Biological Processes Research Articles

Proteomic identification of potential biomarkers for heat tolerance in Caracu beef cattle using high and low thermotolerant groups.

Heat stress has deleterious effects on physiological and performance traits in livestock. Within this context, using tropically adapted cattle breeds in pure herds or terminal crossbreeding schemes to explore heterosis is attractive for increasing animal production in warmer climate regions. This study aimed to identify biological processes, pathways, and potential biomarkers related to thermotolerance in Caracu, a tropically adapted beef cattle breed, by proteomic analysis of blood plasma. To achieve this goal, 61 bulls had their thermotolerance evaluated through a heat tolerance index. A subset of 14 extreme animals, including the seven most thermotolerant (HIGH group) and the seven least thermotolerant (LOW group), had their blood plasma samples used for proteomic analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The differentially regulated proteins detected between HIGH and LOW groups were used to perform functional enrichment analysis and a protein-protein interaction network analysis. A total of 217 proteins were detected only in the HIGH thermotolerant group and 51 only in the LOW thermotolerant group. In addition, 81 and 87 proteins had significantly higher and lower abundancies in the HIGH group, respectively. Regarding proteins with the highest absolute log-fold change values, we highlighted those encoded by DUSP5, IGFALS, ROCK2, RTN4, IRAG1, and NNT genes based on their functions. The functional enrichment analysis detected several biological processes, molecular functions, and pathways related to cellular responses to stress, immune system, complement system, and hemostasis in both HIGH and LOW groups, in addition to terms and pathways related to lipids and calcium only in the HIGH group. Protein-protein interaction (PPI) network revealed as important nodes many proteins with roles in response to stress, hemostasis, immune system, inflammation, and homeostasis. Additionally, proteins with high absolute log-fold change values and proteins detected as essential nodes by PPI analysis highlighted herein are potential biomarkers for thermotolerance, such as ADRA1A, APOA1, APOB, APOC3, C4BPA, CAT, CFB, CFH, CLU, CXADR, DNAJB1, DNAJC13, DUSP5, FGA, FGB, FGG, HBA, HBB, HP, HSPD1, IGFALS, IRAG1, KNG1, NNT, OSGIN1, PROC, PROS1, ROCK2, RTN4, RYR1, TGFB2, VLDLR, VTN, and VWF. Identifying potential biomarkers, molecular mechanisms and pathways that act in response to heat stress in tropically adapted beef cattle contributes to developing strategies to improve performance and welfare traits in livestock under tropical climates.

FgGET3, an ATPase of the GET Pathway, Is Important for the Development and Virulence of Fusarium graminearum

GET3 is an ATPase protein that plays a pivotal role in the guided entry of the tail-anchored (GET) pathway. The protein facilitates the targeting and inserting of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) by interacting with a receptor protein complex on the ER. The role of GET3 in various biological processes has been established in yeast, plants, and mammals but not in filamentous fungi. Fusarium graminearum is the major causal agent of Fusarium head blight (FHB), posing a threat to the yield and quality of wheat. In this study, we found that FgGET3 exhibits a high degree of sequence and structural conservation with its homologs across a wide range of organisms. Ectopic expression of FgGET3 in yeast restored the growth defects of the Saccharomyces cerevisiae ScGET3 knock-out mutant. Furthermore, FgGET3 was found to dimerize and localize to the cytoplasm, similar to its homologs in other species. Deletion of FgGET3 in F. graminearum results in decreased fungal growth, fragmented vacuoles, altered abiotic stress responses, reduced conidia production, delayed conidial germination, weakened virulence on wheat spikes and reduced DON production. Collectively, these findings underscore the critical role of FgGET3 in regulating diverse cellular and biological functions essential for the growth and virulence of F. graminearum.

Distribution and behaviour of reduced sulfur substances in the oligotrophic and hydrothermal waters of the Western Tropical South Pacific

