Tissue Types Research Articles

TauFisher predicts circadian time from a single sample of bulk and single-cell pseudobulk transcriptomic data

As the circadian clock regulates fundamental biological processes, disrupted clocks are often observed in patients and diseased tissues. Determining the circadian time of the patient or the tissue of focus is essential in circadian medicine and research. Here we present tauFisher, a computational pipeline that accurately predicts circadian time from a single transcriptomic sample by finding correlations between rhythmic genes within the sample. We demonstrate tauFisher’s performance in adding timestamps to both bulk and single-cell transcriptomic samples collected from multiple tissue types and experimental settings. Application of tauFisher at a cell-type level in a single-cell RNAseq dataset collected from mouse dermal skin implies that greater circadian phase heterogeneity may explain the dampened rhythm of collective core clock gene expression in dermal immune cells compared to dermal fibroblasts. Given its robustness and generalizability across assay platforms, experimental setups, and tissue types, as well as its potential application in single-cell RNAseq data analysis, tauFisher is a promising tool that facilitates circadian medicine and research.

Immunoengineering Biomaterials for Musculoskeletal Tissue Repair across Lifespan.

Musculoskeletal diseases and injuries are among the leading causes of pain and morbidity worldwide. Broad efforts have focused on developing pro-regenerative biomaterials to treat musculoskeletal conditions; however, these approaches have yet to make a significant clinical impact. Recent studies have demonstrated that the immune system is central in orchestrating tissue repair and that targeting pro-regenerative immune responses can improve biomaterial therapeutic outcomes. However, aging is a critical factor negatively affecting musculoskeletal tissue repair and immune function. Hence, understanding how age affects the response to biomaterials is essential for improving musculoskeletal biomaterial therapies. This review focuses on the intersection of the immune system and aging in response to biomaterials for musculoskeletal tissue repair. The article introduces the general impacts of aging on tissue physiology, the immune system, and the response to biomaterials. Then, it explains how the adaptive immune system guides the response to injury and biomaterial implants in cartilage, muscle, and bone and discusses how aging impacts these processes in each tissue type. The review concludes by highlighting future directions for the development and translation of personalized immunomodulatory biomaterials for musculoskeletal tissue repair.

Identification of hypoxia-induced metabolism-associated genes in canine tumours.

Canine tumours including urothelial carcinoma, lung adenocarcinoma, mammary gland tumour, squamous cell carcinoma, and melanoma have been identified as causes of death, but effective therapies are limited due to insufficient knowledge of the molecular mechanisms involved. Within the tumour microenvironment, hypoxia activates hypoxia-inducible factor 1α (HIF1α) in tumour cells. High HIF1α expression correlates with enhanced glycolysis and poorer outcomes in human cancers. However, the molecular mechanisms underlying hypoxic tumour cells remain elusive in dogs. In our study, we investigated upregulated genes in a canine malignant melanoma cell line during hypoxia using RNA-sequencing analysis. Glycolysis and HIF1 signalling pathways were upregulated in hypoxic melanoma cells. HIF1α knockout melanoma cells revealed that the glycolysis marker MCT4 is regulated by HIF1α activation. Hypoxia induces high lactate secretion due to enhanced glycolysis in canine melanoma cells. Furthermore, we examined monocarboxylate transporter 4 (MCT4) expression in malignant melanoma and eight other types of canine tumour tissues using immunohistochemistry (IHC). Membrane-localized MCT4 protein was mostly detected in urothelial carcinoma and lung adenocarcinoma rather than malignant melanoma. We conclude that canine MCT4 protein plays a role in lactic acid efflux from glycolytic cells and may serve as a marker for hypoxia and glycolysis in canine tumours. These findings could inform future therapeutic strategies targeting MCT4.

Ultrasound-Assisted Extraction of Bioactive Compounds from Broccoli By-Products.

The objective of this work was to gain insight into the operating conditions that affect the efficiency of ultrasound-assisted extraction (UAE) parameters to achieve the best recovery of bioactive compounds from broccoli leaf and floret byproducts. Therefore, total phenolic content (TPC) and the main sulfur bioactive compounds (sulforaphane (SFN) and glucosinolates (GLSs)) were assayed. Distilled water was used as solvent. For each byproduct type, solid/liquid ratio (1:25 and 2:25 g/mL), temperature (25, 40, and 55 °C), and extraction time (2.5, 5, 7.5, 10, 15, and 20 min) were the studied variables to optimize the UAE process by using a kinetic and a cubic regression model. TPC was 12.5-fold higher in broccoli leaves than in florets, while SFN was from 2.5- to 4.5-fold higher in florets regarding the leaf's extracts obtained from the same plants, their precursors (GLS) being in similar amounts for both plant tissues. The most efficient extraction conditions were at 25 °C, ratio 2:25, and during 15 or 20 min according to the target phytochemical to extract. In conclusion, the type of plant tissue and used ratio significantly influenced the extraction of bioactive compounds, the most efficient UAE parameters being those with lower energy consumption.

