Investigating the effects of synbiotic supplementation on functional movement, strength and muscle health in older Australians: a study protocol for a double-blind, randomized, placebo-controlled trial.

BACKGROUND: Aging has been associated with a progressive loss of skeletal muscle quality, quantity and strength, which may result in a condition known as sarcopenia, leading to a decline in physical performance, loss of independence and reduced quality of life. While the cause of impaired physical functioning observed in elderly populations appears to be multifactorial, recent evidence suggests that age-associated alterations in gut microbiota could be a contributing factor. The primary objective will be to assess the effects of a dietary synbiotic formulation on sarcopenia-related functional outcomes such as handgrip strength, gait speed and physical performance within older individuals living independently. The secondary objective will be to examine associations between changes in gut microbiota composition, functional performance and lean muscle mass. METHODS: Seventy-four elderly (60-85 years) participants will be randomized in a double-blind, placebo-controlled fashion to either an intervention or control group. The intervention group (n = 37) will receive oral synbiotic formulation daily for 16 weeks. The control group (n = 37) will receive placebo. Assessments of physical performance (including Short Physical Performance Battery, handgrip strength and timed up-and-go tests) and muscle ultrasonography will be performed at 4 time points (baseline and weeks 8, 16 and 20). Likewise, body composition via bioelectric impedance analysis and blood and stool samples will be collected at each time point. Dual-energy X-ray absorptiometry will be performed at baseline and week 16. The primary outcomes will be between-group changes in physical performance from baseline to 16 weeks. Secondary outcomes include changes in body composition, muscle mass and architecture, fecal microbiota composition and diversity, and fecal and plasma metabolomics. DISCUSSION: Gut-modulating supplements appear to be effective in modifying gut microbiota composition in healthy older adults. However, it is unclear whether these changes translate into functional and/or health improvements. In the present study, we will investigate the effects of a synbiotic formulation on measures of physical performance, strength and muscle health in healthy older populations. TRIAL REGISTRATION: This study was prospectively registered with the Australian New Zealand Clinical Trials Registry (ACTRN12622000652774) in May 2022.

Automated staging of zebrafish embryos with deep learning.

The zebrafish (Danio rerio) is an important biomedical model organism used in many disciplines. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype. However, the detection and quantification of these delays is often achieved through manual observation, which is both time-consuming and subjective. We present KimmelNet, a deep learning model trained to predict embryo age (hours post fertilisation) from 2D brightfield images. KimmelNet's predictions agree closely with established staging methods and can detect developmental delays between populations with high confidence using as few as 100 images. Moreover, KimmelNet generalises to previously unseen data, with transfer learning enhancing its performance. With the ability to analyse tens of thousands of standard brightfield microscopy images on a timescale of minutes, we envisage that KimmelNet will be a valuable resource for the developmental biology community. Furthermore, the approach we have used could easily be adapted to generate models for other organisms.

Bridging imaging users to imaging analysis - A community survey.

The 'Bridging Imaging Users to Imaging Analysis' survey was conducted in 2022 by the Center for Open Bioimage Analysis (COBA), BioImaging North America (BINA) and the Royal Microscopical Society Data Analysis in Imaging Section (RMS DAIM) to understand the needs of the imaging community. Through multichoice and open-ended questions, the survey inquired about demographics, image analysis experiences, future needs and suggestions on the role of tool developers and users. Participants of the survey were from diverse roles and domains of the life and physical sciences. To our knowledge, this is the first attempt to survey cross-community to bridge knowledge gaps between physical and life sciences imaging. Survey results indicate that respondents' overarching needs are documentation, detailed tutorials on the usage of image analysis tools, user-friendly intuitive software, and better solutions for segmentation, ideally in a format tailored to their specific use cases. The tool creators suggested the users familiarise themselves with the fundamentals of image analysis, provide constant feedback and report the issues faced during image analysis while the users would like more documentation and an emphasis on tool friendliness. Regardless of the computational experience, there is a strong preference for 'written tutorials' to acquire knowledge on image analysis. We also observed that the interest in having 'office hours' to get an expert opinion on their image analysis methods has increased over the years. The results also showed less-than-expected usage of online discussion forums in the imaging community for solving image analysis problems. Surprisingly, we also observed a decreased interest among the survey respondents in deep/machine learning despite the increasing adoption of artificial intelligence in biology. In addition, the community suggests the need for a common repository for the available image analysis tools and their applications. The opinions and suggestions of the community, released here in full, will help the image analysis tool creation and education communities to design and deliver the resources accordingly.