Reduced sulfur species (RSS) are involved in essential biological and chemical processes, including metal complexation, yet little is known about their occurrence and behaviour in marine systems. Here, we present a quantitative and qualitative data set of species-specific RSS in open ocean samples collected during the GEOTRACES Tonga GPpr14 cruise. The cruise traversed differing biogeochemical provinces, from the mesotrophic Melanesian waters and the North Fiji Basin, through the hydrothermally active Lau Basin, eastward to the oligotrophic South Pacific Gyre. Using cathodic stripping voltammetry in acidified samples (pH 2), we measured the concentration of two RSS, with peak potentials of -0.18 and -0.09 V in equivalents of thioacetamide (TA) and glutathione (GSH) respectively. GSH-like compounds were only present in the upper 200 m at concentrations up to 6.2 nM eq. GSH, consistent with other cathodic stripping voltammetry as well as chromatography-based studies. In contrast, RSS−0.18 V2 compounds were detected at all depths at concentrations ranging from 48 nM to 980 nM eq. TA. Both RSS−0.18 V2 and GSH-like compounds were present at higher levels in the hydrothermally-impacted region of the Lau Basin relative to other stations. The highest levels, along with high sulfide concentrations, were detected in a hydrothermal plume sample, indicating that hydrothermal vents are a direct or indirect source of these compounds. Elevated levels of RSS−0.18 V2 compounds were detected throughout almost the entire water column at a station located in the North Fiji Basin. We also employed the qualitative technique of cathodic pseudopolarography on unbuffered samples (pH ~ 8.5). Pseudopolarograms of marine RSS were compared to sulfide, GSH and TA standards. Pseudopolarography supports the presence of GSH in marine samples. However, while a compound that is electrochemically similar to TA is often detected in marine samples, TA itself is not thought to be naturally present. This is supported by our pseudopolarograms of RSS−0.52 V8.5 which often lacked the characteristic TA reduction wave but suggested the presence of other unidentified RSS compounds.

Ion channels in acinar cells in acute pancreatitis: crosstalk of calcium, iron, and copper signals

The initial stages of acute pancreatitis (AP) are characterized by a significant event - acinar ductal metaplasia (ADM). This process is a crucial feature of both acute and chronic pancreatitis, serving as the first step in the development of pancreatic cancer. Ion channels are integral transmembrane proteins that play a pivotal role in numerous biological processes by modulating ion flux. In many diseases, the expression and activity of ion channels are often dysregulated. Metal ions, including calcium ions (Ca2+), ferrous ions (Fe2+), and Copper ions (Cu2+), assume a distinctive role in cellular metabolism. These ions possess specific biological properties relevant to cellular function. However, the interactions among these ions exacerbate the imbalance within the intracellular environment, resulting in cellular damage and influencing the progression of AP. A more in-depth investigation into the mechanisms by which these ions interact with acinar cells is essential for elucidating AP’s pathogenesis and identifying novel therapeutic strategies. Currently, treatment for AP primarily focuses on pain relief, complications prevention, and prognosis improvement. There are limited specific treatments targeting acinous cell dedifferentiation or ion imbalance. This study aims to investigate potential therapeutic strategies by examining ion crosstalk within acinar cells in the context of acute pancreatitis.

Tumor Microenvironment in Clear Cell Renal Cell Carcinoma: A Comprehensive Analysis

Background: M6A RNA methylation and the tumor microenvironment (TME) have been reported to play important roles in the progression and prognosis of clear cell renal cell carcinoma (ccRCC). However, whether m6A RNA methylation regulators affect the TME in ccRCC remains unknown. Thus, we aimed to evaluate comprehensively the effect of m6A RNA methylation regulators on the TME in ccRCC. Methods: Transcriptome data of ccRCC were obtained from TCGA database. Consensus clustering analysis was conducted based on the expression of m6A RNA methylation regulators. Survival differences were evaluated by Kaplan–Meier analysis between the clusters. The DESeq2 package was used to analyze the differentially expressed genes (DEGs) between the clusters. GO and KEGG pathway analyses were performed by the ClusterProfiler R package. The CIBERSORT algorithm was used to evaluate immune infiltration. Results: The expression of 15 m6A regulators significantly differed between ccRCC and normal kidney tissues. Based on the expression of these 15 m6A regulators, two clusters were identified by consensus clustering, in which cluster 1 had better overall survival (OS). Overall, 4,429 DEGs were identified between the two clusters and were enriched in immune-related biological processes. Cluster 1 had lower immune and ESTIMATE scores, higher expression of HLA and lower expression of immune checkpoint molecules. Moreover, immune infiltration and expressions of Th1/IFNγ gene signature were also significantly different between the two clusters. Conclusion: Our study revealed m6A regulators were important participants in the development of ccRCC, with a close relationship with the TME.