Cystic soft tissue tumours of the dorsal aspect of the wrist have two distinct histological subtypes.

In total, 38 patients with cystic dorsal wrist tumours managed with surgical excision were prospectively followed up for 2 years. Tissue was examined histologically after primary surgery and at recurrence. Two distinct tissue types were found: ganglion cyst and synovial cyst.

The GH3 Protein from the Clubroot Pathogen Plasmodiophora brassicae Causes Hormone-Related Phenotypes in Arabidopsis thaliana

AbstractIn the genome of the obligate biotrophic protist Plasmodiophora brassicae, which causes clubroot disease in the Brassicaceae family, a single gene was identified with homology to the group of GH3 proteins synthesizing amino acid conjugates with carboxylic acids. In comparison to plant members the P. brassicae GH3 protein seems to be very promiscuous with respect to the substrates converted. In vitro assays showed conversion of three different auxins (indole-3-acetic, IAA; indole-propionic, IPA; and indole-3-butyric acids, IBA), jasmonic acid (JA) and also 12-oxo-phytodienoic acid (OPDA). Auxin and Methyl-JA responsive elements were found in the promoter sequence of PbGH3. For further analysis Arabidopsis thaliana was transformed with PbGH3. Seedlings with confirmed constitutive PbGH3 protein production were grown to adult plants that were shown to produce fewer inflorescences per plant. Growth on 10 µM IAA or JA levels also altered the phenotype of the seedlings, but not so much between wild types and transgenic lines. The hormone profiles in seedlings and adult plants were examined for IAA, JA, OPDA, and respective conjugates. Transgenic seedlings displayed changes that could be partially attributed to the overexpression of a GH3 gene like lower levels of free IAA and the JA precursor cis-OPDA under control conditions, and higher levels of some auxin conjugates accumulated after IAA treatment compared to the corresponding wild types. However, the hormone patterns in adult plants were more complex and varied based on different tissue types analyzed. After inoculation with P. brassicae the transgenic plants did not show any changes in infection rate and disease index, but had higher shoot and lower root weight in infected plants compared to controls. Based on the above findings we discuss a role for PbGH3 during pathogenesis.

77 Healing progression of tail docking and ear tag wound in lambs

Abstract Tail docking and ear tagging are common husbandry practices in lambs, but little is known about the healing process of the resulting wound. Additionally, evidence in other species shows that concurrent injurious procedures may delay wound healing. Thus, the objectives of this study were to 1) describe wound healing following tail docking and ear tagging, and 2) compare healing of ear tag wound in docked and undocked lambs. Within 28 Polypay female twin pairs, one lamb was docked by placing a constrictive rubber ring on the tail 24 to 48 h after birth and the tail of the other lamb was left intact (n = 28/treatment). Plastic-tipped ear tags were applied to both ears of all lambs immediately before tail docking. Wound photos of the ears and tail were taken twice weekly until weaning, resulting in 16 to 22 observations per lamb. Tail wound photos were analyzed to determine when the tail fell off and to score the presence of 6 tissue types: pus, serous exudate, sanguineous exudate, granulation, crust, and slough. A healed tail or ear wound was defined as one that was fully contracted, and no other tissue types were present. Tails took 19 ± 2 d to fall off (mean ± SD; range = 16 to 25 d) and their wound took 41 ± 8 d to heal completely (30 to 60 d). The prevalence of any of the 6 tissue types did not predict how long it took the tail wound to heal (F < 2.50, P > 0.13). Tissue types, indicative of possible infection, such as pus and slough, were present at least once in 89% and 93% of the docked lambs and in 8% and 10% of the total sampling periods, respectively. On average, pus and slough first appeared 12 ± 4 d (range = 5-22) and 11 ± 3 d (5-15) after docking, respectively. Of the 112 ear wounds, only 16% had healed by the time the lambs were weaned (55 to 74 d after tagging). Contrary to our predictions, there was a tendency for more docked lambs to have at least one healed ear wound by the end of the sampling period compared with undocked lambs (11 docked lambs vs 4 undocked lambs; X2 = 3.28, P = 0.07). This may be a result of an increased systemic inflammatory response and possible reduction in activity in the docked lambs. Overall, most ear tag wounds were still not healed after 2 mo, and tail wound took over 1 mo to heal. Further research is needed to determine the welfare implications of routine husbandry practices in sheep, including pain experienced during healing and how other factors may affect the healing process.

