Osteoporosis, the most common bone metabolic disease, impairs the ability of bone to sense and adapt to mechanical stimuli. Mechanical strain distribution plays a crucial role in bone adaptation, yet the molecular mechanisms underlying differential remodeling responses to distinct strain conditions remain unclear. To address this, we compared mouse tibiae subjected to axial compressive (physiological strain distribution) or medial-lateral (non-physiological strain distribution) loading. Strain distributions were calculated by finite element analysis and validated by strain gauge measurements, identifying the posterior-lateral (PL) site as a key region of differential strain between the 2 loading conditions. Loading was optimized to induce equivalent strain magnitude by opposite mode at this site and applied daily for 3 d to 16-wk-old female C57Bl/6J mice. Tibial cross-sections at 37% of the tibial length measured from the tibial plateau were analyzed using the GeoMX Digital Spatial Profiler to assess strain-dependent spatial transcriptomic changes. Medial-lateral loading elicited spatially distinct gene expression patterns at the PL site, with significant downregulation of genes involved in bone remodeling, cellular stress responses, and vasculature. These effects were not observed in axial compressive loading. Notably, the non-physiological tensile strain induced by medial-lateral loading exhibited unique transcriptomic profiles compared to physiological tensile strain, suggesting strain conditioning-dependent activation of bone homeostasis pathways. Our findings highlight the role of strain distribution in spatially regulating upstream signaling pathways driving bone adaptation. This study advances our understanding of how non-physiological strain condition influences bone remodeling and provides a foundation for developing therapeutic strategies for osteoporosis.

Fungal exposure is strongly implicated in the pathogenesis of asthma in horses, but the importance of specific fungi is unknown. Geographic variation in equine asthmatic endotypes is suspected and might be related to different fungal exposures due to different climatological and geographical conditions. This study had two objectives: evaluate the effect of the ecoregion upon BALF inflammatory cells and fungal community composition in horses with asthma and evaluate the effect of BALF fungal community composition upon the likelihood of neutrophilic, mastocytic and eosinophilic inflammation in these horses. Differential cytology counts were obtained from 916 BALF samples submitted from horses with poor performance and/or clinical signs of respiratory disease from five ecoregions. The effect of the ecoregion upon BALF inflammatory cell proportions was modeled using generalized linear models. Seventy banked BALF samples were subjected to sequencing of the internal transcribed spacer regions of fungal DNA. Diversity analysis was performed in QIIME, including alpha diversity metrics and the Bray-Curtis dissimilarity metric. After taxonomy was assigned, differential abundances between ecoregions and inflammatory phenotypes were estimated by generalized linear models in DESeq2. BALF neutrophil (p < 0.0001) and eosinophil (p < 0.0001) proportions varied by ecoregion, while mast cell proportions did not (p = 0.18). Alternaria, Aspergillus, Cladosporium and Epicoccum spp. were found to differ in abundance between regions. These geographical variations in fungal exposure might be responsible for differences in BALF neutrophil and eosinophil proportions between ecoregions.

Introduction/Objective:Single-cell RNA sequencing (scRNA-seq) resolves cell types and molecular phenotypes within heterogeneous specimens but typically requires fresh, high-quality single-cell suspensions processed immediately to preserve transcriptional profiles. This constraint complicates samples with long preparation times and prevents collection at remote sites lacking single-cell instrumentation. Several commercial assays now enable preservation at the point of collection through fixation or cryopreservation, allowing processing to occur months later. The Association of Biomolecular Research Facilities' DNA Sequencing and Genomics and Bioinformatics Research Groups undertook a cross-platform, multisite study to assess the performance and reproducibility of three such platforms: 10x Genomics FLEX, Parse Biosciences Evercode WT v2, and Honeycomb Bio HIVE. Materials and Methods:Total leukocytes and peripheral blood mononuclear cells (PBMCs) were isolated from a single healthy individual, with EasySep reagent used for red blood cell depletion of the leukocyte fraction. Cells were characterized by a 21-color flow cytometry panel to provide a reference, and the remaining material was fixed or cryopreserved according to each platform's protocol. Preserved leukocyte samples were prepared in parallel by two technicians ("A" and "B" replicates) and distributed to multiple ABRF member core facilities for downstream processing, while fresh leukocytes processed with the 10x 3' v3.1 (3pGEX) chemistry served as a reference. Libraries were sequenced at a central site, and performance was evaluated across standard scRNA-seq quality control metrics, gene and transcript detection sensitivity, cell-type discovery and annotation, differential expression, and correlation analyses. Results:Data from all platforms integrated effectively and produced concordant results for cell-type annotation and relative abundance, with cell-type proportions broadly consistent with the flow cytometry reference. However, platform-specific expression signatures were evident for a subset of genes, and cross-site reproducibility varied between methods, with the FLEX workflow showing greater susceptibility to technical variation introduced during on-site sample processing. Preservation-based methods (FLEX and HIVE) showed better retention of fragile granulocyte populations than fresh samples processed with the 10x 3pGEX workflow. Discussion:Improvements to preservation methods are changing how single-cell research is conducted by decoupling sample collection from downstream processing. Our investigation into the performance and reproducibility of each platform provides a resource to help investigators and core facilities select the most appropriate single-cell preservation workflow given their sample type, cell populations of interest, sample collection logistics, and laboratory infrastructure constraints.

