Association between maternal cigarette smoking cessation and risk of preterm birth in Western New York.

BACKGROUND: Although many studies suggested the benefit of smoking cessation among pregnant women in reducing the risk of preterm birth (PTB), the timing of the effect of the cessation remains inconclusive. OBJECTIVES: To examine the association of trimester-specific smoking cessation behaviours with PTB risk. METHODS: We included 199,453 live births in Western New York between 2004 and 2018. Based on self-reported cigarette smoking during preconception and in each trimester, we created six mutually exclusive groups: non-smokers, quitters in each trimester, those who smoked throughout pregnancy, and inconsistent smokers. Risk ratios (RRs) and 95% confidence intervals (CIs) were estimated using Poisson regression to examine the association between smoking cessation and PTB. Effect modification by illegal drug use, maternal age, race and ethnicity and pre-pregnancy body mass index (BMI) was investigated multiplicatively by ratio of relative risk and additively by relative excess risk due to interaction (RERI). RESULTS: Overall, 6.7% of women had a PTB; 14.1% smoked throughout pregnancy and 3.4%, 1.8% and 0.8% reported quitting smoking during the first, second and third trimesters, respectively. Compared to non-smokers, third-trimester cessation (RR 1.20, 95% CI 1.01, 1.43) and smoking throughout pregnancy (RR 1.27, 95% CI 1.21, 1.33) were associated with a higher PTB risk, while quitting smoking during the first or second trimester, or inconsistent smoking was not associated with PTB. A positive additive interaction was identified for maternal age and late smoking cessation or smoking throughout pregnancy on PTB risk (RERI 0.17, 95% CI 0.00, 0.36), and a negative interaction was observed for pre-pregnancy BMI ≥30 kg/m2 (ratio of relative risk 0.70, 95% CI 0.63, 0.78; RERI -0.42, 95% CI -0.56, -0.30). CONCLUSION: Compared to non-smokers, smoking throughout pregnancy and third-trimester smoking cessation are associated with an increased risk of PTB, while quitting before the third trimester may not increase PTB risk.

Leigh syndrome: an adult presentation of a paediatric disease.

A previously healthy 27-year-old man was admitted to the acute neurology ward with events involving his face, throat and upper limb, which video telemetry later confirmed were refractory focal seizures. He also had progressive pyramidal features, dysarthria and ataxia. MR scans of the brain identified progressive bilateral basal ganglia abnormalities, consistent with Leigh syndrome. However, extensive laboratory and genetic panels did not give a unifying diagnosis. A skeletal muscle biopsy showed no histopathological abnormalities on routine stains. Sequencing of the entire mitochondrial genome in skeletal muscle identified a well-characterised pathogenic variant (m.10191T>C in MT-ND3; NC_012920.1) at 85% heteroplasmy in skeletal muscle. We discuss the clinical and molecular diagnosis of an adult presenting with Leigh syndrome, which is more commonly a paediatric presentation of mitochondrial disease, and how early recognition of a mitochondrial cause is important to support patient care.

Health Assessment of Free-Ranging Eastern Indigo Snakes (Drymarchon couperi) from Hydrologic Restoration Construction Sites in South Florida, USA.

There is a paucity of information regarding the health status of free-ranging eastern indigo snakes (EIS; Drymarchon couperi) in heavily modified and developing landscapes. As a component of regional Florida Everglades restoration efforts, several areas occupied by EIS are being converted from agricultural lands to reservoirs. From 2020 to 2022, 28 EIS were opportunistically captured at two of these sites and brought into captivity to join a captive breeding colony; however, 11 snakes died within 5 mo of capture. Health assessments were performed on 28 individuals and included hematology and plasma biochemistry analysis, as well as screening for pesticide contaminant levels, parasites, and other pathogens. Overall, the presence of pathogens was relatively high, suggesting immunosuppression secondary to stress: 25/28 (89.4%) Kalicephalus sp.; 12/28 (42.9%) Raillietiella orientalis; 11/28 (39.2%) Ochetosoma validum; 7/28 (25.0%) Cryptosporidium serpentis; 3/28 (10.7%) snake adenovirus 1; and 1/28 (3.6%) Ferlavirus genotype C. Stress may have been caused by physical displacement, habitat modification, and noise pollution. These potential stressors (including the presence of remnant harmful chemicals from previous land use and the impacts on this federally threatened species) should be considered further when making restoration or construction decisions.