Abstract PS11-03: Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD)

Abstract Introduction. Leptomeningeal disease (LMD) is a devastating complication of advanced malignancies and occurs in 5-15% of patients (pts) with metastatic breast cancer (MBC). Molecular characterization of the primary tumor to identify actionable mutations is standard of care, and targeted therapies have significantly improved survival. In contrast, metastatic tumors to the central nervous system (CNS) are not routinely assessed for the presence of actionable mutations, so the frequency and concordance of actionable mutations centrally vs. peripherally is not well characterized. In this single-center prospective study, we collected cerebrospinal fluid (CSF) in pts with known or suspected LMD and performed next generation sequencing (NGS) to evaluate the status of actionable mutations in the CSF and compared these results with NGS from matched peripheral blood and systemic metastatic tumor samples. Methods. We enrolled sixteen (16) pts with MBC and suspected or confirmed LMD at a single academic center from 2021-2023 in this non-therapeutic prospective study and collected CSF, peripheral blood, and archival systemic tumor samples from each pt. We used NGS to evaluate the status of actionable biomarkers in the CSF, blood, and tissue using the following NGS platforms: CNSide (Biocept) for CSF; Guardant 360, Foundation One CDx and CNSide for blood; and Caris and the UCSF CLIA-validated UCSF500 panel for the systemic metastatic tumor samples. We also evaluated CSF tumor cell enumeration and HER2 status in a subset of pts (CNSide). Results. Of the sixteen pts with MBC and suspected or confirmed LMD, 81% (13/16) were ultimately determined to be LMD-positive based on positive CSF cytology and/or convincing MRI imaging, including 6 pts with HR+/HER2- MBC, 6 pts with metastatic triple negative breast cancer (mTNBC), and 1 pt with HR+/HER2+ MBC; 3 pts were determined to be LMD-negative based on similar criteria. Of the 6 pts with HR+/HER2- MBC and LMD, NGS detected actionable mutations in the CSF in 67% (4/6), including two pts with PIK3CA E545E, one pt with both PIK3CA E542K and ERBB2 amplification, and 1 pt with ESR1 Y537S. Matched NGS analysis of the peripheral blood and/or systemic metastatic tissue showed concordance with PIK3CA E542K detected across tissue types in two pts, but discordance across tissue types in the other pts, including ESR1 detected systemically in two pts but not in the CSF. Of the 6 pts with mTNBC and LMD, CSF NGS detected actionable mutations in the CSF in 17% (1/6) of pts, including one pt with PIK3CA E542K; analysis of blood NGS for this pt is pending. 3 pts (50%) had mutations in TP53 in the CSF (1 pt with R273H and two pts with K132R), which were concordant across tissue types in all 3 pts. NGS did not detect any variants in the CSF of the one pt with HR+/HER2+ MBC and LMD. CSF tumor cell enumeration and central HER2 status will also be reported. Conclusion. This study demonstrates that NGS can detect actionable mutations in the CSF of pts with MBC and LMD. We observed concordance and heterogeneity in the status of actionable mutations between the CSF, peripheral blood, and systemic metastatic tumor tissue. Larger studies are needed to assess the clinical utility of these observations, particularly with the development of several novel targeted agents that are CNS-penetrant. Citation Format: Laura Huppert, Lindy Her, Christine Hodgdon, Susie Brain, Carol Simmons, Jo Chien, Melanie Majure, Hope Rugo, Mark Jesus Magbanua, Ron Balassanian, Michelle Melisko. Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-03.

Abstract PO1-15-01: Digital pathology models reveal case-specific characteristics of the tumor microenvironment

Abstract Background Phenotypic characteristics and genetic content have been observed to vary within individual tumors. However, translational research and some clinical practices often rely on extrapolating single histopathology slides to be representative of whole-tumor biology, even when the extent of intra-tumor biological heterogeneity is unknown. In addition, technical factors in slide preparation and digitization can also contribute to variability. Here, we use digital pathology models to identify cell and tissue types in whole-slide images of breast cancer tumors, and quantify intra- and inter-case heterogeneity in cell and tissue features of the tumor microenvironment. Methods We developed PathExplore, a suite of convolutional neural network models trained on pathologist annotations of cell and tissue types in hematoxylin and eosin-stained slides. The models were deployed on the TCGA breast cancer dataset (n=1083 primary solid tumors). Quantitative human-interpretable features (HIFs) relating to cancer, stromal, and necrotic tissue areas, as well as the abundance and distribution of cancer cells, fibroblasts, and immune cells within these tissues, were extracted for each slide. Similarity between slides on sets of HIFs was assessed using Pearson correlation. Intra- and inter-case variability of individual HIFs was measured using the normalized percent difference (range divided by mean) for each slide pair. Case-specificity was quantified by the AUC of correlation or difference distributions for intra-case vs inter-case slide pairs. Only pairs of slides with matching metadata, including tissue source site, cancer stage, and scanner model, were considered for inter-case analysis. Results A total of 55 cases, comprising 114 slides, were identified as having more than one slide per case. Considering all 123 proportional HIFs, intra-case slide pairs showed high correlation compared to inter-case pairs (r=0.97 vs r=0.90, AUC=0.88), with 29% of slides coming from a multi-slide case most closely correlated with another slide from the same case out of the entire TCGA breast dataset. Subsets of these proportional HIFs relating to each identified cell type were also case-specific, with AUC’s ranging from 0.79 for macrophage HIFs to 0.87 for cancer cell HIFs. All proportion, density, and ratio HIFs (n=297) individually showed case specificity (AUC >0.5), with median intra-case differences ranging from 0.6% to 69%. Area proportion of necrosis was more variable than cancer or stroma tissue, while cancer cell count proportions were less variable than fibroblast or immune cell proportions. Cell count proportions were in general less variable and more specific than their corresponding density HIFs; for instance, the frequency of fibroblasts out of all cells in stroma had a median intra-case difference of 11% compared to 20% for the density of fibroblasts in stroma. Conclusions We quantified heterogeneity of key features of the tumor microenvironment both within and across tumors. These results reveal biological and technical variability that can inform selection and interpretation of biomarkers derived from single slides. The ability to uniquely identify slides from the same case additionally demonstrates the technical robustness of digital pathology models for yielding quantitative insights into tumor biology. Citation Format: Ylaine Gerardin, Christian Kirkup, Archit Khosla, Laura Chambre, Michael Drage, Amaro Taylor-Weiner. Digital pathology models reveal case-specific characteristics of the tumor microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-01.

