Complex Study Designs Research Articles

Integrating gene expression and imaging data across Visium capture areas with visiumStitched.

Visium is a widely-used spatially-resolved transcriptomics assay available from 10x Genomics. Standard Visium capture areas (6.5mm by 6.5mm) limit the survey of larger tissue structures, but combining overlapping images and associated gene expression data allow for more complex study designs. Current software can handle nested or partial image overlaps, but is designed for merging up to two capture areas, and cannot account for some technical scenarios related to capture area alignment. We generated Visium data from a postmortem human tissue sample such that two capture areas were partially overlapping and a third one was adjacent. We developed the R/Bioconductor package visiumStitched, which facilitates stitching the images together with Fiji (ImageJ), and constructing SpatialExperiment R objects with the stitched images and gene expression data. visiumStitched constructs an artificial hexagonal array grid which allows seamless downstream analyses such as spatially-aware clustering without discarding data from overlapping spots. Data stitched with visiumStitched can then be interactively visualized with spatialLIBD. visiumStitched provides a simple, but flexible framework to handle various multi-capture area study design scenarios. Specifically, it resolves a data processing step without disrupting analysis workflows and without discarding data from overlapping spots. visiumStitched relies on affine transformations by Fiji, which have limitations and are less accurate when aligning against an atlas or other situations. visiumStitched provides an easy-to-use solution which expands possibilities for designing multi-capture area study designs.

Flavored Tobacco User Characteristics in U.S. young Adults: Wave 5 of the Population Assessment of Tobacco and Health Study

Introduction: Flavorings are one of the major appealing factors of current tobacco products, especially for young adults. They constantly evolve, and new flavorings are introduced to the market every year, but little is known about user profiles of flavored tobacco products (FTPs). Methods: We analyzed young adult (ages 18–24) tobacco product users (N = 4,707) from Wave 5 of the Population Assessment of Tobacco and Health Study. We applied bivariate analyses to compare individuals who use FTP and non-FTPs by different flavor types (i.e., menthol/mint, fruit, candy/sweets, and tobacco). Parameter estimates were weighted by applying the sample and replicate weights for the complex study design to produce population-representative estimates. Results: Sixty percent of the current combustible tobacco users and 90% of electronic nicotine delivery systems (ENDS) users were FTP users. FTP users were more likely to be multiple product users than non-FTP users. Being non-Hispanic White, drinking alcohol in the past year, experiencing internalizing problems in the past month, and having tobacco dependence were associated with FTP use. Flavored ENDS users had different sociodemographic characteristics by flavor types but did not differ significantly in vaping-related behaviors (e.g., frequency). Also, preferred flavors differed among tobacco user groups (i.e., ENDS, hookah, and cigars). Conclusions: FTPs appear to be gaining popularity among young adults and tend to show co-use with multiple tobacco products and other substances. Future studies are warranted to monitor the risk factors of young adult FTP use, especially in the context of flavor ban on ENDS products.

Integrating gene expression and imaging data across Visium capture areas with visiumStitched.

Visium is a widely-used spatially-resolved transcriptomics assay available from 10x Genomics. Standard Visium capture areas (6.5mm by 6.5mm) limit the survey of larger tissue structures, but combining overlapping images and associated gene expression data allow for more complex study designs. Current software can handle nested or partial image overlaps, but is designed for merging up to two capture areas, and cannot account for some technical scenarios related to capture area alignment. We generated Visium data from a postmortem human tissue sample such that two capture areas were partially overlapping and a third one was adjacent. We developed the R/Bioconductor package visiumStitched, which facilitates stitching the images together with Fiji (ImageJ), and constructing SpatialExperiment R objects with the stitched images and gene expression data. visiumStitched constructs an artificial hexagonal array grid which allows seamless downstream analyses such as spatially-aware clustering without discarding data from overlapping spots. Data stitched with visiumStitched can then be interactively visualized with spatialLIBD. visiumStitched provides a simple, but flexible framework to handle various multi-capture area study design scenarios. Specifically, it resolves a data processing step without disrupting analysis workflows and without discarding data from overlapping spots. visiumStiched relies on affine transformations by Fiji, which have limitations and are less accurate when aligning against an atlas or other situations. visiumStiched provides an easy-to-use solution which expands possibilities for designing multi-capture area study designs.