De novo variants in RNF213 are associated with a clinical spectrum ranging from Leigh syndrome to early-onset stroke.

PURPOSE: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations. METHODS: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts. RESULTS: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect. CONCLUSION: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.

Transgenerational epigenetic inheritance and immunity in chickens that vary in Marek's disease resistance.

Marek's disease virus (MDV), a naturally oncogenic, highly contagious alpha herpesvirus, induces a T cell lymphoma in chickens that causes severe economic loss. Marek's disease (MD) outcome in an individual is attributed to genetic and environmental factors. Further investigation of the host-virus interaction mechanisms that impact MD resistance is needed to achieve greater MD control. This study analyzed genome-wide DNA methylation patterns in 2 highly inbred parental lines 63 and 72 and 5 recombinant congenic strains (RCS) C, L, M, N, and X strains from those parents. Lines 63 and 72, are MD resistant and susceptible, respectively, whereas the RCS have different combinations of 87.5% Line 63 and 12.5% Line 72. Our DNA methylation cluster showed a strong association with MD incidence. Differentially methylated regions (DMRs) between the parental lines and the 5 RCS were captured. MD-resistant and MD-susceptible markers of DNA methylation were identified as transgenerational epigenetic inheritable. In addition, the growth of v-src DNA tumors and antibody response against sheep red blood cells differed among the 2 parental lines and the RCS. Overall, our results provide very solid evidence that DNA methylation patterns are transgenerational epigenetic inheritance (TEI) in chickens and also play a vital role in MD tumorigenesis and other immune responses; the specific methylated regions may be important modulators of general immunity.

TNFα-Induced Altered miRNA Expression Links to NF-κB Signaling Pathway in Endometriosis.

Endometriosis is a common gynecological inflammatory disorder characterized by immune system dysregulation, which is involved in lesion initiation and progression. Studies have demonstrated that several cytokines are associated with the evolution of endometriosis, including tumor necrosis factor-α (TNFα). TNFα is a non-glycosylated cytokine protein with potent inflammatory, cytotoxic, and angiogenic potential. In the current study, we examined the ability of TNFα to induce dysregulation of microRNAs (miRNAs) linked to NFkB signaling pathways, thus contributing to the pathogenesis of endometriosis. Using RT-qPCR, the expression of several miRNAs was quantified in primary cells derived from eutopic endometrium of endometriosis subjects (EESC) and normal endometrial stromal cells (NESC), and also TNFα-treated NESCs. The phosphorylation of the pro-inflammatory molecule NF-κB and the candidates of the survival pathways PI3K, AKT, and ERK was measured by western blot analysis. The elevated secretion of TNFα in EESCs downregulates the expression level of several miRNAs significantly in EESCs compared to NESCs. Also, treatment of NESCs with exogenous TNFα significantly reduced the expression of miRNAs in a dose-dependent manner to levels similar to EESCs. In addition, TNFα significantly increased the phosphorylation of the PI3K, AKT, ERK, and NF-κB signaling pathways. Notably, treatment with curcumin (CUR, diferuloylmethane), an anti-inflammatory polyphenol, significantly increased the expression of dysregulated miRNAs in EESC in a dose-dependent manner. Our findings demonstrate that TNFα is upregulated in EESCs, which subsequently dysregulates the expression of miRNAs, contributing to the pathophysiology of endometriotic cells. CUR effectively inhibits the expression of TNFα, subsequently altering miRNA levels and suppressing the phosphorylation of AKT, ERK, and NF-κB.

Chondrosarcoma of the hyoid bone: a case of mistaken identity in the submandibular region.