Tissue-Specific Distribution and Maternal Transfer of Persistent Organic Halogenated Pollutants in Frogs.

Persistent organic pollutants pose a great threat to amphibian populations, but information on the bioaccumulation of contaminants in amphibians remains scarce. To examine the tissue distribution and maternal transfer of organic halogenated pollutants (OHPs) in frogs, seven types of tissues from black-spotted frog (muscle, liver, kidney, stomach, intestine, heart, and egg) were collected from an e-waste-polluted area in South China. Among the seven frog tissues, median total OHP concentrations of 2.3 to 9.7 μg/g lipid weight were found (in 31 polychlorinated biphenyl [PCB] individuals and 15 polybrominated diphenyl ether [PBDE], dechlorane plus [syn-DP and anti-DP], bexabromobenzene [HBB], polybrominated biphenyl] PBB153 and -209], and decabromodiphenyl ethane [DBDPE] individuals). Sex-specific differences in contaminant concentration and compound compositions were observed among the frog tissues, and eggs had a significantly higher contaminant burden on the whole body of female frogs. In addition, a significant sex difference in the concentration ratios of other tissues to the liver was observed in most tissues except for muscle. These results suggest that egg production may involve the mobilization of other maternal tissues besides muscle, which resulted in the sex-specific distribution. Different parental tissues had similar maternal transfer mechanisms; factors other than lipophilicity (e.g., molecular size and proteinophilic characteristics) could influence the maternal transfer of OHPs in frogs. Environ Toxicol Chem 2024;43:1557-1568. © 2024 SETAC.

Hyperbaric oxygen therapy: future prospects in regenerative therapy and anti-aging.

Hyperbaric Oxygen Therapy (HBOT) utilizes 100% oxygen at high atmospheric pressure for clinical applications. HBOT has proven to be an effective supplementary treatment for a variety of clinical and pathological disorders. HBOT's therapeutic results are based on the physiological effects of increased tissue oxygenation, or improved oxygen bioavailability. HBOT's current indications in illnesses like as wound healing, thermal or radiation burns, and tissue necrosis point to its function in facilitating the regeneration process. Various research has revealed that HBOT plays a function in vascularization, angiogenesis, and collagen production augmentation. Individual regeneration capacity is influenced by both environmental and genetic factors. Furthermore, the regenerating ability of different types of tissues varies, and this ability declines with age. HBOT affects physiological processes at the genetic level by altering gene expression, delaying cell senescence, and assisting in telomere length enhancement. The positive results in a variety of indications, ranging from tissue regeneration to better cognitive function, indicate that it has enormous potential in regenerative and anti-aging therapy.

Abstract PO5-09-03: Multi-tissue transcriptome-wide association studies identified genes for intrinsic subtypes of breast cancer