Assessment of Cognitive Function in European Adults Aged 50+in Relation to Their Handgrip Strength and Physical Inactivity: The SHARE Study During 2019-2020.

Cognitive function is crucial during aging. This study assessed the cognitive function of European adults aged 50 and over in relation to handgrip strength and physical inactivity. Study Design: This was a cross-sectional survey. Data were collected from 41,395 adults from 27 European countries participating in the Survey of Health, Ageing, and Retirement in Europe (SHARE) during 2019-2020. Cognitive function was assessed based on five tests, and cognitive impairment was defined using 3+tests. Handgrip strength and physical inactivity were also correlated through the analysis of covariance using a complex study design. The majority of participants were female (56.6%), with a mean age of 70.9 years, and 22.6% presented multimorbidity. Furthermore, 51.1% had a normal cognitive function, while 13.3% had cognitive impairment (The estimated population was 21,944,722). Moreover, cognitive impairment was more prevalent in females than in males (14.4% vs. 12.0%, P<0.001) in patients with no years of education (P<0.001) and origin from southern European countries (P<0.001). Additionally, participants with cognitive impairment had lower mean handgrip strength compared to those with cognitive impairment in 1-2 criteria or with normal cognitive function (29.3 vs. 33.4 and 35.1 kg, respectively, P<0.001). Physically inactive participants had higher odds ratio (OR) of cognitive impairment than those engaging in moderate/vigorous physical activity, both in 1-2 tests (OR:1.73, 95% confidence interval (CI): 1.32-2.26) and in 3+tests (OR: 3.36, 95% CI: 2.57-4.40). Cognitive impairment presented low prevalence and was associated with low levels of handgrip strength and physical inactivity. These specific factors may play a special role in early detection, diagnosis, and treatment or may slow down the progression of cognitive impairment.

MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation.

We introduce MetaboAnalyst version 6.0 as a unified platform for processing, analyzing, and interpreting data from targeted as well as untargeted metabolomics studies using liquid chromatography - mass spectrometry (LC-MS). The two main objectives in developing version 6.0 are to support tandem MS (MS2) data processing and annotation, as well as to support the analysis of data from exposomics studies and related experiments. Key features of MetaboAnalyst 6.0 include: (i) a significantly enhanced Spectra Processing module with support for MS2 data and the asari algorithm; (ii) a MS2 Peak Annotation module based on comprehensive MS2 reference databases with fragment-level annotation; (iii) a new Statistical Analysis module dedicated for handling complex study design withmultiple factors orphenotypic descriptors; (iv) a Causal Analysis module for estimating metabolite - phenotype causal relations based on two-sample Mendelian randomization,and (v) a Dose-Response Analysis module for benchmark dose calculations. In addition, we have also improved MetaboAnalyst's visualization functions, updated its compound database and metabolite sets, and significantly expanded its pathway analysis support to around 130 species. MetaboAnalyst 6.0 is freely available at https://www.metaboanalyst.ca.

An analysis of published study designs in PubMed prisoner health abstracts from 1963 to 2023: a text mining study

BackgroundThe challenging nature of studies with incarcerated populations and other offender groups can impede the conduct of research, particularly that involving complex study designs such as randomised control trials and clinical interventions. Providing an overview of study designs employed in this area can offer insights into this issue and how research quality may impact on health and justice outcomes.MethodsWe used a rule-based approach to extract study designs from a sample of 34,481 PubMed abstracts related to epidemiological criminology published between 1963 and 2023. The results were compared against an accepted hierarchy of scientific evidence.ResultsWe evaluated our method in a random sample of 100 PubMed abstracts. An F1-Score of 92.2% was returned. Of 34,481 study abstracts, almost 40.0% (13,671) had an extracted study design. The most common study design was observational (37.3%; 5101) while experimental research in the form of trials (randomised, non-randomised) was present in 16.9% (2319). Mapped against the current hierarchy of scientific evidence, 13.7% (1874) of extracted study designs could not be categorised. Among the remaining studies, most were observational (17.2%; 2343) followed by systematic reviews (10.5%; 1432) with randomised controlled trials accounting for 8.7% (1196) of studies and meta-analysis for 1.4% (190) of studies.ConclusionsIt is possible to extract epidemiological study designs from a large-scale PubMed sample computationally. However, the number of trials, systematic reviews, and meta-analysis is relatively small – just 1 in 5 articles. Despite an increase over time in the total number of articles, study design details in the abstracts were missing. Epidemiological criminology still lacks the experimental evidence needed to address the health needs of the marginalized and isolated population that is prisoners and offenders.