We describe a case of woman in her 60s who presented with a painless mass in her left submandibular region that biopsies and imaging suggested was a pleomorphic adenoma. Intraoperative findings showed a submandibular mass originating from the hyoid bone, and subsequent histopathological examination showed a grade 2 chondrosarcoma of the hyoid bone. Chondrosarcomas in the head and neck region are uncommon and presentations in the hyoid bone are very rare with only approximately 27 previously published cases. Presentations that pose as more benign pathology can cause diagnostic dilemmas for clinicians.

Combining supervised machine learning with statistics reveals differential gene expression patterns related to energy metabolism in the jejuna of chickens divergently selected for antibody response to sheep red blood cells.

Since the 1970s, 2 lines of White Leghorn chickens, HAS and LAS, have been continuously divergently selected for 5-day postinjection antibody titer to injection with sheep red blood cells (SRBC). Antibody response is a complex genetic trait and characterizing differences in gene expression could facilitate better understanding of physiological changes due to selection and antigen exposure. At 41 d of age, randomly selected HAS and LAS chickens, which had been coraised from hatch, were either injected with SRBC (HASI and LASI) or kept as the noninjected cohort (HASN and LASN). Five days later, all were euthanized, and samples collected from the jejunum for RNA isolation and sequencing. Resulting gene expression data were analyzed combining traditional statistics with machine learning to obtain signature gene lists for functional analysis. Differences in ATP production and cellular processes were observed in the jejunum between lines and following SRBC injection. HASN vs. LASN exhibited upregulation of ATP production, immune cell motility, and inflammation. LASI exhibits upregulation of ATP production and protein synthesis vs. LASN, reflective of what was observed in HASN vs. LASN. In contrast, no corresponding upregulation of ATP production was observed in HASI vs. HASN, and most other cellular processes appear inhibited. Without exposure to SRBC, gene expression in the jejunum indicates HAS generates more ATP than LAS, suggesting HAS maintains a "primed" system; and gene expression of HASI vs. HASN further suggests this basal ATP production is sufficient for robust antibody responses. Conversely, LASI vs. LASN jejunal gene expression implies a physiological need for increased ATP production with only minimal correlating antibody production. The results of this experiment provide insight into energetic resource needs and allocations in the jejunum in response to genetic selection and antigen exposure in HAS and LAS which may help explain phenotypic differences observed in antibody response.

TNFα-induced altered miRNA expression links to NF-κB signaling pathway in endometriosis.

Endometriosis is a common gynecological inflammatory disorder characterized by immune system dysregulation, which is involved in lesion initiation and progression. Studies have demonstrated that several cytokines are associated with the evolution of endometriosis, including tumor necrosis factor-α (TNFα). TNFα is a non-glycosylated cytokine protein with potent inflammatory, cytotoxic, and angiogenic potential. In the current study, we examined the ability of TNFα to induce dysregulation of microRNAs (miRNAs) linked to NFkB-signaling pathways, thus contributing to the pathogenesis of endometriosis. Using RT-QPCR, the expression of several miRNAs were quantified in primary cells derived from eutopic endometrium of endometriosis subjects (EESC) and normal endometrial stromal cells (NESC) and also TNFα treated NESCs. The phosphorylation of the pro-inflammatory molecule NF-κB and the candidates of the survival pathways PI3K, AKT and ERK was measured by westernblot analysis. The elevated secretion of TNFα in EESCs downregulates the expression level of several miRNAs significantly (p < 0.05) in EESCs compared to NESC. Also treatment of NESCs with exogenous TNFα significantly reduced the expression of miRNAs in a dose-dependent manner to levels similar to EESCs. In addition, TNFα significantly increased the phosphorylation of the PI3K, AKT, ERK, and NF-κB signaling pathways. Notably, treatment with curcumin (CUR, diferuloylmethane), an anti-inflammatory polyphenol, significantly increased the expression of dysregulated miRNAs in EESC in a dose-dependent manner. Our findings demonstrate that TNFα is upregulated in EESCs, which subsequently dysregulates the expression of miRNAs, contributing to the pathophysiology of endometriotic cells. CUR effectively inhibits the expression of TNFα, subsequently altering miRNA levels and suppresses the phosphorylation of AKT, ERK, and NF-κB.