Epi-microRNA mediated metabolic reprogramming ensures affinity maturation.

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centres (GCs). Among those, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by relying on oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)-155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Transcriptome examination and mass spectrometry analysis revealed that miR-155 regulates H3K36me2 levels by directly repressing hypoxia-induced histone lysine demethylase, Kdm2a. This is indispensable for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia. The miR-155-Kdm2a interaction is crucial to prevent excessive production of reactive oxygen species and apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity clones, ensuring their expansion and consequently affinity maturation.

Bridging Imaging Users to Imaging Analysis - A community survey.

The "Bridging Imaging Users to Imaging Analysis" survey was conducted in 2022 by the Center for Open Bioimage Analysis (COBA), Bioimaging North America (BINA), and the Royal Microscopical Society Data Analysis in Imaging Section (RMS DAIM) to understand the needs of the imaging community. Through multi-choice and open-ended questions, the survey inquired about demographics, image analysis experiences, future needs, and suggestions on the role of tool developers and users. Participants of the survey were from diverse roles and domains of the life and physical sciences. To our knowledge, this is the first attempt to survey cross-community to bridge knowledge gaps between physical and life sciences imaging. Survey results indicate that respondents' overarching needs are documentation, detailed tutorials on the usage of image analysis tools, user-friendly intuitive software, and better solutions for segmentation, ideally in a format tailored to their specific use cases. The tool creators suggested the users familiarize themselves with the fundamentals of image analysis, provide constant feedback, and report the issues faced during image analysis while the users would like more documentation and an emphasis on tool friendliness. Regardless of the computational experience, there is a strong preference for 'written tutorials' to acquire knowledge on image analysis. We also observed that the interest in having 'office hours' to get an expert opinion on their image analysis methods has increased over the years. In addition, the community suggests the need for a common repository for the available image analysis tools and their applications. The opinions and suggestions of the community, released here in full, will help the image analysis tool creation and education communities to design and deliver the resources accordingly.

WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

Measuring "pain load" during general anesthesia.

Introduction: Functional near-infrared spectroscopy (fNIRS) allows for ongoing measures of brain functions during surgery. The ability to evaluate cumulative effects of painful/nociceptive events under general anesthesia remains a challenge. Through observing signal differences and setting boundaries for when observed events are known to produce pain/nociception, a program can trigger when the concentration of oxygenated hemoglobin goes beyond ±0.3 mM from 25 s after standardization. Method: fNIRS signals were retrieved from patients undergoing knee surgery for anterior cruciate ligament repair under general anesthesia. Continuous fNIRS measures were measured from the primary somatosensory cortex (S1), which is known to be involved in evaluation of nociception, and the medial polar frontal cortex (mPFC), which are both involved in higher cortical functions (viz. cognition and emotion). Results: A ±0.3 mM threshold for painful/nociceptive events was observed during surgical incisions at least twice, forming a basis for a potential near-real-time recording of pain/nociceptive events. Evidence through observed true positives in S1 and true negatives in mPFC are linked through statistically significant correlations and this threshold. Conclusion: Our results show that standardizing and observing concentrations over 25 s using the ±0.3 mM threshold can be an arbiter of the continuous number of incisions performed on a patient, contributing to a potential intraoperative pain load index that correlates with post-operative levels of pain and potential pain chronification.

GIANI - open-source software for automated analysis of 3D microscopy images.

The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.

CD33 Expression on Peripheral Blood Monocytes Predicts Efficacy of Anti-PD-1 Immunotherapy Against Non-Small Cell Lung Cancer.