Evaluation of noninvasive biospecimens for transcriptome studies

Transcriptome studies disentangle functional mechanisms of gene expression regulation and may elucidate the underlying biology of disease processes. However, the types of tissues currently collected typically assay a single post-mortem timepoint or are limited to investigating cell types found in blood. Noninvasive tissues may improve disease-relevant discovery by enabling more complex longitudinal study designs, by capturing different and potentially more applicable cell types, and by increasing sample sizes due to reduced collection costs and possible higher enrollment from vulnerable populations. Here, we develop methods for sampling noninvasive biospecimens, investigate their performance across commercial and in-house library preparations, characterize their biology, and assess the feasibility of using noninvasive tissues in a multitude of transcriptomic applications. We collected buccal swabs, hair follicles, saliva, and urine cell pellets from 19 individuals over three to four timepoints, for a total of 300 unique biological samples, which we then prepared with replicates across three library preparations, for a final tally of 472 transcriptomes. Of the four tissues we studied, we found hair follicles and urine cell pellets to be most promising due to the consistency of sample quality, the cell types and expression profiles we observed, and their performance in disease-relevant applications. This is the first study to thoroughly delineate biological and technical features of noninvasive samples and demonstrate their use in a wide array of transcriptomic and clinical analyses. We anticipate future use of these biospecimens will facilitate discovery and development of clinical applications.

Sample size planning for complex study designs: A tutorial for the mlpwr package.

A common challenge in designing empirical studies is determining an appropriate sample size. When more complex models are used, estimates of power can only be obtained using Monte Carlo simulations. In this tutorial, we introduce the R package mlpwr to perform simulation-based power analysis based on surrogate modeling. Surrogate modeling is a powerful tool in guiding the search for study design parameters that imply a desired power or meet a cost threshold (e.g., in terms of monetary cost). mlpwr can be used to search for the optimal allocation when there are multiple design parameters, e.g., when balancing the number of participants and the number of groups in multilevel modeling. At the same time, the approach can take into account the cost of each design parameter, and aims to find a cost-efficient design. We introduce the basic functionality of the package, which can be applied to a wide range of statistical models and study designs. Additionally, we provide two examples based on empirical studies for illustration: one for sample size planning when using an item response theory model, and one for assigning the number of participants and the number of countries for a study using multilevel modeling.

Trends and determinants of early initiation of breastfeeding in Indonesia: A multivariate decomposition analysis.

Early initiation of breastfeeding (EIBF) is key to reducing neonatal morbidity and mortality, however, little is known about the determinants of the trends of EIBF prevalence in Indonesia. This study aims to assess the contributing factors to the changes in the prevalence of EIBF between 2007 and 2017. We analysed data from the 2007, 2012, and 2017 Indonesia Demographic and Health Surveys to estimate the trends in EIBF. A multivariate logistic decomposition model was fitted to examine variables associated with changes in the percentage of EIBF from 2007 to 2017. The contributing factors to changes in EIBF prevalence were categorized into either compositional or behavioural changes, with each of them divided into portions or percentages of contribution (pct) of the independent variables. The former refers to the changes in the distribution of samples, while the latter refers to the changes in the behavioural responses toward EIBF in both surveys. All analyses accounted for the complex study design and potential confounding factors. An increase in the prevalence of EIBF from 49.9% to 56.5% was recorded between 2012 and 2017, with an overall increase of 16.9 percentage points from 2007 to 2017. At the aggregate level, the compositional differences did not significantly contribute to the changes in the percentage of EIBF, while 98.3pct (p < 0.001) was associated with changes in mothers' behavioural response towards EIBF. The composition changes in the geographical region of Sumatra, and caesarean delivery negatively contributed to the changes in EIBF prevalence with -0.6pct and -14.2pct, respectively. However, the compositional differences in those living in Kalimantan & Sulawesi, first-time mothers, and small-born infants positively contributed to the change. Behaviour changes in mothers with higher education (8.8pct), from higher income households (-17.5pct), and those residing in Sumatra (-8.2pct) and Kalimantan & Sulawesi (-10.2pct) significantly contributed to the upward trend in EIBF prevalence. Almost half of the newborns experienced delayed breastfeeding initiation despite the improvement in the prevalence of EIBF. Therefore, further research and interventions on behaviour change in mother's attitudes towards EIBF, especially among those undergoing caesarean delivery, living in Kalimantan or Sulawesi, and from wealthier households, are recommended to close this gap.