Identification, Measurement, and Assessment of the Clinical Utility of Human Pancreatic Polypeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Human pancreatic polypeptide (HPP) is a 36 amino acid peptide hormone that plays a role in the bidirectional communication between the digestive system and the brain. HPP measurements are used to assess vagal nerve function following sham feeding and to detect gastroenteropancreatic-neuroendocrine tumors. These tests have historically been conducted by radioimmunoassays, but liquid chromatography-tandem mass spectrometry (LC-MS/MS) has several advantages such as improved specificity and elimination of radioactive molecules. Here, we present our LC-MS/MS method. Initially, samples were immunopurified and subjected to LC-high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) to identify circulating forms of the peptide in human plasma. We identified 23 forms of HPP, including several glycosylated forms. The most abundant peptides then were used for targeted LC-MS/MS measurements. LC-MS/MS performance for precision, accuracy, linearity, recovery, limit of detection, and carryover met our acceptance criteria based on CLIA regulations. Additionally, we observed the expected physiological rise in HPP in response to sham feeding. Our results indicate that HPP measurement by LC-MS/MS produces clinically equivalent results to our established immunoassay when several peptides are monitored, making it a suitable replacement. The measurement of peptide fragments, including modified species, might have additional clinical value.

Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.

A multidisciplinary approach to the laboratory diagnosis of mitochondrial disease has long been applied, with crucial information provided by deep clinical phenotyping, blood investigations, and biomarker screening as well as histopathological and biochemical testing of biopsy material to support molecular genetic screening. In an era of second and third generation sequencing technologies, traditional diagnostic algorithms for mitochondrial disease have been replaced by gene agnostic, genomic strategies including whole-exome sequencing (WES) and whole-genome sequencing (WGS), increasingly supported by other 'omics technologies (Alston et al., 2021). Whether a primary testing strategy, or one used to validate and interpret candidate genetic variants, the availability of a range of tests aimed at determining mitochondrial function (i.e., the assessment of individual respiratory chain enzyme activities in a tissue biopsy or cellular respiration in a patient cell line) remains an important part of the diagnostic armory. In this chapter, we summarize several disciplines used in the laboratory investigation of suspected mitochondrial disease, including the histopathological and biochemical assessment of mitochondrial function, as well as protein-based techniques to assess the steady-state levels of oxidative phosphorylation (OXPHOS) subunits and assembly of OXPHOS complexes via traditional (immunoblotting) and cutting-edge (quantitative proteomic) approaches.

Genetic testing for mitochondrial disease: the United Kingdom best practice guidelines.

Primary mitochondrial disease describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation. Diagnosis is challenging; >350 genes, both nuclear and mitochondrial DNA (mtDNA) encoded, are known to cause mitochondrial disease, leading to all possible inheritance patterns and further complicated by heteroplasmy of the multicopy mitochondrial genome. Technological advances, particularly next-generation sequencing, have driven a shift in diagnostic practice from 'biopsy first' to genome-wide analyses of blood and/or urine DNA. This has led to the need for a reference framework for laboratories involved in mitochondrial genetic testing to facilitate a consistent high-quality service. In the United Kingdom, consensus guidelines have been prepared by a working group of Clinical Scientists from the NHS Highly Specialised Service followed by national laboratory consultation. These guidelines summarise current recommended technologies and methodologies for the analysis of mtDNA and nuclear-encoded genes in patients with suspected mitochondrial disease. Genetic testing strategies for diagnosis, family testing and reproductive options including prenatal diagnosis are outlined. Importantly, recommendations for the minimum levels of mtDNA testing for the most common referral reasons are included, as well as guidance on appropriate referrals and information on the minimal appropriate gene content of panels when analysing nuclear mitochondrial genes. Finally, variant interpretation and recommendations for reporting of results are discussed, focussing particularly on the challenges of interpreting and reporting mtDNA variants.

Changing faces of mitochondrial disease: autosomal recessive POLG disease mimicking myasthenia gravis and progressive supranuclear palsy.