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related deaths globally. Immune checkpoint blockade (ICB) has transformed cancer medicine, with anti-programmed cell death protein 1 (anti-PD-1) therapy now well-utilized for treating NSCLC. Still, not all patients with NSCLC respond positively to anti-PD-1 therapy, and some patients acquire resistance to treatment. There remains an urgent need to find markers predictive of anti-PD-1 responsiveness. To this end, we performed mass cytometry on peripheral blood mononuclear cells from 26 patients with NSCLC during anti-PD-1 treatment. Patients who responded to anti-PD-1 ICB displayed significantly higher levels of antigen-presenting myeloid cells, including CD9+ nonclassical monocytes, and CD33hi classical monocytes. Using matched pre-post treatment samples, we found that the baseline pre-treatment frequencies of CD33hi monocytes predicted patient responsiveness to anti-PD-1 therapy. Moreover, some of these classical and nonclassical monocyte subsets were associated with reduced immunosuppression by T regulatory (CD4+FOXP3+CD25+) cells in the same patients. Our use of machine learning corroborated the association of specific monocyte markers with responsiveness to ICB. Our work provides a high-dimensional profile of monocytes in NSCLC and links CD33 expression on monocytes with anti-PD-1 effectiveness in patients with NSCLC.

Automated staging of zebrafish embryos using machine learning.

The zebrafish ( Danio rerio), is an important biomedical model organism used in many disciplines, including development, disease modeling and toxicology, to better understand vertebrate biology. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype, and accurate characterization of such delays is imperative. Despite this, the only way at present to identify and quantify these delays is through manual observation, which is both time-consuming and subjective. Machine learning approaches in biology are rapidly becoming part of the toolkit used by researchers to address complex questions. In this work, we introduce a machine learning-based classifier that has been trained to detect temporal developmental differences across groups of zebrafish embryos. Our classifier is capable of rapidly analyzing thousands of images, allowing comparisons of developmental temporal rates to be assessed across and between experimental groups of embryos. Finally, as our classifier uses images obtained from a standard live-imaging widefield microscope and camera set-up, we envisage it will be readily accessible to the zebrafish community, and prove to be a valuable resource.

RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering.

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes.

The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.

A FIJI macro for quantifying pattern in extracellular matrix.

Diverse extracellular matrix patterns are observed in both normal and pathological tissue. However, most current tools for quantitative analysis focus on a single aspect of matrix patterning. Thus, an automated pipeline that simultaneously quantifies a broad range of metrics and enables a comprehensive description of varied matrix patterns is needed. To this end, we have developed an ImageJ plugin called TWOMBLI, which stands for The Workflow Of Matrix BioLogy Informatics. This pipeline includes metrics of matrix alignment, length, branching, end points, gaps, fractal dimension, curvature, and the distribution of fibre thickness. TWOMBLI is designed to be quick, versatile and easy-to-use particularly for non-computational scientists. TWOMBLI can be downloaded from https://github.com/wershofe/TWOMBLI together with detailed documentation and tutorial video. Although developed with the extracellular matrix in mind, TWOMBLI is versatile and can be applied to vascular and cytoskeletal networks. Here we present an overview of the pipeline together with examples from a wide range of contexts where matrix patterns are generated.

Fission yeast condensin contributes to interphase chromatin organization and prevents transcription-coupled DNA damage.

BACKGROUND: Structural maintenance of chromosomes (SMC) complexes are central organizers of chromatin architecture throughout the cell cycle. The SMC family member condensin is best known for establishing long-range chromatin interactions in mitosis. These compact chromatin and create mechanically stable chromosomes. How condensin contributes to chromatin organization in interphase is less well understood. RESULTS: Here, we use efficient conditional depletion of fission yeast condensin to determine its contribution to interphase chromatin organization. We deplete condensin in G2-arrested cells to preempt confounding effects from cell cycle progression without condensin. Genome-wide chromatin interaction mapping, using Hi-C, reveals condensin-mediated chromatin interactions in interphase that are qualitatively similar to those observed in mitosis, but quantitatively far less prevalent. Despite their low abundance, chromatin mobility tracking shows that condensin markedly confines interphase chromatin movements. Without condensin, chromatin behaves as an unconstrained Rouse polymer with excluded volume, while condensin constrains its mobility. Unexpectedly, we find that condensin is required during interphase to prevent ongoing transcription from eliciting a DNA damage response. CONCLUSIONS: In addition to establishing mitotic chromosome architecture, condensin-mediated long-range chromatin interactions contribute to shaping chromatin organization in interphase. The resulting structure confines chromatin mobility and protects the genome from transcription-induced DNA damage. This adds to the important roles of condensin in maintaining chromosome stability.