Successful implementation of a stakeholder engagement program for pharmacoepidemiologic research.

Rigorously conducted pharmacoepidemiologic research requires methodologically complex study designs and analysis yet evaluates problems of high importance to patients and clinicians. Despite this, participation in and mechanisms for stakeholder engagement in pharmacoepidemiologic research are not well-described. Here, we describe our approach and lessons learned from engaging stakeholders, of varying familiarity with research methods, in a rigorous multi-year pharmacoepidemiologic research program evaluating the comparative effectiveness of diabetes medications. We recruited 5 patient and 4 clinician stakeholders; each was compensated for their time. Stakeholders received initial formal training in observational research and pharmacoepidemiologic methods sufficient to enable contribution to the research project. After onboarding, stakeholder engagement meetings were held virtually, in the evening, 2-3 times annually. Each was approximately 90 min and focused on 1-2 specific questions about the project, with preparatory materials sent in advance. Stakeholder meeting attendance was high (89%-100%), and all stakeholders engaged with the research project, both during and between meetings. Stakeholders reported positive experiences with meetings, satisfaction, and interest in the research project and its findings, and dedication to the success of the project's goals. They affirmed the value of receiving materials to review in advance and the effectiveness of a virtual platform. Their contributions included prioritizing and suggesting research questions, optimizing written evidence briefs for a lay audience, and guidance on broader topics such as research audience and methods of dissemination. Stakeholder engagement in pharmacoepidemiologic research using complex study designs and analysis is feasible, acceptable, and positively impacts the research project.

Double-Blind Randomized Study to Evaluate the Effect of Hydroxycarbamide on Albuminuria in Adults with Sickle Cell Disease: Sikamic (SIklos on Kidney Function and AlbuMInuria Clinical Trial)

Background Sickle cell disease (SCD) is associated with a high frequency of chronic kidney disease, which is an independent risk factor for mortality in this population. SCD-associated nephropathy (SCAN) is an emerging concern, characterized by significant albuminuria and progressive deterioration of renal function with a prevalence of up to 26% to 68% in SCD adult patients (Falk 1992, Powars 2005, Yeruva 2016, Ataga 202). Because of rapid decline of estimated glomerular filtration rate (eGFR) is a frequent finding in this population (Derebail 2019, Ataga KI 2021), early diagnosis of SCAN and identification of associated clinical and biologic risk factors are crucial for initiating kidney-protective therapy at early stages of renal impairment. Albuminuria serves as a relevant biomarker for detecting early glomerular damage in SCD patients (Audard 2017). To date, Hydroxycarbamide (HU) remains the cornerstone treatment for managing SCD patients, and its efficacy is primarily attributed to its ability to increase fetal hemoglobin (HbF) levels (Segal 2008). Several observational studies conducted in adult SCD patients have shown that HU may have renal protective effects, making it a promising therapeutic option, but until now, randomized clinical trials confirming this hypothesis are missing (Bartolucci 2015, Laurin 2014). Method SIKAMIC (SIklos on Kidney Function and AlbuMInuria Clinical Trial) is an international (France and Africa), phase IIb, double-blind, randomized, placebo-controlled study. The main objective is to assess the effect of HU on albuminuria levels in SCD adult patients after 6 months of treatment. Eligible patients are of the SS or Sβ0 genotype with a mean albuminuria value (assessed by albumin-to-creatinine ratio: ACR) above 3 and less than 100 mg/mmol, on three urine samples taken one day apart. Patients should not have received HU treatment within the previous 6 months and should not be taking conventional kidney-protecting measures (ACE inhibitors or ARA2) and having blood transfusion in the last 3 months. Patient with an eGFR rate &amp;lt; 60 and ≥ 140 ml/min/1,73m² are excluded from the study. Patients receive either HU at a dosage of 15mg/kg/day or placebo for 6 months, and responders (patient achieving at least a 30% decrease of the ACR baseline at 6 months) have the option to continue the study for an additional six months. Recruitment started in August 2019 in France and in October 2021 in Africa and was extended until June 2023 due to challenge in finding HU-Naïve patients. The end of the study is planned in June 2024 with safety monitoring conducted by an Independent Data Monitoring Committee throughout. Results During the recruitment period, 179 patients were screened, 99 were randomized, 34 in France, 65 in Africa (Senegal: 31, Mali: 23, Ivory Coast: 11) ( Figure 1). The main reason for screen failure was mean ACR &amp;lt; 3mg/mmol. Two patients were excluded from the analysis due to non-compliance with exclusion criteria. Among the 97 patients analyzed, 32 completed the study by June 30, 2023, while 46 were still ongoing treatment, and 19 withdrew before the end of the 6 months follow-up period. The study includes a higher proportion of women (68%), which can be attributed to challenges faced in conducting semen analysis in African countries and the hesitancy to initiate HU treatment without prior semen analysis. The mean age at enrolment was 29.7 ± 9.2 years. Recruitment significantly improved after opening African sites. The study encountered several difficulties, primarily stemming from the challenge of finding HU-naïve patients in France and the high rate of screen failure attributed to non-expected ACR measurement variability and eGFR outside the accepted limits. No safety issues were reported, with 15 well-known adverse events related to HU occurring in 11 patients. Results of the study are expected by the end of 2024. Conclusion This study is the first randomized placebo-controlled study investigating the effect of HU on albuminuria in SCD patients. By employing a complex and robust study design, with ACR measurements at multiple time points, the study will provide valuable insights into the impact of HU on albuminuria levels. Moreover, the collaboration with African countries has highlighted the significance of considering country-specific factors and fostering partnerships to address the unique challenges faced by patients in these regions.