Background: Mitochondrial disorders are known to cause diverse neurological phenotypes which cause a diagnostic challenge to most neurologists. Pathogenic polymerase gamma (POLG) variants have been described as a cause of chronic progressive external ophthalmoplegia, which manifests with ptosis, horizontal and vertical eye movement restriction and myopathy. Autosomal dominant progressive external ophthalmoplegia is rarely associated with Parkinsonism responsive to levodopa. Methods: We report a case of a 58-year-old man who presented with an eye movement disorder then Parkinsonism who made his way through the myasthenia then the movement disorder clinic. Results: A diagnostic right tibialis anterior biopsy revealed classical hallmarks of mitochondrial disease, and genetic testing identified compound heterozygous pathogenic gene variants in the POLG gene. The patient was diagnosed with autosomal recessive POLG disease. Conclusions: It is important to maintain a high index of suspicion of pathogenic POLG variants in patients presenting with atypical Parkinsonism and ophthalmoplegia. Patients with POLG-related disease will usually have ptosis, and downgaze is typically preserved until late in the disease. Accurate diagnosis is essential for appropriate prognosis and genetic counselling.

Testing of drugs using human feto-maternal interface organ-on-chips provide insights into pharmacokinetics and efficacy.

Objectives: To improve preclinical drug testing during pregnancy, we developed multiple microfluidic organ-on-chip (OOC) devices that represent the structure, functions, and responses of the two feto-maternal interfaces (FMis) in humans (fetal membrane [FMi-OOC] and placenta [PLA-OOC]). This study utilized feto-maternal interface OOCs to test the kinetics and efficacy of drugs during pregnancy. Study design: The FMi-OOC contained amnion epithelial, mesenchymal, chorion trophoblast, and decidual cells. The PLA-OOC contained cytotrophoblasts (BeWo), syncytiotrophoblasts (BeWo + forskolin), and human umbilical vein endothelial cell lines. Therapeutic concentrations of either pravastatin or rosuvastatin (200 ng mL-1), a model drug for these experiments, were applied to either decidua (in FMi-OOC) and syncytiotrophoblasts (in PLA-OOC) chambers under normal and oxidative stress conditions (induced by cigarette smoke extract [CSE 1 : 25]) to evaluate maternal drug exposure during normal pregnancy or oxidative stress (OS) associated pathologies, respectively. We determined statin pharmacokinetics and metabolism (LC-MS/MS), drug-induced cytotoxicity (LDH assay), and efficacy to reduce OS-induced inflammation (multiplex cytokine assay). Results: Both OOCs mimicked two distinct human feto-maternal interfaces. The drugs tested permeated the maternal-fetal cell layers of the FMi-OOC and PLA-OOC within 4 hours and generated cell and time-specific statin metabolites from various cell types without causing any cytotoxicity. OS-induced pro-inflammatory cytokines were effectively reduced by statins by increasing anti-inflammatory cytokine response across the FMi-OOC and PLA-OOC. Conclusion: Two distinct feto-maternal interface OOCs were developed, tested, and validated for their utility to conduct preclinical trials during pregnancy. We demonstrated that the placenta and fetal membranes-decidual interface both are able to transport and metabolize drugs and that the safety and efficacy of a drug can be determined using the anatomical structures recreated on OOCs.

Nonstop mRNAs generate a ground state of mitochondrial gene expression noise.

A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating nonstop ribosome complexes. Here, we use deep sequencing to investigate sources of nonstop mRNAs generated from the human mitochondrial genome. We identify diverse types of nonstop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Failure to resolve these aberrations by the mitochondrial release factor in rescue (MTRFR) imparts a negative regulatory effect on protein synthesis that is associated with human disease. Our findings reveal a source of underlying noise in mitochondrial gene expression and the importance of responsive ribosome quality control mechanisms for cell fitness and human health.

Acipimox in Mitochondrial Myopathy (AIMM): study protocol for a randomised, double-blinded, placebo-controlled, adaptive design trial of the efficacy of acipimox in adult patients with mitochondrial myopathy.