Abstract Background: Though several genome-wide association studies (GWAS) of breast cancer (BC) have identified common variants which differ between intrinsic subtypes, the genes through which these variants act through to impact BC risk have not been fully established. Furthermore, transcriptome-wide association studies (TWAS) have thus far been primarily employed to identify genes associated with overall BC risk, while overlooking how the influence of common variation on gene expression may contribute to subtype-specific differences. Methods: In this study, we performed two complementary multi-tissue TWASs for each of the following BC intrinsic subtypes: Luminal A-like, Luminal B-like, Luminal B/HER2-negative-like, HER2-enriched-like, Triple Negative BC. These two approaches included 1) an expression-based approach that collated TWAS signals from expression quantitative trait loci (eQTLs) across multiple tissues using the Aggregated Cauchy Association Test (ACAT) and 2) a splicing-based approach that utilized two applications of ACAT to collate splicing QTLs (sQTLs) for a given gene in a tissue and then across tissues. To perform these two TWASs, we utilized e/sQTL models trained in 11 tissues from the Genotype-Tissue Expression Project including breast, ovary, uterus, vagina, EBV-transformed lymphocytes, whole blood, spleen, liver, subcutaneous adipose, visceral adipose, and cell-cultured fibroblasts. GWAS summary statistics were previously generated from 133,384 BC cases and 113,789 controls who were participants in the Breast Cancer Association Consortium (BCAC). We further performed our TWAS while conditioning e/sQTL effect sizes on nearby GWAS index SNPs. Additionally, we utilized gene-based fine-mapping of eQTLs and sQTL to identify candidate causal genes for each intrinsic subtype. Results: Overall, we identified 164 genes in 69 loci that were associated with Luminal A-like, 19 genes in 9 loci with Luminal B-like, 18 genes in 11 loci with Luminal B/HER2-negative-like, 10 genes in 7 loci with HER2-enriched-like, and 29 genes in 12 loci with TNBC. Among these genes, 17 genes had not been reported in previous TWAS of BC, and 140 genes, 1 gene, 2 genes, 2 genes, and 16 genes were uniquely associated with each of the intrinsic subtypes, respectively. Additionally, we identified one gene associated with Luminal A-like and one gene with Luminal B-like BC that were each in a locus located at least 1.4 Mb from published GWAS hits. Furthermore, we identified 106, 11, 10, 5, and 21 candidate causal genes for each of the intrinsic subtypes, respectively, that had a posterior inclusion probability of 0.9 in at least one e/sQTL. Conclusion: In summary, our multi-tissue TWAS corroborated previous GWAS loci for overall BC risk and intrinsic subtypes, while underscoring how common variation impacts BC etiology by modulating the expression and splicing of genes in multiple tissue types. Citation Format: James Li, Julian McClellan, Haoyu Zhang, Guimin Gao, Dezheng Huo. Multi-tissue transcriptome-wide association studies identified genes for intrinsic subtypes of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-03.

BraNet: a mobil application for breast image classification based on deep learning algorithms.

Mobile health apps are widely used for breast cancer detection using artificial intelligence algorithms, providing radiologists with second opinions and reducing false diagnoses. This study aims to develop an open-source mobile app named "BraNet" for 2D breast imaging segmentation and classification using deep learning algorithms. During the phase off-line, an SNGAN model was previously trained for synthetic image generation, and subsequently, these images were used to pre-trained SAM and ResNet18 segmentation and classification models. During phase online, the BraNet app was developed using the react native framework, offering a modular deep-learning pipeline for mammography (DM) and ultrasound (US) breast imaging classification. This application operates on a client-server architecture and was implemented in Python for iOS and Android devices. Then, two diagnostic radiologists were given a reading test of 290 total original RoI images to assign the perceived breast tissue type. The reader's agreement was assessed using the kappa coefficient. The BraNet App Mobil exhibited the highest accuracy in benign and malignant US images (94.7%/93.6%) classification compared to DM during training I (80.9%/76.9%) and training II (73.7/72.3%). The information contrasts with radiological experts' accuracy, with DM classification being 29%, concerning US 70% for both readers, because they achieved a higher accuracy in US ROI classification than DM images. The kappa value indicates a fair agreement (0.3) for DM images and moderate agreement (0.4) for US images in both readers. It means that not only the amount of data is essential in training deep learning algorithms. Also, it is vital to consider the variety of abnormalities, especially in the mammography data, where several BI-RADS categories are present (microcalcifications, nodules, mass, asymmetry, and dense breasts) and can affect the API accuracy model.

Flow Cytometry for Estimating Plant Genome Size: Revisiting Assumptions, Sources of Variation, Reference Standards, and Best Practices