CircFOXP1: a potential diagnostic and therapeutic target in human diseases

Circular RNA (circRNA) are a unique class of non-coding RNAs characterized by their covalently closed loop structures, which grant them properties such as stability and conservation. Among these, circFOXP1 has been implicated in various diseases, including cancers, respiratory, skeletal, and cardiovascular disorders. This review systematically examines circFOXP1’s role in disease progression, highlighting its involvement in critical biological processes, including cell proliferation, invasion, apoptosis, and autophagy. Mechanistically, circFOXP1 functions through miRNA sponging, protein interactions, and modulation of key signaling pathways such as Wnt and PI3K/AKT. We discuss its potential as a diagnostic and therapeutic target. Our analysis also identifies key unresolved questions, such as the precise regulatory networks involving circFOXP1 and its translation potential, offering pathways for future research.

Regulation of Milk Fat Synthesis: Key Genes and Microbial Functions

Milk is rich in a variety of essential nutrients, including fats, proteins, and trace elements that are important for human health. In particular, milk fat has an alleviating effect on diseases such as heart disease and diabetes. Fatty acids, the basic units of milk fat, play an important role in many biological reactions in the body, including the involvement of glycerophospholipids and sphingolipids in the formation of cell membranes. However, milk fat synthesis is a complex biological process involving multiple organs and tissues, and how to improve milk fat of dairy cows has been a hot research issue in the industry. There exists a close relationship between milk fat synthesis, genes, and microbial functions, as a result of the organic integration between the different tissues of the cow’s organism and the external environment. This review paper aims (1) to highlight the synthesis and regulation of milk fat by the first and second genomes (gastrointestinal microbial genome) and (2) to discuss the effects of ruminal microorganisms and host metabolites on milk fat synthesis. Through exploring the interactions between the first and second genomes, and discovering the relationship between microbial and host metabolite in the milk fat synthesis pathway, it may become a new direction for future research on the mechanism of milk fat synthesis in dairy cows.

Integrating Metabolomic Analysis, Network Pharmacology, and Molecular Docking to Underlying Pharmacological Mechanism and Ethnobotanical Rationalization for Diabetes Mellitus: Study on Medicinal Plant Fibraurea tinctoria Lour.

Fibraurea tinctoria Lour. has long been used in traditional medicine to treat diabetes mellitus (DM). However, a comprehensive scientific understanding of its potential active compounds and underlying pharmacological mechanisms still needs to be unveiled. This study, therefore, presents a novel approach by integrating metabolomic profiling, pharmacological network, and molecular docking analysis to investigate the potential of F. tinctoria as antidiabetes mellitus. Active compounds were obtained through analysis using ultrahigh-performance liquid chromatography-quadrupole-orbital ion trap-high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) and screening of active compounds using Lipinski rule of five and ADMET parameters. Potential targets of F. tinctoria compounds and DM-related targets were retrieved from public databases, such as DisGeNET, GeneCards, OMIM, PharmaGKB, and TTD. The targets' gene ontology (GO) was created using DAVID and protein-protein interactions using STRING. The plant-organ-compound-target-disease network was constructed using Cytoscape. Then, molecular docking analysis predicted and verified the interactions of essential bioactive compounds of F. tinctoria and DM core targets. The network pharmacology approach identified 35 active compounds, 565 compound-related targets, and 17,289 DM-related targets. EGFR, HSP90AA1, ESR1, HSP90AB1, and GSK3B were the core targets, whereas isolariciresinol, cubebin, corypalmine, (-)-8-oxocanadine, and (+)-N-methylcoclaurine were the most active compounds of F. tinctoria with DM potential. GO functional enrichment analysis revealed 483 biological processes, 485 cellular components, and 463 molecular functions. REACTOME pathway enrichment analysis yielded 463 significantly enriched signaling pathways. Of these pathways, the cytokine signaling in the immune system pathway may play a key role in treating DM. The results of molecular docking analysis showed that the core targets of DM, such as 5gnk, 3o0i, 6psj, 5ucj, and 1q5k, bind stably to the analyzed bioactive compounds of F. tinctoria. This study provides significant insights into the potential mechanism of F. tinctoria in treating DM. The main active compounds of F. tinctoria were found to interact with the core targets (EGFR, HSP90AA1, ESR1, HSP90AB1, and GSK3B) through the cytokine signaling pathway in the immune system, suggesting a potential therapeutic pathway for DM. However, it is essential to note that these findings are preliminary, and further research is necessary to validate them. Those research studies could involve invitro and invivo studies to confirm the bioactivity of the identified compounds and their interactions with the core targets. When the findings are confirmed, they could have significant clinical implications, potentially leading to developing new therapeutic strategies for DM.