Using ExpressAnalyst for Comprehensive Gene Expression Analysis in Model and Non-Model Organisms.

ExpressAnalyst is a web-based platform that enables intuitive, end-to-end transcriptomics and proteomics data analysis. Users can start from FASTQ files, gene/protein abundance tables, or gene/protein lists. ExpressAnalyst will perform read quantification, gene expression table processing and normalization, differential expression analysis, or meta-analysis with complex study designs. The results are presented via various interactive visualizations such as volcano plots, heatmaps, networks, and ridgeline charts, with built-in functional enrichment analysis to allow flexible data exploration and understanding. ExpressAnalyst currently contains built-in support for 29 common organisms. For non-model organisms without good reference genomes, it can perform comprehensive transcriptome profiling directly from RNA-seq reads. These common tasks are covered in 11 Basic Protocols. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: RNA-seq count table uploading, processing, and normalization Basic Protocol 2: Differential expression analysis with linear models Basic Protocol 3: Functional analysis with volcano plot, enrichment network, and ridgeline visualization Basic Protocol 4: Hierarchical clustering analysis of transcriptomics data using interactive heatmaps Basic Protocol 5: Cross-species gene expression analysis based on ortholog mapping results Basic Protocol 6: Proteomics and microarray data processing and normalization Basic Protocol 7: Preparing multiple gene expression tables for meta-analysis Basic Protocol 8: Statistical and functional meta-analysis of gene expression data Basic Protocol 9: Functional analysis of transcriptomics signatures Basic Protocol 10: Dose-response and time-series data analysis Basic Protocol 11: RNA-seq reads processing and quantification with and without reference transcriptomes.

Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus.

Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and library construction, raising the need for efficient sample preservation methods which are compatible with scRNA-seq downstream analysis. Several studies evaluated the effect of methanol fixation as preservation method, yet none of them deeply assessed its effect on adult primary dissociated brain tissue. Here, we evaluated its effect on murine dentate gyrus (DG) single cell suspensions and on subsequent scRNA-seq downstream analysis by performing SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CEL-seq2 protocol. Our results show that MeOH fixation preserves RNA integrity and has no apparent effects on cDNA library construction. They also suggest that fixation protects from sorting-induced cell stress and increases the proportion of high-quality cells. Despite evidence of mRNA leakage in fixed cells, their relative gene expression levels correlate well with those of fresh cells and fixation does not significantly affect the variance of the dataset. Moreover, it allows the identification of all major DG cell populations, including neural precursors, granule neurons and different glial cell types, with a tendency to preserve more neurons that are underrepresented in fresh samples. Overall, our data show that MeOH fixation is suitable for preserving primary neural cells for subsequent single-cell RNA profiling, helping to overcome challenges arising from complex workflows, improve experimental flexibility and facilitate scientific collaboration.