Flow cytometry has been widely used to estimate relative and absolute genome sizes (DNA contents) of plants for more than 50 years. However, the accuracy of these estimates can vary widely because of many factors, including errors in the genome size estimates of reference standards and various experimental methods. The objectives of this study were to reassess genome sizes of commonly used reference standards and quantify sources of variation and error in estimating plant genome sizes that arise from buffers, confounding plant tissues, tissue types, and plant reference standards using both 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) fluorochromes. Five separate studies were performed to elucidate these objectives. Revised estimates of genome sizes of commonly used plant reference standards were determined using human male leukocytes as a primary standard with an updated genome size (6.15 pg; 12.14% lower than that of earlier studies) using both DAPI and PI fluorochromes. Comparison of six different buffers (Galbraith’s, LB01, MB01, MgSO4, Otto’s, and Sysmex) resulted in variations in genome size estimates by as much as 18.1% for a given taxon, depending on the buffer–fluorochrome combination. The addition of different confounding plant tissues (representing 10 diverse taxa and associated secondary metabolites) resulted in variations in genome size estimates by as much as 10.3%, depending on the tissue–fluorochrome combination. Different plant tissue types (leaf color/exposure and roots) resulted in a variation in genome size estimates of 10.7%, independent of the fluorochrome. The selection of different internal reference standards introduced an additional variation in genome size estimates of 5.9%, depending on the standard–fluorochrome combination. The choice of fluorochrome (DAPI vs. PI) had one of the largest impacts on genome size estimates and differed by as much as 32.9% for Glycine max ‘Polanka’ when using human male leukocytes as an internal standard. A portion of this variation (∼10.0%) can be attributed to the base pair (bp) bias of DAPI and variations in Guanine-Cytosine (GC):Adenine-Thymine (AT) ratios between the sample and standard. However, as much as 22.9% of the variation in genome size estimates may result from how effectively these fluorochromes stain and report the genome. The combined variation/error from all these factors (excluding variation from bp bias for different fluorochromes, and assuming variations from confounding tissues and tissue types to both result from secondary metabolites) totaled 57.6%. Additional details of how selected factors impact accuracy, precision, and the interaction of these factors are presented. Overall, flow cytometry can be precise, repeatable, and extremely valuable for determining the relative genome size and ploidy of closely related plants when using consistent methods, regardless of fluorochrome. However, accurate determination of the absolute genome size by flow cytometry remains elusive, and estimates of genome size using flow cytometry should be considered gross approximations that may vary by ±29% or more as a function of experimental methods and plant environments. Additional recommendations of best practices are provided.

Gut-muscle axis: establishing the connection between microbial signaling and skeletal muscle function in a mouse model (mdx) of Duchenne muscular dystrophy

The metabolic and endocrine nature of skeletal muscle allows for systemic signaling and influence over the enteric environment influencing gut microbial populations. Concomitantly, commensal microbial signaling can alter the composition and development of skeletal muscle forming a bidirectional gut-muscle axis. Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by skeletal muscle atrophy, fibrosis, and ultimately cardiorespiratory failure. Previous studies have shown that gut microbial signaling can alter the pathways involved in muscle atrophy, enhancing atrophy when dysbiotic, or blunting the atrophic effect by restoring gut microbial populations to a healthy status; thus, we sought to characterize the metabolic signaling between the gut and the diaphragm in a mouse model of DMD ( mdx) to identify potential therapeutic targets. The study was divided into two arms and conducted following institutional ethical approval. The first arm investigated metabolomic signalling. 6-month-old male BL10 wildtype (WT; n = 12) and mdx (n = 12) mice were anaesthetized (induction by 5% isoflurane) and decapitated. Following collection of trunk blood, the diaphragm was excised, and colonic/cecal contents were collected. The samples were then processed by mass-spectrometry to isolate the metabolomic profiles of semi-polar, secondary bile acid, and short chain fatty acid (SCFA) targets in the gut, plasma and diaphragm of WT and mdx mice. The second arm of the study explored the gut microbial profile and diaphragm gene expression, structure and function. 6-month-old male WT (n = 15) and mdx (n = 15) were anesthetized (induction by 5% isoflurane) followed by cervical dislocation. The diaphragm was excised, and longitudinal strips were isolated for structural analysis (i.e., histological staining and immunohistochemistry for fibre type and size), PCR gene expression, and isometric force assessment ex vivo. Additionally, faecal pellets were collected and analysed via shotgun metagenomic sequencing to assess microbial populations. Diaphragm twitch and tetanic force in mdx were profoundly decreased compared to WT. Metabolic analysis of faecal metabolites indicated impairment in the three main tryptophan metabolic pathways in mdx mice: kynurenine, indole and serotonin. Metagenomic analysis indicated a profound genotype effect between WT and mdx gut microbial populations. Two main producers of the short chain fatty acid acetate (Lactobacillus and Bifidobacterium) were significantly decreased within the mdx group. Acetate serves as a substate in the TCA cycle contributing to ATP production and positively affects skeletal muscle via numerous pathways including the down regulation of key atrogenes associated with atrophy. Pathway enrichment analysis showed a global disruption to both purine and pyrimidine metabolism in all three tissue types. Purine/pyrimidine metabolism have been tightly linked with microbial metabolism and affect skeletal muscle health and function. Further understanding of the gut-muscle axis in dystrophic models may pave the way for novel microbiota-directed therapeutic strategies to ameliorate muscle dysfunction in DMD. Funded by Science Foundation Ireland SFI/19/6628 INSPIRE DMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypoxia impairs Kv7 channel function in the coronary microcirculation