Fluorescent Sensing for the Detection and Quantification of Sulfur-Containing Gases.

Sulfur-containing gases, such as H2S and SO2, play significant roles in a multitude of biological processes affecting human life and health. Precise and efficient detection of these gases is therefore crucial for advancing one's understanding of their biological roles and developing effective diagnostic strategies. Fluorescent sensing offers a highly sensitive and versatile approach for detecting these gases. This Review examines the recent advances in the fluorescent detection of H2S and SO2, highlighting the key mechanisms involved in fluorescence signal transduction, including changes in intensity and wavelength shifts. The diverse array of probe molecules employed for this purpose, including those utilizing mechanisms such as nucleophilic reactions, Förster resonance energy transfer (FRET), and sulfur affinity interactions are explored. In additional to organic sensors, the focus of the Review is particularly directed toward quantum dot (QD) systems, emphasizing their tunable optical properties that hold immense potential for fluorescence sensing. Beyond the traditional III-V QDs, we delve into the emerging fluorescence sensors based on halide perovskite QDs, upconversion nanocrystals, and other novel materials. These advanced QD systems hold promise for the development of highly sensitive and cost-effective gas detectors, paving the way for significant advances in biomedical and environmental monitoring. This Review provides a comprehensive overview of the current state-of-the-art in QD-based fluorescence sensing of sulfur-containing gases and provides a multifaceted discussion comparing organic fluorescent sensors with QD sensors, highlighting the key challenges and opportunities for the integration of fluorescence sensing as it evolves. The Review aims to facilitate further research and development of innovative sensing platforms to enable more accurate and sensitive detection of these important gases.

Proteome-Wide Association Studies for Blood Lipids and Comparison with Transcriptome-Wide Association Studies.

Blood lipid traits are treatable and heritable risk factors for heart disease, a leading cause of mortality worldwide. Although genome-wide association studies (GWAS) have discovered hundreds of variants associated with lipids in humans, most of the causal mechanisms of lipids remain unknown. To better understand the biological processes underlying lipid metabolism, we investigated the associations of plasma protein levels with total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) in blood. We trained protein prediction models based on samples in the Multi-Ethnic Study of Atherosclerosis (MESA) and applied them to conduct proteome-wide association studies (PWAS) for lipids using the Global Lipids Genetics Consortium (GLGC) data. Of the 749 proteins tested, 42 were significantly associated with at least one lipid trait. Furthermore, we performed transcriptome-wide association studies (TWAS) for lipids using 9,714 gene expression prediction models trained on samples from peripheral blood mononuclear cells (PBMCs) in MESA and 49 tissues in the Genotype-Tissue Expression (GTEx) project. We found that although PWAS and TWAS can show different directions of associations in an individual gene, 40 out of 49 tissues showed a positive correlation between PWAS and TWAS signed p-values across all the genes, which suggests a high-level consistency between proteome-lipid associations and transcriptome-lipid associations.

Identification of Goat Supernumerary Teat Phenotype Using Wide-Genomic Copy Number Variants

Supernumerary teats (SNTs) or nipples often emerge around the mammary line. This study performed a genome-wide selective sweep analysis (GWS) at the copy number variant (CNV) level using two selected signal calculation methods (VST and FST) to identify candidate genes associated with SNTs in goats. A total of 12,310 CNVs were identified from 37 animals and 123 CNVs, with the top 1% VST values including 84 candidate genes (CDGs). Of these CDGs, minichromosome maintenance complex component 3, ectodysplasin A receptor associated via death domain, and cullin 5 demonstrated functions closely related to mammary gland development. In addition, 123 CNVs with the top 1% FST values were annotated to 97 CDGs. 5-Hydroxytryptamine receptor 2A, CCAAT/enhancer-binding protein alpha, and the polymeric immunoglobulin receptor affect colostrum secretion through multiple signaling pathways. Two genes, namely, RNA-binding motif protein 46 and β-1,3-galactosyltransferase 5, showed a close relation to mammary gland development. Six CNVs were identified and annotated to five genes by intersecting the top 1% of candidate CNVs with both parameters. These genes include LOC102185621, LOC102190481, and UDP-glucose pyrophosphorylase 2, which potentially affect the occurrence of BC through multiple biological processes, such as cell detoxification, glycogen synthesis, and phospholipid metabolism. In conclusion, we discovered numerous genes related to mammary development and breast cancer (BC) through a GWS, which suggests the mechanism of SNTs in goats and a certain association between mammary cancer and SNTs.