Single cell RNA sequencing (scRNA-seq) is a revolutionary technique to identify cell types and their molecular phenotype in heterogeneous biological specimens. ScRNA-seq typically requires fresh, high quality single cell suspensions that are processed immediately to preserve their molecular profiles. This presents a challenge for samples with long preparation times and prevents collection at remote sites lacking the required instrumentation for sample processing. Recently, several commercial assays have been released that enable sample preservation at the time of collection either via fixation or cryopreservation, allowing for sample processing to occur months after the initial collection. The Association of Biomolecular Research Facilities' (ABRF) DNA Sequencing (DSRG) and Genomics Bioinformatics (GBiRG) Research Groups have undertaken a cross-platform, multi-site study to assess the performance and reproducibility of three platforms: a) 10x Genomics FLEX, b) Parse Bioscience Evercode WT v2 and c) Honeycomb Bio HIVE. Total leukocytes were isolated from a single healthy individual using the EasySep RBC depletion reagent. Cells were then characterized by collecting a 21-color flow cytometry dataset for reference and the remaining material was used for scRNA-seq procedures where different sites then processed either the fixed or cryopreserved cells for each method. We evaluated performance of each method across traditional scRNA-seq quality control metrics and analysis applications, including gene/transcript detection sensitivity, cell type discovery and annotation, and differential expression. We demonstrate that data from the methods tested can be effectively integrated and produce concordant results with regard to cell type annotation and relative abundance, though we observe platform-specific differences in the expression of a subset of genes. Preservation-based methods also show better retention of fragile granulocyte populations compared with fresh samples processed using the 10× 3' workflow. The improvements to preservation methods are changing the way research is conducted and our thorough investigation into the performance of each method provides a valuable resource to help scientists determine the most appropriate single cell preservation workflow given their sample collection logistics and laboratory infrastructure constraints.

In their natural environment, plants experience temperature fluctuations, including intensified heat waves driven by climate change, which pose significant threats to their productivity. To adapt, plants have evolved diverse mechanisms to withstand heat stress (HS), minimizing potential damage and ensuring survival. One such adaptation is acquired thermotolerance (AT), where prior exposure to HS primes plants to withstand subsequent severe HS. AT can persist for several days and involves a recovery period during which plants establish heat stress memory (HSM), reorganize cellular processes, and strengthen stress resilience. The molecular mechanisms underlying HSM remain the subject of active investigation. In this study, we employ a high-throughput comparative multi-omics approach to unravel the transcriptome, metabolome, and proteome of Arabidopsis thaliana seedlings during distinct intervals of the HSM phase. Our findings provide insights into the intricacies of HS recovery and the memory process. Notably, distinct temporal responses emerge at both the transcriptional and protein levels during the early and late recovery phases. Transcripts associated with HSM are upregulated during the early HS recovery phase, indicating a rapid response crucial for initial memory formation, while corresponding protein levels remain elevated throughout the recovery period, supporting memory consolidation. Additionally, metabolite profiles reveal distinctive patterns across the HS memory phase. This marks the first detailed multi-omic analysis of the HSM phase in Arabidopsis seedlings, providing insights into the multifaceted nature of this complex process. These comprehensive datasets hold promise in elucidating regulators of HS resilience, thereby enhancing efforts in breeding HS-tolerant crops ### Competing Interest Statement The authors have declared no competing interest.