Prevalence and determinants of hysterectomy in India

Increase in the prevalence of hysterectomy among low-and middle-income countries (LMICs) such as India has become a significant concern. Reports based on either a particular group or region show an increasing trend in hysterectomy, but there is a dearth of national-level data in this domain. Hence, there seems to be an urgent need to garner evidence on the prevalence and determinants of hysterectomy, which could pave the way for future programs and policies. We aimed to estimate the prevalence of hysterectomy and assess its determinants using a nationally representative sample. An observational analysis was conducted using data from Longitudinal Aging Study in India (LASI), 2017–2018. 38,154 women aged > 18 years were included. A multivariable logistic regression, presented as an adjusted odds ratio (AOR) with a 95% confidence interval (CI), was used to predict the association between various socio-demographic characteristics and hysterectomy. A separate multivariable logistic regression model was executed to determine the association between selected non-communicable diseases (NCDs) and hysterectomy. Survey weights compensated the complex study design. The overall prevalence of hysterectomy was around 11.35%. Excessive menstrual bleeding followed by fibroids emerged as the leading causes of hysterectomy. The various determinants of hysterectomy were urban residents [AOR: 1.54 (1.21–1.96)], other backward class [AOR: 2.19 (1.72–2.78], working women [AOR: 1.19(1–1.42)] and the most affluent (rich) group [AOR: 2.06 (1.62–2.63)]. Hysterectomy was associated with cancer [AOR: 4.83 (2.51–9.29)], diabetes [AOR: 1.79 (1.25–2.57)], hypertension [AOR: 1.48 (1.27–1.71)] and joint diseases [AOR: 1.43 (1.09–1.88)]. Hysterectomy is considerably prevalent in India, which cannot be overlooked. Health promotion regarding hysterectomy and its implications is needed especially among urban residents, affluent groups and those with a higher body mass index. Health programmes aimed at women should follow a life course approach by prioritizing health and overall well-being even after reproductive years.

ESMira: A decentralized open-source application for collecting experience sampling data.

This paper introduces ESMira, a server and mobile app (Android, iOS) developed for research projects using experience sampling method (ESM) designs. ESMira offers a very simple setup process and ease of use, while being free, decentralized, and open-source (source code is available on GitHub). The ongoing development of ESMira started in early 2019, with a focus on scientific requirements (e.g., informed consent, ethical considerations), data security (e.g., encryption), and data anonymity (e.g., completely anonymous data workflow). ESMira sets itself apart from other platforms by both being free of charge and providing study administrators with full control over study data without the need for specific technological skills (e.g., programming). This means that study administrators can have ESMira running on their own webspace without needing much technical knowledge, allowing them to remain independent from any third-party service. Furthermore, ESMira offers an extensive list of features (e.g., an anonymous built-in chat to contact participants; a reward system that allows participant incentivization without breaching anonymity; live graphical feedback for participants) and can deal with complex study designs (e.g., nested time-based sampling). In this paper, we illustrate the basic structure of ESMira, explain how to set up a new server and create studies, and introduce the platform's basic functionalities.

P-155 MicroRNA-155 (miR-155) expression profile and developmental characteristics of bovine early embryos are altered when cultured in group versus individually