Cohort Studies and Multi-omics Approaches to Low-Dose Ionizing Radiation-Induced Cardiovascular Disease: A Comprehensive Review.

The effect of low-dose ionizing radiation exposure on the risk of cardiovascular disease (CVD) represents a significant concern in the field of radiation protection. The prevailing approach to mitigating the adverse effects of low-dose or low-dose-rate radiation does not currently incorporate the potential risk of CVD, despite the possibility that such risk may be a substantial contributor to overall health hazards. Current evidence suggests a potential association between radiation exposure and CVD; however, the overall findings remain inconclusive. This is particularly due to the uncertainty surrounding the influence of significant non-radiation risk factors on the associations reported in epidemiological studies. It is difficult to discern the underlying connection in observational epidemiology when there is substantial variation in baseline risk factors. The paucity of epidemiological research in this domain is being partially offset by the advancement of multi-omics approaches. These methods assist in identifying radiosensitive targets, comprehending underlying biological processes, and pinpointing biomarkers. This, in turn, fortifies the evidence gleaned from epidemiological studies. In this review, we delve into the body of epidemiological research pertaining to CVD induced by low-dose ionizing radiation and the application of multi-omics techniques. The integration of these two methodologies holds the promise of identifying specific molecules or biological pathways that can be employed to validate endpoints related to radiation risk assessment.

Abstract B036: Pathway repression in progression on osimertinib therapy in NSCLC via a comprehensive genomic and epigenomic circulating tumor DNA (ctDNA) assay

Abstract Background: Osimertinib is currently the most common precision therapy for patients with non- small cell lung cancer (NSCLC) with EGFR mutations. While it prolongs progression-free survival, resistance eventually develops in many, leading to disease progression. Somatic NGS has identified resistance mutations in the EGFR pathway and other oncogenes, but these account for only 40-45% of cases, suggesting non-genetic factors, such as epigenetic changes, may be involved. This study investigates the epigenetic landscape at diagnosis and progression on first- line osimertinib, focusing on cases with and without canonical resistance mutations. Methods: We defined two cohorts from GuardantINFORM, a clinico-genomics database, restricting to only patients with a test on the Guardant Infinity platform, a comprehensive blood-based NGS assay, that analyzes DNA sequence variants and performs extensive methylation profiling (∼15Mb) in >20,000 differentially methylated regions (DMRs) assessed for sample-specific methylation patterns. (A) Diagnosis Cohort: Comprised of 34 NSCLC patients with blood samples collected within six days prior to the initiation of first-line osimertinib therapy. (B) Progression Cohort: Comprised of 36 patients who were on first-line osimertinib therapy, with blood samples collected within 60 days of discontinuation. The progression cohort was further stratified based on the presence or absence of known resistance mutations identified through somatic variant analysis by Guardant360. Gene set enrichment analysis was conducted on these methylation profiles using the Reactome database to define pathway-level differences between the cohorts. Results: Pathway enrichment analysis in the diagnosis and progression cohorts found expected biological processes in NSCLC such as extracellular matrix organization, G- protein coupled receptor (GPCR) signaling, and FGFR pathways. Additionally, the progression cohort lacking canonical resistance mutations (n=23) exhibited unique enrichment in two specific pathways: Glucose-dependent Insulinotropic Polypeptide and Physiological Factors. These pathways include genes such as GATA4, which has been implicated in the epithelial- mesenchymal transition (EMT)—a process associated with transforming to a small cell phenotype, a known mechanism of resistance to EGFR inhibition, including osimertinib. Conclusion: This study demonstrates the utility of epigenetic profiling via liquid biopsies in uncovering non-genetic mechanisms underlying osimertinib resistance in advanced NSCLC. Identification of unique epigenetic pathways in progressing patients without canonical resistance mutations highlights the potential for discovering novel biomarkers of resistance. Further investigation into these findings may advance our understanding of resistance mechanisms across various cancer therapies, particularly for patients without identified genomic resistance, and inform clinical decision-making and trial design. Citation Format: Aaron Hardin, Leslie Bucheit, Amar Das, Craig Eagel, Helmy Eltoukhy. Pathway repression in progression on osimertinib therapy in NSCLC via a comprehensive genomic and epigenomic circulating tumor DNA (ctDNA) assay [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B036.