Ischemic heart disease (IHD) is a multifaceted pathological manifestation that continues to press an extensive burden on individuals and healthcare resources. The strenuous weight of this disease affects more than 80 million people in the United States, with an annual economic cost of more than $300 billion in healthcare expenses and lost productivity. Understanding the progression of this disease and the underlying regulatory mechanisms is essential for dampening the substantial socio-economic burden it poses by supporting the development of novel therapeutic strategies for afflicted patients. Myocardial perfusion is largely controlled by the microcirculation, consisting of small arteries and arterioles. Under physiological conditions, an increase in myocardial metabolic activity stimulates coronary vasodilation and increased vascular perfusion. However, pathological coronary stenosis of the conduit arteries impairs downstream flow and O2 delivery that must be compensated by collateral supply from neighboring nonoccluded regions. The ability of the vasculature to dilate in response to a reduction in flow is paramount for improved perfusion from the collateral circulation. Voltage-gated potassium (Kv) channels present as significant end effectors of vasodilation for many signaling axes, including reactive oxygen species, which are reported to be upregulated under ischemic conditions. Using the clinically relevant swine model for the current study, we isolate a reduction in oxygen tension, ex vivo, as a single characteristic of an otherwise complicated disease and test the hypothesis that hypoxic insult impairs Kv7 channel function. Isometric tension wire myography and perforated patch voltage clamp were used to investigate Kv7 channel contribution to H2O2-mediated vasodilation and Kv channel currents in response to hypoxic insult. Our data reveal that hypoxic conditions impair H2O2-mediated vasodilation and that this impairment is attributable, at least in part, to a loss of Kv7 channel activation. Further, protein kinases are known to associate with and activate Kv7 channels in other tissue types. Inhibition of protein kinase A (PKA) yields a similar inhibitory response to blockade of Kv7 channels. Taken together, these studies reveal Kv7 channels as potential targets of hypoxic insult, and their reduced contribution may be responsible for impaired vasoreactivity in the coronary microcirculation in response to hypoxic insult. Funding: NIH RO1 HL139903. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Fibrostricturing Crohn's disease is marked by an increase in active eosinophils in the deeper layers.

Approximately 50% of Crohn's disease (CD) patients develop intestinal strictures necessitating surgery. The immune cell distribution in these strictures remains uncharacterized. We aimed to identify the immune cells in intestinal strictures of CD patients. During ileocolonic resections, transmural sections of terminal ileum were sampled from 25 CD patients and 10 non-inflammatory bowel disease (IBD) controls. Macroscopically, unaffected, fibrostenotic and inflamed ileum was collected and analysed for immune cell distribution (flow cytometry) and protein expression. Collagen deposition was assessed via a Masson's Trichrome staining. Eosinophil and fibroblast co-localization was assessed through immunohistochemistry. The Masson's Trichrome staining confirmed augmented collagen deposition in both the fibrotic as the inflamed region, though with a significant increased collagen deposition in fibrotic compared to inflamed tissue. Distinct Th1, Th2, regulatory T cells, dendritic cells and monocytes were identified in fibrotic and inflamed CD ileum compared to unaffected ileum of CD patients as non-IBD controls. Only minor differences were observed between fibrotic and inflamed tissue, with more active eosinophils in fibrotic deeper layers and increased Eosinophil Cationic Protein (ECP) protein expression in inflamed deeper layers. Lastly, no differences in eosinophil and fibroblast co-localization was observed between the different regions. This study characterized immune cell distribution and protein expression in fibrotic and inflamed ileal tissue of CD patients. Immunologic, proteomic and histological data suggest inflammation and fibrosis are intertwined, with large overlap between both tissue types. However strikingly, we did identify an increased presence of active eosinophils only in the fibrotic deeper layers, suggesting their potential role in fibrosis development.

Vimentin Expression Following Mechanical Overload Induced Skeletal Muscle Hypertrophy Appears To Be Satellite Cell Dependent

Introduction: Vimentin (VIM) is an intermediate filament that plays a key role in development and regeneration of various tissue types. However, the expression of VIM during skeletal muscle hypertrophy has not been fully elucidated. Therefore, the purpose of this study was to determine the response of VIM following mechanical overload (MOV) with or without the presence of satellite cells. Methods: Pax7-DTA mice (age 4mo) were subjected to 3, 10 or 20 days of synergist ablation to induce overload of the plantaris muscle, time matched sham surgery mice served as controls. Following overload, the plantaris was removed and either sectioned for immunohistochemistry (IHC) or placed in Trizol for later RNA/protein isolation. Satellite cell depletion was confirmed via IHC to determine Pax7+ cells. VIM expression was also examined via RT-qPCR, western blotting, and IHC. Data were checked for normality via Shapiro-Wilks tests and two-way ANOVAs were performed with Tukey post-hoc tests. Results: In the Pax7-DTA mice, VIM expression revealed a significant Group, Time, and Group x time interaction (all P < 0.001). Further analysis revealed VIM expression was significantly upregulated in satellite cell intact mice after 3, 10, and 20 days of overload (all P < 0.001). VIM expression in the satellite cell depleted mice was non-responsive to 3 and 10 days of MOV (P = 0.964, and P = 0.984, respectively), however VIM expression following 20 days of MOV in satellite cell depleted mice was significantly increased compared to sham mice (P = 0.0047). Conclusion: VIM expression is responsive to hypertrophic stimuli and appears to be satellite cell dependent in the early stages of skeletal muscle hypertrophy. Funding was provided through discretionary funds for Michael Roberts at Auburn University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TAK1 in Vascular Signaling: "Friend or Foe"?