Candida auris is an emerging multidrug-resistant fungal pathogen that preferentially colonizes and persists in skin tissue, yet the host immune factors that regulate the skin colonization of C. auris in vivo are unknown. In this study, we employed unbiased single-cell transcriptomics of murine skin infected with C. auris to understand the cell type-specific immune response to C. auris. C. auris skin infection results in the accumulation of immune cells such as neutrophils, inflammatory monocytes, macrophages, dendritic cells, T cells, and NK cells at the site of infection. We identified fibroblasts as a major non-immune cell accumulated in the C. auris infected skin tissue. The comprehensive single-cell profiling revealed the transcriptomic signatures in cytokines, chemokines, host receptors (TLRs, C-type lectin receptors, NOD receptors), antimicrobial peptides, and immune signaling pathways in individual immune and non-immune cells during C. auris skin infection. Our analysis revealed that C. auris infection upregulates the expression of the IL-1RN gene (encoding IL-1R antagonist protein) in different cell types. We found IL-1Ra produced by macrophages during C. auris skin infection decreases the killing activity of neutrophils. Furthermore, C. auris uses a unique cell wall mannan outer layer to evade IL-1R-signaling mediated host defense. Collectively, our single-cell RNA seq profiling identified the transcriptomic signatures in immune and non-immune cells during C. auris skin infection. Our results demonstrate the IL-1Ra and IL-1R-mediated immune evasion mechanisms employed by C. auris to persist in the skin. These results enhance our understanding of host defense and immune evasion mechanisms during C. auris skin infection and identify potential targets for novel antifungal therapeutics.

Aims To perform an integrated comparative analysis of metabolic pathway to understand coenzyme Q10 (CoQ10) production in Agrobacterium tumefaciens. Methods and results Comparative analysis of the CoQ10 metabolic pathway in 10 organisms using a genome to KEGG orthology program (G2KO) and the KEGG database elucidated the completeness of the production pathway in A. tumefaciens. The specific roles of the key precursors and the enzymes in the metabolic network were subsequently confirmed using pathway inhibitors and enhancers. While the use of fosmidomycin and glyphosate was found to inhibit CoQ10 production by 54.54% to 99%, the supplementation of polyprenyl pyrophosphate of the methylerythritol 4-phosphate pathway and 4-hydroxybenzoate precursor of the shikimate pathway did increse the production of CoQ10 by 2.3-fold. Conclusions The present study provides a comprehensive understanding of the CoQ10 biosynthetic pathway in A. tumefaciens, which would assist rational metabolic engineering strategies for augmenting CoQ10 biosynthesis.

The porcine immune system has an important role in pre-clinical studies together with understanding the biological response mechanisms before entering into clinical trials. The size distribution of the Korean minipig is an important feature that make this breed ideal for biomedical research and safe practice in post clinical studies. The extremely tiny (ET) minipig serves as an excellent model for various biomedical research studies, but the comparatively frail and vulnerable immune response to the environment over its Large (L) size minipig breed leads to additional after born care. To overcome this pitfall, comparative analysis of the genomic regions under selection in the L type breed could provide a better understanding at the molecular level and lead to the development of an enhanced variety of ET type minipig. In this study, we utilized whole genome sequencing (WGS) to identify traces of artificial selection and integrated them with transcriptome data generated from blood samples to find strongly selected and differentially expressed genes of interest. We identified a total of 35 common genes among which 7 were differentially expressed and showed selective sweep in the L type over the ET type minipig breed. The stabilization of these genes were further confirmed using nucleotide diversity analysis, and these genes could serve as potential biomarkers for the development of a better variety of ET type pig breed.

Wireless Sensor Network (WSN) is built with the wireless interconnection of Sensor Nodes (SNs) generally deployed to monitor the changes within the environment of hostile, rugged, and unreachable target regions. The optimal placement of SNs is very important for the efficient and effective operation of any WSN. Unlike small and reachable regions, the deployment of the SNs in large-scale regions (e.g., forest regions, nuclear radiation affected regions, international border regions, natural calamity affected regions, etc.) is substantially challenging. Present paper deals with an autonomous air-bone scheme for the precise placement of SNs in such large-scale regions. It uses an Omni-directional Circular Glider (OCG) per SN. After being aerially dropped, SN pilots the OCG to glide itself to the predetermined locations (PL) within a target region. The major advantage of using OCG is its capability to quickly update the direction, during the flight (with turning radius = 0) toward its PL. The proposed uses a recursive path correction model to maintain the orientation of the gliding SN towards the PL. The simulation results, and the hardware implementation, indicate that the proposed model is effectively operational in the environmental winds. It is time-efficient and more accurate in the deployment of the SNs in comparison to existing state of art SN deployment models.