Abstract PR018: Plasma proteomic biomarkers reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade

Abstract Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses, stressing a critical need to identify early non-invasive biomarkers of response. Circulating biomarkers provide easy access for serial monitoring and can provide insight on the mechanisms of response to ICB. We performed plasma proteomics (2943 proteins) on 250 metastatic melanoma patients before and on ICB treatment and performed analysis to identify response- and time point-associated peripheral protein biomarkers. We next generated patient- matched peripheral blood lymphocyte (PBL) surface proteomic data to measure the proportions of immune cell subsets in circulation. We further obtained 13 tumor samples that were processed for single-cell RNA-sequencing (scRNA-seq). We fit linear mixed effects (LME) models to predict plasma protein abundances, using the sample time point, response status and interaction term between time point and response as covariates. We identified 397 proteins with a significant time point, response, or interaction effect. For proteins upregulated post-ICB, gene ontology analysis suggested these proteins were associated with inflammatory pathways involving the activation of effector immune functions. LME models fitted on the circulating immune cell proportions and parallel analysis of immune-related plasma proteins supported that post-ICB samples may be defined by increased immune activity, including T cell infiltration. Moreover, expression of genes corresponding to these post-ICB proteins in the tumor scRNA-seq data tended to be favored by immune subsets, namely those involved in immune cell recruitment and tumor reactivity, such as CXCL10+ macrophages and CXCL13+ T cells, respectively. On the other hand, expression of genes corresponding to plasma proteins upregulated in non-responders in the TME tended to be favored by suppressive myeloid subsets such as SPP1+ macrophages as well as malignant cells, suggesting the potential of immunosuppressive and pro-tumor biological processes to be involved in ICB resistance. Inference of cell-cell interactions in the TME further showed the involvement of these non-responder genes in potentially immunosuppressive and pro-tumor processes, including the PTPRC-MRC1 axis, involved in myeloid cell plasticity, the IGF1/2-IGF1R axis, involved in growth and survival of normal and neoplastic cells, and the GAS6-TYRO3 axis, associated with carcinogenetic mechanisms in multiple malignancies. We further identified a correlation between IL13 protein abundance in circulation and GAS6 expression in the tumor, suggesting that IL13 may reach the tumor from circulation to turn on the STAT6 pathway in receiving cells and upregulate GAS6 in turn to participate in this interaction with TYRO3. Together, these data represent one of the deepest peripheral biomarker studies using paired samples in melanoma patients treated with anti-PD1 therapy, and begin to elucidate the complex interplay between tumors and the systemic immune response within the host. Citation Format: Samuel J. Wright, Izabella Zamora, Milan Parikh, Deepika Yeramosu, Lynn Bi, Sarah Kang, Marijana Rucevic, Moshe Sade-Feldman, Baolin Liu, Ngan Nguyen, Jamey Guess, Alexis Schneider, David Lieb, Elliot Woods, William Michaud, Aleigha R. Lawless, Tatyana Sharova, Gyulnara Kasumova, Michelle S. Kim, Ryan J. Park, Russell W. Jenkins, Samuel J. Klempner, Ryan J. Sullivan, Keith T. Flaherty, Nir Hacohen, Genevieve M. Boland, Arnav Mehta. Plasma proteomic biomarkers reveal biological insights about the tumor microenvironment in melanoma patients after PD1 blockade [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR018.

CircRNA profiling of skeletal muscle satellite cells in goats reveals circTGFβ2 promotes myoblast differentiation.

