If there are insufficient studies for meta-analysis, we will conduct a narrative synthesis. Study quality will be assessed using the MethodologicAl STandards for Epidemiological Research scale., Ethics and Dissemination: Our findings will be disseminated in a peer-reviewed journal, in presentations at academic conferences and in a brief report for policy makers and service providers. We do not require ethics approval as this review will use data that have been previously published., Prospero Registration Number: CRD42022357565., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Issues: Despite long-standing recommendations to integrate mental health care and alcohol and other drug (AOD) treatment, no prior study has synthesised evidence on the impact of physically co-locating these specialist services on health outcomes., Approach: We searched Medline, PsycINFO, Embase, Web of Science and CINAHL for studies examining health outcomes associated with co-located outpatient mental health care and AOD specialist treatment for adults with a dual diagnosis of substance use disorder and mental illness. Due to diversity in study designs, patient populations and outcome measures among the included studies, we conducted a narrative synthesis. Risk of bias was assessed using the MASTER scale., Key Findings: Twenty-eight studies met our inclusion criteria. We found provisional evidence that integrated care that includes co-located mental health care and AOD specialist treatment is associated with reductions in substance use and related harms and mental health symptom severity, improved quality of life, decreased emergency department presentations/hospital admissions and reduced health system expenditure. Many studies had a relatively high risk of bias and it was not possible to disaggregate the independent effect of physical co-location from other common aspects of integrated care models such as care coordination and the integration of service processes., Implications: There are few high-quality, peer-reviewed studies establishing the impact of co-located mental health care and AOD specialist treatment on health outcomes. Further research is required to inform policy, guide implementation and optimise practice., Conclusion: Integrated care that includes the co-location of mental health care and AOD specialist treatment may yield health and economic benefits., (© 2023 The Authors. Drug and Alcohol Review published by John Wiley & Sons Australia, Ltd on behalf of Australasian Professional Society on Alcohol and other Drugs.)

Purpose of Review: This review aims to summarize the current knowledge regarding the pharmacological interventions studied in both experimental and clinical trials for secondary lymphedema., Recent Findings: Lymphedema is a progressive disease that results in tissue swelling, pain, and functional disability. The most common cause of secondary lymphedema in developed countries is an iatrogenic injury to the lymphatic system during cancer treatment. Despite its high incidence and severe sequelae, lymphedema is usually treated with palliative options such as compression and physical therapy. However, recent studies on the pathophysiology of lymphedema have explored pharmacological treatments in preclinical and early phase clinical trials., Summary: Many potential treatment options for lymphedema have been explored throughout the past two decades including systemic agents and topical approaches to decrease the potential toxicity of systemic treatment. Treatment strategies including lymphangiogenic factors, anti-inflammatory agents, and anti-fibrotic therapies may be used independently or in conjunction with surgical approaches., Competing Interests: Conflict of InterestBabak J. Mehrara, MD, is the recipient of investigator-initiated research grants from PureTech and Regeneron and has received royalty payments from PureTech; he also has served as a consultant for Pfizer Corp. Joseph H. Dayan, MD, is a paid consultant for the Stryker Corporation, has intellectual property rights with Elucida Oncology and equity interest in Welwaze Medical, LLC, and has a royalty agreement with Springer Publishers for Multimodal Management of Upper and Lower Extremity Lymphedema. All other authors report no potential conflicts of interest., (© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Lymphedema is a chronic condition that commonly occur from lymphatic injury following surgical resection of solid malignancies. While many studies have centered on the molecular and immune pathways that perpetuate lymphatic dysfunction, the role of the skin microbiome in lymphedema development remains unclear. In this study, skin swabs collected from normal and lymphedema forearms of 30 patients with unilateral upper extremity lymphedema were analyzed by 16S ribosomal RNA sequencing. Statistical models for microbiome data were utilized to correlate clinical variables with microbial profiles. Overall, 872 bacterial taxa were identified. There were no significant differences in microbial alpha diversity of the colonizing bacteria between normal and lymphedema skin samples (p = 0.25). Notably, for patients without a history of infection, a one-fold change in relative limb volume was significantly associated with a 0.58-unit increase in Bray-Curtis microbial distance between paired limbs (95%CI = 0.11,1.05, p = 0.02). Additionally, several genera, including Propionibacterium and Streptococcus, demonstrated high variability between paired samples. In summary, we demonstrate high compositional heterogeneity in the skin microbiome in upper extremity secondary lymphedema, supporting future studies into the role of host-microbe interactions on lymphedema pathophysiology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Campbell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

β2-adrenoreceptor (β2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of β2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD., (© 2022. The Author(s).)