BACKGROUND: Mitochondrial disease is a heterogenous group of rare, complex neurometabolic disorders. Despite their individual rarity, collectively mitochondrial diseases represent the most common cause of inherited metabolic disorders in the UK; they affect 1 in every 4300 individuals, up to 15,000 adults (and a similar number of children) in the UK. Mitochondrial disease manifests multisystem and isolated organ involvement, commonly affecting those tissues with high energy demands, such as skeletal muscle. Myopathy manifesting as fatigue, muscle weakness and exercise intolerance is common and debilitating in patients with mitochondrial disease. Currently, there are no effective licensed treatments and consequently, there is an urgent clinical need to find an effective drug therapy. AIM: To investigate the efficacy of 12-week treatment with acipimox on the adenosine triphosphate (ATP) content of skeletal muscle in patients with mitochondrial disease and myopathy. METHODS: AIMM is a single-centre, double blind, placebo-controlled, adaptive designed trial, evaluating the efficacy of 12 weeks' administration of acipimox on skeletal muscle ATP content in patients with mitochondrial myopathy. Eligible patients will receive the trial investigational medicinal product (IMP), either acipimox or matched placebo. Participants will also be prescribed low dose aspirin as a non-investigational medical product (nIMP) in order to protect the blinding of the treatment assignment. Eighty to 120 participants will be recruited as required, with an interim analysis for sample size re-estimation and futility assessment being undertaken once the primary outcome for 50 participants has been obtained. Randomisation will be on a 1:1 basis, stratified by Fatigue Impact Scale (FIS) (dichotomised as < 40, ≥ 40). Participants will take part in the trial for up to 20 weeks, from screening visits through to follow-up at 16 weeks post randomisation. The primary outcome of change in ATP content in skeletal muscle and secondary outcomes relating to quality of life, perceived fatigue, disease burden, limb function, balance and walking, skeletal muscle analysis and symptom-limited cardiopulmonary fitness (optional) will be assessed between baseline and 12 weeks. DISCUSSION: The AIMM trial will investigate the effect of acipimox on modulating muscle ATP content and whether it can be repurposed as a new treatment for mitochondrial disease with myopathy. TRIAL REGISTRATION: EudraCT2018-002721-29 . Registered on 24 December 2018, ISRCTN 12895613. Registered on 03 January 2019, https://www.isrctn.com/search?q=aimm.

ENDOCELL-Seud: a Delphi protocol to harmonise methods in endometrial cell culturing.

In vitro: culturing of endometrial cells obtained from the uterine mucosa or ectopic sites is used to study molecular and cellular signalling relevant to physiologic and pathologic reproductive conditions. However, the lack of consensus on standard operating procedures for deriving, characterising and maintaining primary cells in two- or three-dimensional cultures from eutopic or ectopic endometrium may be hindering progress in this area of research. Guidance for unbiased in vitro research methodologies in the field of reproductive science remains essential to increase confidence in the reliability of in vitro models. We present herein the protocol for a Delphi process to develop a consensus on in vitro methodologies using endometrial cells (ENDOCELL-Seud Project). A steering committee composed of leading scientists will select critical methodologies, topics and items that need to be harmonised and that will be included in a survey. An enlarged panel of experts (ENDOCELL-Seud Working Group) will be invited to participate in the survey and provide their ratings to the items to be harmonised. According to Delphi, an iterative investigation method will be adopted. Recommended measures will be finalised by the steering committee. The study received full ethical approval from the Ethical Committee of the Maastricht University (ref. FHML-REC/2021/103). The study findings will be available in both peer-reviewed articles and will also be disseminated to appropriate audiences at relevant conferences. Lay summary: Patient-derived cells cultured in the lab are simple and cost-effective methods used to study biological and dysfunctional or disease processes. These tools are frequently used in the field of reproductive medicine. However, the lack of clear recommendations and standardised methodology to guide the laboratory work of researchers can produce results that are not always reproducible and sometimes are incorrect. To remedy this situation, we define here a method to ascertain if researchers who routinely culture cells in the lab agree or disagree on the optimal laboratory techniques. This method will be used to make recommendations for future researchers working in the field of reproductive biology to reproducibly culture endometrial cells in the laboratory.

A Participatory-Based Research Approach for Assessing Exposure to Lead-Contaminated Drinking Water in the Houston Neighborhood of the Greater Fifth Ward.