Abstract Study question Culturing bovine preimplantation embryos in group versus individually has an impact on the expression profile of miR-155 and developmental characteristics. Summary answer MiR-155 was significantly greater in its expression when embryos were cultured in groups versus singly. Developmental characteristics of blastocysts were similar regardless of culture condition. What is known already MiRNAs are short non-coding RNAs that play important roles in many biological pathways including embryogenesis and are potent effectors of post-transcriptional gene silencing. It has been estimated that approximately 60% of all proteins-coding genes are regulated by miRNAs. MiR-155 is involved in early events of embryogenesis; however, it has not been characterized in bovine embryos and this knowledge has not yet been clinically translated. Existing literature indicates that group culture yields superior embryo development as embryos can benefit from autocrine signaling and paracrine communication but embryos in groups may be exposed to negative effects of dying or delayed embryos. Study design, size, duration The study consisted of two groups: zygotes cultured to blastocysts in group and those cultured individually. Each sample consists of 3 blastocysts. Droplet digital PCR (dd-PCR) was used to examine the expression of miR-155. Gene Set Enrichment Analysis (GSEA) of miRNAs was carried out to identify functional Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are enriched for the targets of one or more miRNAs. Participants/materials, setting, methods Oocytes were aspirated from antral follicles of bovine ovaries and matured in vitro. Twenty-four hours post retrieval, matured oocytes were inseminated with sperm from similar bull. Presumptive zygotes were cultured either in groups or individually until blastocyst stage, Blastocyst were collected and purified for RNA. Analysis for KEGG and GO Biological Process, Cellular Component and Molecular Functions were carried out on the identified target genes. Results were plotted according to level of enrichment for miR-155. Main results and the role of chance Five hundred and seventy-five oocytes were retrieved from 120 bovine ovaries. Cleavage rate and blastocyst formation after IVF were compared between those blastocysts cultured in groups versus individually. There was no significant difference among the two groups with regards to cleavage rate (69% vs. 54%) and blastocyst formation (42% vs.35%). ddPCR assessment showed significantly greater MiR-155 expression in those blastocysts cultured in groups (p = 0.002). The highest level of miR-151 expressed was 20 copies/sample. Every sample, regardless of the culture conditions, had detectable miR-155 level. The interaction of predicted miR-155 target clusters based on the GO Biological Processes shows that miR-155 plays a role in DNA binding, transcription factor binding, regulates transcription and affects cell adhesion. KEGG analysis revealed an association between miR-155 to steroidogenesis and controls apoptosis pathways. Limitations, reasons for caution The number of copies expressed for this miRNA was relatively low, which could suggest that miR-155 may not have a functional role at the blastocyst stage as the effectiveness of a miRNA depends on the relative number of binding site and target molecules. Blastocysts were pooled based on morphological assessment. Wider implications of the findings MiRNAs offer a novel mean to assess embryonic fitness. Further validation and more complex study designs are required to implement the profiling of embryonic miRNAs as a diagnostic test for embryo selection. Group culture increased miR-155 expression, possibly reflecting differences in cellular behavior between group cultured embryos and individually ones. Trial registration number Not applicable

Building Capacity for Research Ethics Committees in Uganda.

Timely communication from Research Ethics Committees (REC) to researchers is essential to meet deadlines. We conducted a capacity building program for REC members, REC and research administrators, and researchers from seven RECs in Uganda in order to improve the research regulatory approval cycle. The training was delivered from March 2020 to July 2021. Trainees were evaluated using pre and post-training tests. There was an increase in the average score from 38% to 53% in pre and post-training test respectively for the personal effectiveness and leadership programme for REC and research administrators. There was an increase in the average score of from 53.9% to 70.1% in pre and post-training test respectively for training on emerging and complex study designs. We achieved shift in knowledge and skills in use of the National Research Information Management System. We recommend regular training of REC members and administrators for efficient review of research protocols.