Circular RNAs (circRNAs) function as essential regulatory elements with pivotal roles in various biological processes. However, their expression profiles and functional regulation during the differentiation of goat myoblasts have not been thoroughly explored. This study conducts an analysis of circRNA expression profiles during the proliferation phase (cultured in growth medium, GM) and differentiation phase (cultured in differentiation medium, DM1/DM5) of skeletal muscle satellite cells (MuSCs) in goats. A total of 2,094 circRNAs were identified, among which 84 were differentially expressed as determined by pairwise comparisons across three distinct groups. Validation of the expression levels of six randomly selected circRNAs was performed using reverse transcription PCR (RT-PCR) and quantitative RT-PCR (qRT-PCR), with confirmation of their back-splicing junction sites. Enrichment analysis of the host genes associated with differentially expressed circRNAs (DEcircRNAs) indicated significant involvement in biological processes such as muscle contraction, muscle hypertrophy, and muscle tissue development. Additionally, these host genes were implicated in key signaling pathways, including Hippo, TGF-beta, and MAPK pathways. Subsequently, employing Cytoscape, we developed a circRNA-miRNA interaction network to elucidate the complex regulatory mechanisms underlying goat muscle development, encompassing 21 circRNAs and 47 miRNAs. Functional assays demonstrated that circTGFβ2 enhances myogenic differentiation in goats, potentially through a miRNA sponge mechanism. In conclusion, we identified the genome-wide expression profiles of circRNAs in goat MuSCs during both proliferation and differentiation phases, and established that circTGFβ2 plays a role in the regulation of myogenesis. This study offers a significant resource for the advanced exploration of the biological functions and mechanisms of circRNAs in the myogenesis of goats.

Pil1 Co-tethering Assay to Detect Protein-Protein Interactions in the Fission Yeast Schizosaccharomyces pombe.

Protein-protein interactions play critical roles in biological processes. We previously developed the Pil1 co-tethering assay, an imaging-based method to detect protein-protein interactions in living Schizosaccharomyces pombe cells. This assay leverages the distinct localization pattern of the Pil1 protein by fusing a bait protein to Pil1 and examining whether a prey protein co-localize with the Pil1-fused bait. Here, we present an improved protocol of the Pil1 co-tethering assay. In this protocol, modified stable integration vectors (SIVs) with a NotI site as the linearization site are used to express bait and prey proteins. We expect that this protocol will enhance the application of the Pil1 co-tethering assay for studying protein-protein interactions.

Effects of iron overload in human joint tissue explant cultures and animal models.

Osteoarthritis (OA) is a prevalent degenerative joint disease affecting over 530 million individuals worldwide. Recent studies suggest a potential link between iron overload, a condition characterised by the excessive accumulation of iron in the body, and the onset of OA. Iron is essential for various biological processes, and any disruption in its homeostasis can trigger significant health effects, including OA. This study aimed to elucidate the effects of excess iron on joint tissue and the underlying mechanisms associated with excess iron and OA development. Human articular cartilage (n = 6) and synovium (n = 4) were collected from patients who underwent total knee arthroplasty. Cartilage and synovium explants were incubated with a gradually increasing concentration of ferric ammonium citrate for 3days respectively. The effects of iron homeostasis in tissue explants were analysed using a Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). To further study the effects of iron excess on OA initiation and development, male 3-week-old Hfe-/- and 5-week-old Tfr2-/- mice, animal models of hereditary haemochromatosis were established. Littermate wild-type mice were fed a high-iron diet to induce dietary overload. All animals were sacrificed at 8weeks of age, and knee joints were harvested for histological analysis. The LA-ICP-MS analysis unveiled changes in the elemental composition related to iron metabolism, which included alterations in FTH1, FPN1, and HAMP within iron(III)-treated cartilage explants. While chondrocyte viability remained stable under different iron concentrations, ex vivo treatment with a high concentration of Fe3+ increased the catabolic gene expression of MMP13. Similar alterations were observed in the synovium, with added increases in GAG content and inflammation markers. In vivo studies further supported the role of iron overload in OA development as evidenced by spontaneous OA symptoms, proteoglycan loss, increased Mankin scores, synovial thickening, and enhanced immunohistochemical expression of MMP13, ADAMTS5, and P21 in Hfe-/-, Tfr2-/-, and diet-induced iron overload mouse models. Our findings elucidate the specific pathways through which excess iron accelerates OA progression and highlights potential targets for therapeutic intervention aimed at modulating iron levels to mitigate OA symptoms. KEY MESSAGES: Iron overload alters joint iron metabolism, increasing OA markers in cartilage and synovium. High iron levels in mice accelerate OA, highlighting genetic and dietary impacts. Excess iron prompts chondrocyte iron storage response, signalling potential OA pathways. Iron dysregulation linked to increased cartilage degradation and synovial inflammation. Our findings support targeted therapies for OA based on iron modulation strategies.

The Immunohistochemical and Bioinformatics Analysis of the Placental Expressions of Vascular Cell Adhesion Protein 1 (VCAM-1) and High Mobility Group Box 1 (HMGB1) Proteins in Gestational Diabetic Mothers.