To address community-driven concerns about lead-contaminated drinking water in residential homes in the Greater Fifth Ward neighborhood in Northeast Houston, Texas utilizing participatory-based research. The study collected survey data and performed lead analysis on drinking water from residents' homes. The Greater Fifth Ward is characterized as a majority-minority environmental justice community and is located within two confirmed cancer clusters. The residents of 172 homes completed a survey and had detectable lead levels in their water samples. Survey results indicated that more than half of the residents (58.2%) were concerned with the water quality and 42.9% rated the drinking water as poor. Water lead levels detected ranged from 0.01 to 22 µg/L. 10.9% of homes exceeding 1 µg/L, and one located exceeded the USEPA's action limit of 15 µg/L. Homes built prior to 1978 without major renovation had significantly higher levels of lead in their drinking water compared to homes built after 1978 (p-value < 0.05). These findings demonstrate the need for lead testing of residential water in low socioeconomic-status communities, as well as demonstrating the benefits of community engagement and participatory research to address environmental health concerns.

Rapid identification of human muscle disease with fibre optic Raman spectroscopy.

The diagnosis of muscle disorders ("myopathies") can be challenging and new biomarkers of disease are required to enhance clinical practice and research. Despite advances in areas such as imaging and genomic medicine, muscle biopsy remains an important but time-consuming investigation. Raman spectroscopy is a vibrational spectroscopy application that could provide a rapid analysis of muscle tissue, as it requires no sample preparation and is simple to perform. Here, we investigated the feasibility of using a miniaturised, portable fibre optic Raman system for the rapid identification of muscle disease. Samples were assessed from 27 patients with a final clinico-pathological diagnosis of a myopathy and 17 patients in whom investigations and clinical follow-up excluded myopathy. Multivariate classification techniques achieved accuracies ranging between 71-77%. To explore the potential of Raman spectroscopy to identify different myopathies, patients were subdivided into mitochondrial and non-mitochondrial myopathy groups. Classification accuracies were between 74-89%. Observed spectral changes were related to changes in protein structure. These data indicate fibre optic Raman spectroscopy is a promising technique for the rapid identification of muscle disease that could provide real time diagnostic information. The application of fibre optic Raman technology raises the prospect of in vivo bedside testing for muscle diseases which would significantly streamline the diagnostic pathway of these disorders.

One- and Two-Year Analysis of a Five-Year Prospective Multicenter Study Assessing Radiographic and Patient-Reported Outcomes Following Triplanar First Tarsometatarsal Arthrodesis With Early Weightbearing for Symptomatic Hallux Valgus.

We report one- and 2-year results of a prospective, 5-year, multicenter study of radiographic, clinical, and patient-reported outcomes following triplanar first tarsometatarsal arthrodesis with early weightbearing. One-hundred and seventeen patients were included with a mean (95% confidence interval [CI]) follow-up time of 16.6 (15.5, 17.7) months. Mean (95% CI) time to weightbearing in a boot walker was 7.8 (6.6, 9.1) days, mean time to return to athletic shoes was 45.0 (43.5, 46.6) days, and mean time to return to unrestricted activity was 121.0 (114.5, 127.5) days. There was a significant improvement in radiographic measures with a mean corrective change of -18.0° (-19.6, -16.4) for hallux valgus angle, -8.3° (-8.9, -7.8) for intermetatarsal angle and -2.9 (-3.2, -2.7) for tibial sesamoid position at 12 months (n = 108). Additionally, there was a significant improvement in patient-reported outcomes (Visual Analog Scale, Manchester-Oxford Foot Questionnaire, and Patient-Reported Outcomes Measurement Information System) and changes were maintained at 12 and 24 months postoperatively. There was 1/117 (0.9%) reported recurrence of hallux valgus at 12 months. There were 16/117 (13.7%) subjects who experienced clinical complications of which 10/117 (8.5%) were related to hardware. Of the 7/117 (6.0%) who underwent reoperation, only 1/117 (0.9%) underwent surgery for a nonunion. The results of the interim report of this prospective, multicenter study demonstrate favorable clinical and radiographic improvement of the HV deformity, early return to weightbearing, low recurrence, and low rate of complications.