Creating a Practice-Based Implementation Network: Facilitating Practice Change Across Health Care Systems.

A proof-of-concept practice-based implementation network was developed in the US Departments of Veteran Affairs (VA) and Defense to increase the speed of implementation of mental health practices, derive lessons learned prior to larger-scale implementation, and facilitate organizational learning. One hundred thirty-four clinicians in 18 VA clinics received brief training in the use of the PTSD checklist (PCL) in clinical care. Two implementation strategies, external facilitation and technical assistance, were used to encourage the use of outcomes data to inform treatment decisions and increase discussion of results with patients. There were mixed results for changes in the frequency of PCL administration, but consistent increases in clinician use of data and incorporation into the treatment process via discussion. Programs and clinicians were successfully recruited to participate in a 2-year initiative, suggesting the feasibility of using this organizational structure to facilitate the implementation of new practices in treatment systems.

Development of a Leader Tool for Assessing and Mitigating Suicide Risk Factors.

INTRODUCTION: Despite efforts in prevention, suicide rates in the US military remain unchanged. This article describes the development of a tool for leaders to identify and mitigate suicide risk factors. METHODS: A seven-item measure, the Leader Suicide Risk Assessment Tool (LSRAT), was constructed to allow leaders to assess and mitigate suicide drivers. During a 6-mo pilot, unit leaders completed the LSRAT for 161 at-risk soldiers. The LSRAT data were compared to clinical data from a subset of these soldiers. RESULTS: The LSRAT showed good test-retest reliability. The LSRAT scores showed significant correlations with both clinical and screening measures of suicidality. Command actions mitigated or partially mitigated 89% of risk factors identified on the LSRAT. CONCLUSIONS: This study provides initial psychometric data on a tool that prescribes concrete responses to mitigate risk. The LSRAT may be a valid and feasible tool to assist front-line commanders in identifying potential area's risk mitigation. Synchronization efforts between commanders, clinicians, and support services are crucial to ensure effective intervention to prevent suicide behavior.

Mitigation of impact of a major benzene spill into a river through flow control and in-situ activated carbon absorption.

Benzene is a toxic contaminant and can harm many aquatic species and cause serious damages to the river eco-system, if released to rivers. In 2012, a major spill accident occurred on the Huaihe River in Eastern China with 3 tons of benzene released to the river section 70 km upstream of a natural reserve. Two emergency measures were taken to minimize the impact of the accident on the natural reserve: 1) flow control by adjusting upstream sluices to delay the arrival of the contaminant plume at the reserve and 2) in-situ treatment using activated carbons to reduce the contaminant concentration. Here we develop a process-based mathematical model to analyze the monitoring data collected shortly after the accident, and explore not only how effective the adopted measures were over the incident but more importantly the mechanisms and critical conditions underlying the effectiveness of these measures. The model can be used as a tool for designing optimal management responses to similar spill accidents in regulated river systems, combining flow control and in-situ treatment.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

CD9 identifies pancreatic cancer stem cells and modulates glutamine metabolism to fuel tumour growth.

Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9high cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9high cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9low cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRasG12D; p53F/F mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation.

Matrix feedback enables diverse higher-order patterning of the extracellular matrix.

The higher-order patterning of extra-cellular matrix in normal and pathological tissues has profound consequences on tissue function. Whilst studies have documented both how fibroblasts create and maintain individual matrix fibers and how cell migration is altered by the fibers they interact with, a model unifying these two aspects of tissue organization is lacking. Here we use computational modelling to understand the effect of this interconnectivity between fibroblasts and matrix at the mesoscale level. We created a unique adaptation to the Vicsek flocking model to include feedback from a second layer representing the matrix, and use experimentation to parameterize our model and validate model-driven hypotheses. Our two-layer model demonstrates that feedback between fibroblasts and matrix increases matrix diversity creating higher-order patterns. The model can quantitatively recapitulate matrix patterns of tissues in vivo. Cells follow matrix fibers irrespective of when the matrix fibers were deposited, resulting in feedback with the matrix acting as temporal 'memory' to collective behaviour, which creates diversity in topology. We also establish conditions under which matrix can be remodelled from one pattern to another. Our model elucidates how simple rules defining fibroblast-matrix interactions are sufficient to generate complex tissue patterns.