Background: There is a range of magneto-inertial measurement unit (MIMU) systems commercially available, however sensor specifications and fusion methods vary considerably between manufacturers. Such variability can influence the concurrent validity of MIMUs relative to reference standard measurement devices. Different MIMUs have been compared during static or low-velocity conditions, with higher-velocity movements assessed in robotic-based studies. However, there is a need for the concurrent validity of higher-velocity movements to be established in human-based studies., Research Question: This study aimed to assess the concurrent validity of two commercial MIMU systems (Noraxon and Xsens), relative to a 'gold-standard' retro-reflective motion capture system, when measuring trunk angles during uni-planar range of motion (ROM) and cricket bowling, which involves high-speed, multi-planar movements., Methods: For this criterion-based validity study, both MIMU systems incorporated comparable sensor specifications and employed Kalman filter sensor fusion algorithms. The MIMU based angles were compared with angles derived from concurrently captured three-dimensional retro-reflective data for 10 fast-medium bowlers. Statistical parametric mapping and root mean squared differences (RMSD) were computed for both MIMU systems., Results: One-dimensional statistical parametric mapping showed no significant differences for angles from both MIMU systems when compared with retro-reflective based angle outputs. The MIMU systems produced ROM RMSDs between 1.4 ± 1.0° and 2.6 ± 1.5°. One system displayed RMSDs between 4.6 ± 1.4° and 7.4 ± 1.9° during bowling, indicating functionally relevant differences to retro-reflective derived angles. There were some small but statistically significant differences in RMSDs between the MIMU systems., Significance: MIMU-based angle accuracy is poorer during high-speed, multi-planar movement than uni-planar tasks. Comparable MIMU systems can produce varying measurements during ROM and bowling tasks. It is likely that varying sample rates and sensor fusion algorithm parameters contributed to the differences., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Proline dehydrogenase (PRODH) is a flavoenzyme that catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ 1 -pyrroline-5-carboxylate. PRODH has emerged as a cancer therapy target because of its involvement in the metabolic reprogramming of cancer cells. Here, we report the discovery of a new class of PRODH inactivator, which covalently and irreversibly modifies the FAD in a light-dependent manner. Two examples, 1,3-dithiolane-2-carboxylate and tetrahydrothiophene-2-carboxylate, have been characterized using X-ray crystallography (1.52-1.85 Å resolution), absorbance spectroscopy, and enzyme kinetics. The structures reveal that in the dark, these compounds function as classical reversible, proline analogue inhibitors. However, exposure of enzyme-inhibitor cocrystals to bright white light induces decarboxylation of the inhibitor and covalent attachment of the residual S-heterocycle to the FAD N5 atom, locking the cofactor into a reduced, inactive state. Spectroscopic measurements of the inactivation process in solution confirm the requirement for light and show that blue light is preferred. Enzyme activity assays show that the rate of inactivation is enhanced by light and that the inactivation is irreversible. We also demonstrate the photosensitivity of cancer cells to one of these compounds. A possible mechanism is proposed involving photoexcitation of the FAD, while the inhibitor is noncovalently bound in the active site, followed by electron transfer, decarboxylation, and radical combination steps. Our results could lead to the development of photopharmacological drugs targeting PRODH.

Magneto-inertial measurement unit (MIMU) systems allow calculation of simple sensor-to-sensor Euler angles, though this process does not address sensor-to-segment alignment, which is important for deriving meaningful MIMU-based kinematics. Functional sensor-to-segment calibrations have improved concurrent validity for elbow and knee angle measurements but have not yet been comprehensively investigated for trunk or sport-specific movements. This study aimed to determine the influence of MIMU functional calibration on thorax and lumbar joint angles during uni-planar and multi-planar, sport-specific tasks. It was hypothesised that functionally calibrating segment axes prior to angle decomposition would produce smaller differences than a non-functional method when both approaches were compared with concurrently collected 3D retro-reflective derived angles. Movements of 10 fast-medium cricket bowlers were simultaneously recorded by MIMUs and retro-reflective motion capture. Joint angles derived from four different segment definitions were compared, with three incorporating functionally defined axes. Statistical parametric mapping and root mean squared differences (RMSD) quantified measurement differences one-dimensionally and zero-dimensionally, respectively. Statistical parametric mapping found no significant differences between MIMU and retro-reflective data for any method across bowling and uni-planar trunk movements. The RMSDs for the functionally calibrated methods and non-functional method were not significantly different. Functional segment calibration may be unnecessary for MIMU-based measurement of thorax and lumbar joint angles., (© 2021. Crown.)

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using spatially-separated proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that l-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: d-proline, trans-4-hydroxy-d-proline, cis-4-hydroxy-d-proline, l-proline, and trans-4-hydroxy-l-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD + or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone., (Copyright © 2020 Elsevier Inc. All rights reserved.)

OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material., (© 2013-2021 The Journal of Biological Methods, All rights reserved.)

The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus . Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-l-ornithine and NADP + . Crystal structures were determined for M101A in the resting state and complexed with NADP + . The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A-NADP + complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP + in preparation for a new catalytic cycle.

Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD + -dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition. Two crystal structures of ALDH9A1 are reported, including the first of the enzyme complexed with NAD + . One of the structures reveals the active conformation of the enzyme, in which the Rossmann dinucleotide-binding domain is fully ordered and the inter-domain linker adopts the canonical β-hairpin observed in other ALDH structures. The oligomeric structure of ALDH9A1 was investigated using analytical ultracentrifugation, small-angle X-ray scattering, and negative stain electron microscopy. These data show that ALDH9A1 forms the classic ALDH superfamily dimer-of-dimers tetramer in solution. Our results suggest that the presence of an aldehyde substrate and NAD + promotes isomerization of the enzyme into the active conformation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N -monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1-'C2' and N10-C1' bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Campbell et al.)

Allicin is a component of the characteristic smell and flavor of garlic ( Allium sativum ). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S -allyl-l-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with l-cysteine, N -acetyl-l-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly ( K D of ∼2 μm) to AsFMO and that the hydride transfer occurs with pro- R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP + , reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the k cat The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the k cat The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Valentino et al.)

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ 1 -pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analogue thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabolic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling l-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a substrate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Objectives: In 2012, British Columbia (BC) became the first Canadian province to implement an influenza prevention policy requiring healthcare workers (HCW) to either be vaccinated annually against influenza or wear a mask in patient care areas during the influenza season. This study describes an evaluation of influenza policy implementation processes and identifies supports and challenges related to successful policy implementation at the level of healthcare facilities, during the second policy year (2013/14)., Methods: Implementation leaders from 262 long-term care (LTC) and acute care facilities, mostly in three of BC's five regional Health Authorities, were invited to participate in an online survey following the 2013/14 influenza season. Descriptive quantitative and qualitative analyses identified common and effective strategies for improving vaccination coverage and policy compliance., Results: A total of 127 respondents completed the survey on behalf of 33 acute care and 99 LTC facilities, representing 36% of acute care and 27% of LTC facilities in BC. Respondents agreed that the policy was successfully implemented at 89% of facilities, and implementation was reported to be easy at 52% of facilities. The findings elaborate on communication and leadership strategies, campaign logistics and enforcement approaches involved in policy implementation., Conclusion: Implementation of a vaccinate-or-mask influenza policy is complex. This study provides insight for other jurisdictions considering implementing such a policy and offers practical recommendations for facilities and health authorities., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2-HAH was shown to be monomeric in solution and contained a non-covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox-neutral, 2-HAH was active only in the reduced state. A covalent flavin-substrate intermediate, consistent with the flavin-acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small-angle X-ray scattering was consistent with 2-HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2-HAH as a novel noncanonical flavoenzyme., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Young children (ages 3 to 5) are using mobile touchscreen technology, including tablet computers, yet little is known on the potential musculoskeletal and physical activity implications of its use. This within-subject laboratory study (n = 10) examined head, trunk and arm postures, upper trapezius muscle activity, and total body and upper limb physical activity during playing with tablets compared to during TV watching and playing with non-screen toys. Overall, this study found that during tablet play children had greater mean head, trunk and upper arm angles compared to both TV watching and toy play. Conversely, compared to toy play, children playing with tablets had lesser trunk, upper arm and elbow postural variation, lesser trapezius activity, more time sitting and lesser physical activity. Thus, to minimize potential musculoskeletal and sedentary risks, non-screen toy play should be encouraged and education and guidelines provided for parents and caretakers to support wise use of tablets., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ 1 -pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Context: Eccentric exercises are increasingly being used to treat lower-limb musculoskeletal conditions such as Achilles tendinopathy. Despite widespread clinical application and documented efficacy, mechanisms underpinning clinical benefit remain unclear. Positive adaptations in motor performance are a potential mechanism., Objective: To investigate how an eccentric loading intervention influences measures of stretch-shortening-cycle (SSC) behavior during a hopping task., Design: Within-subjects repeated-measures observational study., Setting: University motion-analysis laboratory., Participants: Healthy adults., Interventions: A single intervention of 5 sets of 10 eccentric plantar-flexion contractions at 6 repetitions maximum using a commercial seated calf-raise machine., Main Outcome Measures: Lower-limb stiffness, sagittal-plane ankle kinematics, and temporal muscle activity of the agonist (soleus) and antagonist (tibialis anterior) muscles, measured during submaximal hopping on a custom-built sledge-jump system., Results: Eccentric loading altered ankle kinematics during submaximal hopping; peak angle shifted to a less dorsiflexed position by 2.9° and ankle angle precontact shifted by 4.4° (P < .001). Lower-limb stiffness increased from 5.9 to 6.8 N/m (P < .001), while surface EMG measures of soleus occurred 14-44% earlier (P < .001) after the loading intervention., Conclusions: These findings suggest that eccentric loading alters SSC behavior in a manner reflective of improved motor performance. Decreased ankle excursion, increased lower-limb stiffness, and alterations in motor control may represent a positive adaptive response to eccentric loading. These findings support the theory that mechanisms underpinning eccentric loading for tendinopathy may in part be due to improved "buffering" of the tendon by the neuromuscular system.