Globally, lipstatin/orlistat has been prescribed as anti-obesity drug since long with the various trade names like xenical, and alli. Recently, it evolved with other medical usages such as anti-tumor, anti-viral and, anti-stress. The main objective of this research is to study the different pathway modeling and simulation aspects along with carbon conserving non-oxidative glycolysis pathway (NOG) for more lipstatin biosynthesis in Streptomyces toxytricini using system biology approach. NOG pathway, have potential to give more acetyl-CoA which will further enhanced the fatty acid synthesis resulted into high lipstatin production. Lipstatin biosynthesis pathway constructed without NOG gives less amount of lipstatin (2.18 mg/mL) compared to model with NOG (8.25 mg/ mL). Topological analysis reveals constructed model without NOG have 154 nodes and 177 edges and model with NOG contain 185 nodes and 223 edges. The finding of present study based on modeling and simulation of lipstatin biosynthetic map should be useful in the future in metabolic engineering of lipstatin biosynthesis pathway.

There is a growing interest to use in vitro BBB cell assays in early safety assessment of compounds. By modifying a well-validated co-culture model of brain capillary endothelial and glial cells, developed by Dehouck et al. [Dehouck, M.P., Meresse, S., Delorme, P., Fruchart, J.C., Cecchelli, R., 1990. An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. Journal of Neurochemistry 54 (5), 1798-1801], it has been possible to develop a new in vitro BBB system suitable for high throughput screening (HTS). In addition, this new procedure substantially reduces the use of experimental animals and considerably facilitates the process of obtaining a functional in vitro BBB model. The model is ready to use after only 4 days of culture and then shows the typical expression and localization of tight junction proteins. The function of the P-glycoprotein and the transcriptional expression of other efflux transporters such as MRP 1, 4 and 5 have been demonstrated. In addition, the model produces a good in vitro/in vivo correlation for 10 compounds (R2=0.81). Furthermore, studies were undertaken within the European ACuteTox consortium with the objective to assess BBB toxicity and make risk assessments of potentially toxic compounds according to their predicted ability to reach the CNS compartment. These investigations demonstrated that the results produced in the HTS BBB model were similar to the standard co-culture model.

Rice is an important staple food grain consumed by most of the population around the world. With climate and environmental changes, rice has undergone a tremendous stress state which has impacted crop production and productivity. Plant growth hormones are essential component that controls the overall outcome of the growth and development of the plant. Cytokinin is a hormone that plays an important role in plant immunity and defense systems. Trans-zeatin is an active form of cytokinin that can affect plant growth which is mediated by a multi-step two-component phosphorelay system that has different roles in various developmental stages. Systems biology is an approach for pathway analysis to trans-zeatin treated rice that could provide a deep understanding of different molecules associated with them. In this study, we have used a weighted gene co-expression network analysis method to identify the functional modules and hub genes involved in the cytokinin pathway. We have identified nine functional modules comprising of different hub genes which contribute to the cytokinin signaling route. The biological significance of these identified hub genes has been tested by applying well-proven statistical techniques to establish the association with the experimentally validated QTLs and annotated by the DAVID server. The establishment of key genes in different pathways has been confirmed. These results will be useful to design new stress-resistant cultivars which can provide sustainable yield in stress-specific conditions.

A series of bifunctionalized bis-1,2,4-triazolylcarbodithioates/carbamodithioates having metal ions as linkers were synthesized based on the multitargeted strategy against phytopathogenic fungi under resistance management measure. All the synthesized compounds showed significant enhancement in potential against Fusarium verticillioides and &polaris oryzae, synergized by zinc covalency. Zinc bis(1H-1,2,4-triazole-1-carbodithioate) inflicted the best potential against the test fungi with ED50 values <5.0 mu g/ml which was much lower than their mono analogs, other metal analogs as well as standard commercial fungicides. Well fit structure into the active site of 14-alpha demethylase inhibitors and favorable physical parameters due to presence of zinc as linker were the rationale behind the augmented bioactivity. The ultramicroscopic details revealed fungal cell wall distortions caused by the compounds. Toxicity analysis of the molecules is another most prominent feature needed to be analyzed for their use as plant protection material. In silico toxicity analysis using ProTox-II webserver, frontier molecular orbital approach, and actual cell cytotoxicity assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) revealed that zinc bis(1H-1,2,4-triazole-1-carbodithioate) belongs to "Class IV" of pesticides which corresponded well with the actual toxicity analysis, on most sensitive mammalian stem cells, confirming the hypotoxicity of these compounds, recommending their further exploration for bioapplication.