The maintenance of normal vascular function and homeostasis is largely dependent on the signaling mechanisms that occur within and between cells of the vasculature. TGF-β-activated kinase 1 (TAK1), a multifaceted signaling molecule, has been shown to play critical roles in various tissue types. Although the precise function of TAK1 in the vasculature remains largely unknown, emerging evidence suggests its potential involvement in both physiological and pathological processes. A comprehensive search strategy was employed to identify relevant studies, PubMed, Web of Science, and other relevant databases were systematically searched using keywords related to TAK1, TABs and MAP3K7.In this review, we discussed the role of TAK1 in vascular signaling, with a focus on its function, activation, and related signaling pathways. Specifically, we highlight the TA1-TABs complex is a key factor, regulating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) involved in the processes of inflammation, vascular proliferation and angiogenesis. This mini review aims to elucidate the evidence supporting TAK1 signaling in the vasculature, in order to better comprehend its beneficial and potential harmful effects upon TAK1 activation in vascular tissue.

Radiomics analysis of the tissue content of the esophageal hiatus of normal subjects without esophageal symptoms (establishing control values)

Introduction: The opening in the diaphragm through which the esophagus enters from the thorax into the abdomen, i.e., esophageal hiatus is formed by the right crus of the diaphragm. During esophageal peristalsis, the lower end of the esophagus, i.e., lower esophageal sphincter (LES), slides from the abdomen into chest, which we suspect is due to the presence of fat in the hiatus that serves as a lubricant. A recent study from our laboratory found that in patients with achalasia esophagus, the LES and crus of diaphragm are firmly anchored together, which we suspect is due to the loss of fat from the hiatus. Gray scale intensity analysis of the radiological images can be used to determine the nature of tissue, i.e., fat, connective tissue, muscle etc. Goal: To establish gray scale values of the esophageal hiatus from the CT scan images in controls. Methods: 42 patients who had undergone an abdominal CT scan for symptoms unrelated to upper gastrointestinal pathology were selected for this analysis. These patients had no esophageal symptoms and were not taking any medications related to the esophagus and stomach, which included acid inhibition medications. The CT scan was visualized in the Visage (San Diego, CA), a software program that allows rotation of images in X, Y and Z planes to visualize the esophageal hiatus in a 2D image. A software (Segcrus) was built to perform the grayscale intensity analysis of the 3 regions of equal size in the image, 1) subcutaneous fat, 2) muscle, and 3) esophageal hiatus. The 3 regions of interest were demarcated manually in the 2D image. The inner margin of the hiatus was selected as the region of interest for the hiatus image. The software generated a statistical non-parametric method approximation using kernel density estimation (KDE) of the grey scale intensity distributions of the 3 regions of interest. The median values were computed to represent the central tendencies of the distributions, providing an insight into the typical greyscale values of the tissue types. Results: distinct patterns in the region intensity distributions for hiatal region, muscle, and fat were seen. For the hiatus, the values were (Median = 105), interquartile range (IQR = 80.3), and range (Min = 61.5, Max = 137.95), providing a comprehensive description of central tendency, variability, and data spread. For muscle, (Median = 139.92), interquartile range (IQR = 71.1), and range (Min = 119.67, Max = 157.17), and for fat, (Median = 48.24), interquartile range (IQR = 52.9), and range (Min = 23.99, Max = 67.79. Hiatus to fat tissue intensity overlap was not related to BMI (r=0.17, p=0.29), or Age (r=-0.04, p=0.82). There was a notable correlation between the overlap of Hiatal-Muscle tissue intensity and Age (r=0.38, p=0.012). However, such correlation was not observed with BMI (r=-0.3, p=0.06). Additionally, there was no significant correlation between the overlap of Hiatal-Fat tissue intensity and gender (r=-0.26, p=0.09), and similarly, the overlap of Hiatal-Muscle intensity showed no correlation with sex (r=0.09, p=0.58). The results highlight the variability and central tendencies within these tissue types across the normal subjects. Conclusion: These analysis in subjects with no esophageal symptoms may be used as control values to determine if there are difference in the tissue content of the esophageal hiatus between normal and patients with various esophageal motility disorders. R01 DK109376. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.