0501 Manganese and sleep health outcomes in U.S. adults: analysis of the NHANES 2017-2020

Abstract Introduction Manganese (Mn) is an essential trace mineral required for normal growth and physiological processes. However, inadequate or excess Mn can have a detrimental impact on human health. Despite the essentiality, little is known about the relationship between Mn and sleep. The objective of this study is to examine the relationships between blood Mn levels and sleep patterns (duration, midpoint of sleep, obstructive sleep apnea [OSA] symptoms, and trouble sleeping) in U.S. adults. Methods Data from the 2017 to 2020 National Health and Nutrition Examination Survey (NHANES) was used for the analysis. Adults aged 18 or older with both blood Mn levels and sleep parameters were included (n=8356). Self-reported fall asleep time and wake up time in the past week were used to calculate sleep duration and midpoint. Trouble sleeping and symptoms of OSA were also collected. Blood Mn levels were divided into quintiles for analysis. Multivariable logistic regressions that accounted for complex study design were used. Results The mean (SE) blood Mn concentration was 9.7 (0.1) µg/L, and was higher among participants who were younger, female, identified as non-Hispanic Asian, had lower income, were non-smokers, and had lower iron levels (P&amp;lt; 0.05, for all). About 25% of adults had short sleep duration (&amp;lt; 7 hours) on weekdays. OSA symptoms were noted in 49% of adults. In men, the odds of short sleep duration on weekdays were higher among those with the lowest Mn level (Q1: &amp;lt; 7.0 µg/L) than the reference group (Q3: 8.5-10.0 µg/L) after adjusting for age, race, income, smoking, and iron (OR: 1.5, 95% CI: 1.1-2.1, P=0.03). Among women, Q2 (7.0-8.4 µg/L) was the related to the lowest odds of OSA symptoms compared to all other groups after adjusting for covariates (P=0.049). Conclusion Lower or higher Mn levels in blood were related to poor sleep health in U.S. adults, which suggests that inadequate or excessive Mn intake might be harmful to sleep. Future studies on the mechanisms of these relationships are needed. Support (if any) This work was supported by National Institutes of Health R01DK123022, R21NS112974 to YAS, and K01HL151673 to ECJ.

Association between inflammatory markers and bone mineral density: a cross-sectional study from NHANES 2007–2010

PurposeMonocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) are acknowledged as novel inflammatory markers. However, studies investigating the correlation between inflammatory markers and osteoporosis (OP) remain scarce. We aimed to investigate the relationship between NLR, MLR, PLR and bone mineral density (BMD).MethodsA total of 9054 participants from the National Health and Nutrition Examination Survey were included in the study. MLR, NLR and PLR were calculated for each patient based on routine blood tests. Given the complex study design and sample weights, the relationship between inflammatory markers and BMD was evaluated through weighted multivariable-adjusted logistic regression and smooth curve fittings. In addition, several subgroup analyses were conducted to assess the robustness of the outcomes.ResultsThis study observed no significant relationship between MLR and lumbar spine BMD (P = 0.604). However, NLR was positively correlated with lumbar spine BMD (β = 0.004, 95% CI: 0.001 to 0.006, P = 0.001) and PLR was negatively linked to lumbar spine BMD (β = − 0.001, 95% CI: − 0.001 to − 0.000, P = 0.002) after accounting for covariates. When bone density measurements were changed to the total femur and femoral neck, PLR was still significantly positively correlated with total femur (β = − 0.001, 95% CI: − 0.001, − 0.000, P = 0.001) and femoral neck BMD (β = − 0.001, 95% CI: − 0.002, − 0.001, P < 0.001). After converting PLR to a categorical variable (quartiles), participants in the highest PLR quartile had a 0.011/cm2 lower BMD than those in the lowest PLR quartile (β = − 0.011, 95% CI: − 0.019, − 0.004, P = 0.005). According to subgroup analyses stratified by gender and age, the negative correlation with PLR and lumbar spine BMD remained in males and age < 18 groups, but not in female and other age groups.ConclusionsNLR and PLR were positively and negatively correlated with lumbar BMD, respectively. And PLR might serve as a potential inflammatory predictor of osteoporosis outperforming MLR and NLR. The complex correlation between the inflammation markers and bone metabolism requires further evaluation in large prospective studies.

MiGut: A scalable in vitro platform for simulating the human gut microbiome-Development, validation and simulation of antibiotic-induced dysbiosis.

In vitro models of the human colon have been used extensively in understanding the human gut microbiome (GM) and evaluating how internal and external factors affect the residing bacterial populations. Such models have been shown to be highly predictive of in vivo outcomes and have a number of advantages over animal models. The complexity required by in vitro models to closely mimic the physiology of the colon poses practical limits on their scalability. The scalable Mini Gut (MiGut) platform presented in this paper allows considerable expansion of model replicates and enables complex study design, without compromising on in vivo reflectiveness as is often the case with other model systems. MiGut has been benchmarked against a validated gut model in a demanding 9-week study. MiGut showed excellent repeatability between model replicates and results were consistent with those of the benchmark system. The novel technology presented in this paper makes it conceivable that tens of models could be run simultaneously, allowing complex microbiome-xenobiotic interactions to be explored in far greater detail, with minimal added resources or complexity. This platform expands the capacity to generate clinically relevant data to support our understanding of the cause-effect relationships that govern the GM.