Chicken is important livestock that serves as a vital food source which remain largely affected by heat stress. Therefore, we performed the transcriptome analysis to help understand the mechanisms of heat stress response in chickens. In the animal experiments, we grouped them into a normal and severe at 21 and 33 °C, with identified physiologic parameters for 2-weeks. Subsequently, RNA-seq analysis was performed to identify DEGs with a false discovery rate < 0.05 and a fold change ≥ 1.5. In the physiological parameters, we observed average daily gain was declined, rectal temperature and respiration rate was increased in severe group. Among total 245 DEGs, 230 and 15 genes were upregulated and downregulated, respectively. In upregulated DEGs, HSPs, MYLK2, and BDKRB1 genes were identified as key genes in heat stress. The KEGG pathway analysis showed involvement in the ATP metabolic process, MAPK signaling pathway and calcium signaling pathway with related protein processing and synthesis. In conclusion, with induced heat stress, such changes in physiologic parameters alter the neuroendocrine system, and we observed that the heat stress environment regulates such Heat shock protein genes to protect the cells and proteins from an altered metabolism. These findings provide a more comprehensive understanding of the heat stress response in poultry.

Pig as a food source serves daily dietary demand to a wide population around the world. Preference of meat depends on various factors with muscle play the central role. In this regards, selective breeding abled us to develop “Nanchukmacdon” a pig breeds with an enhanced variety of meat and high fertility rate. To identify genomic regions under selection we performed whole-genome resequencing, transcriptome, and whole-genome bisulfite sequencing from Nanchukmacdon muscles samples and used published data for three other breeds such as Landrace, Duroc, Jeju native pig and analyzed the functional characterization of candidate genes. In this study, we present a comprehensive approach to identify candidate genes by using multi-omics approaches. We performed two different methods XP-EHH, XP-CLR to identify traces of artificial selection for traits of economic importance. Moreover, RNAseq analysis was done to identify differentially expressed genes in the crossed breed population. Several genes (UGT8, ZGRF1, NDUFA10, EBF3, ELN, UBE2L6, NCALD, MELK, SERP2, GDPD5, and FHL2) were identified as selective sweep and differentially expressed in muscles related pathways. Furthermore, nucleotide diversity analysis revealed low genetic diversity in Nanchukmacdon for identified genes in comparison to related breeds and whole-genome bisulfite sequencing data shows the critical role of DNA methylation pattern in identified genes that leads to enhanced variety of meat. This work demonstrates a way to identify the molecular signature and lays a foundation for future genomic enabled pig breeding.

The growing need for sustainable technologies has attracted considerable interest in the synthesis of ecofriendly materials. This paper reports the anti-inflammatory and antibacterial activities of sustainable silver nanoparticles (AgNPs) fabricated using endophytic fungus extracted from a medicinal plant, Cassia fistula. Fourier transform-infrared and UV-visible were used for AgNPs characterisation. X-ray diffraction, scanning electron microscope, energy dispersive X-ray analysis, atomic force microscope (AFM), transmission electron microscope and dynamic light scattering analysis revealed that the biosynthesised AgNPs were within the size of similar to 4-54 nm. The synthesised AgNPs displayed considerable antibacterial activity against Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia bacterial strains. Additionally, synthesised AgNPs showed significant anti-inflammatory potential.

BACKGROUND:DNA methylation and demethylation at CpG islands is one of the main regulatory factors that allow cells to respond to different stimuli. These regulatory mechanisms help in developing tissue without affecting the genomic composition or undergoing selection. Liver and backfat play important roles in regulating lipid metabolism and control various pathways involved in reproductive performance, meat quality, and immunity. Genes inside these tissue store a plethora of information and an understanding of these genes is required to enhance tissue characteristics in the future generation.RESULTS:A total of 16 CpG islands were identified, and they were involved in differentially methylation regions (DMRs) as well as differentially expressed genes (DEGs) of liver and backfat tissue samples. The genes C7orf50, ACTB and MLC1 in backfat and TNNT3, SIX2, SDK1, CLSTN3, LTBP4, CFAP74, SLC22A23, FOXC1, GMDS, GSC, GATA4, SEMA5A and HOXA5 in the liver, were categorized as differentially-methylated. Subsequently, Motif analysis for DMRs was performed to understand the role of the methylated motif for tissue-specific differentiation. Gene ontology studies revealed association with collagen fibril organization, the Bone Morphogenetic Proteins (BMP) signaling pathway in backfat and cholesterol biosynthesis, bile acid and bile salt transport, and immunity-related pathways in methylated genes expressed in the liver.CONCLUSIONS:In this study, to understand the role of genes in the differentiation process, we have performed whole-genome bisulfite sequencing (WGBS) and RNA-seq analysis of Nanchukmacdon pigs. Methylation and motif analysis reveals the critical role of CpG islands and transcriptional factors binding site (TFBS) in guiding the differential patterns. Our findings could help in understanding how methylation of certain genes plays an important role and can be used as biomarkers to study tissue specific characteristics.