We aimed to examine both the expression levels of high mobility group box 1 (HMGB1) and vascular cell adhesion molecule-1 (VCAM-1) proteins in the placentas of pregnant women with gestational diabetes mellitus (GDM) and control groups by immunohistochemical (IHC) method. An experimental case-control study was conducted, including 40 pregnant women complicated with GDM and 40 healthy pregnant women. Placental tissues obtained following cesarean delivery were subjected to routine tissue monitoring. The placental sections were stained with VCAM-1 and HMGB1 immunostains and subjected to IHC examination under a light microscope. H-score (HS) was used to evaluate the results of IHC staining by semi-quantitative analysis. Pathway analysis in Cytoscape software identified GDM-associated proteins within HMGB1 and VCAM-1 interaction networks, followed by GO analysis to explore associated biological processes. Placental HGMB1 expression was significantly increased in the GDM group compared to the control group (p<0.001). However, placental VCAM-1 expression was found to be statistically similar in GDM and control groups (p=0.584). The shared 19 proteins were identified between HMGB1 and GDM, and 13 between VCAM-1 and GDM, with notable GO biological process terms such as immune system activation for HMGB1 and interleukin-6 regulation for VCAM-1 associated with GDM. We consider that GDM-related inflammation and oxidative stress may contribute to tissue damage and inflammation by increasing placental HMGB1 expression. The blockade of HMGB1 and its receptors might represent a promising therapeutic approach to control inflammation in GDM. Understanding the distinct roles of HMGB1 and VCAM-1 may provide valuable insights for the development of targeted therapies aimed at mitigating the inflammatory processes associated with GDM and improving maternal and fetal outcomes.

Functional and Practical Insights Into the Genetic Basis of Takayasu Arteritis.

Takayasu arteritis (TAK) is a rare vasculitis characterized by inflammation of large arteries. Although the exact etiology of TAK remains unclear, a genetic predisposition to the disease has been established. Large-scale genetic studies have significantly contributed to the identification of genetic variation associated with immune-mediated diseases. To date, five genome-wide association studies (GWAS) have been performed in TAK, identifying multiple genetic susceptibility loci across the genome. Here, we summarize the major findings from GWAS in TAK and provide an in silico functional evaluation of the associated loci (P < 5 × 10-8) and variants in high linkage disequilibrium with them (r2 > 0.8). By exploring gene expression and chromatin interaction data, we identified candidate causal genes in TAK, some of them with well-known functional implications. The analysis of transcription factor motifs within TAK-associated loci revealed enrichment of the STAT and RUNX families, both characterized by their role in immune functions and inflammatory responses. The enrichment in biological processes in susceptibility loci confirmed the involvement of specific immune-related pathways in TAK. Further, we devised and calculated a cumulative genetic risk score for TAK and confirmed differences in genetic risk for the disease among ancestries. Finally, we provide a practical guide to communicate genetic information for TAK to patients and families.

Abstract 4138334: Identification and Immunological Characterization of Arrhythmia-Related Molecular Clusters in Ischemic Cardiomyopathy

Ischemic cardiomyopathy, a severe cardiac condition resulting from prolonged myocardial ischemia, is characterized by ventricular dilation, dysfunction, and an increased risk of life-threatening arrhythmias. Arrhythmia, a common complication in ischemic cardiomyopathy, is associated with poor clinical outcomes. Recent studies have indicated that the molecular heterogeneity of ischemic cardiomyopathy may contribute to the development of arrhythmias. Therefore, our study aimed to explore arrhythmia-related molecular clusters in ischemic cardiomyopathy and establish a prediction model for risk stratification. Based on gene expression profiles of ischemic cardiomyopathy patients, we analyzed the dysregulation of arrhythmia-associated genes and the associated immune characteristics (Figure 1A-C). Using unsupervised clustering analysis, we identified distinct arrhythmia-related molecular clusters in ischemic cardiomyopathy patients (Figure 1D-F). Cluster-specific differentially expressed genes were further identified using weighted gene co-expression network analysis (WGCNA). Functional enrichment analysis revealed significant differences in biological processes and pathways between the clusters (Figure 1G-H). Subsequently, we developed a prediction model using a random forest algorithm, which showed superior performance in distinguishing patients with different arrhythmia risks (Figure 1I-J). This forest model demonstrated excellent performance in one external validation datasets with AUC values of 0.891. In conclusion, our study provides novel insights into the molecular basis of arrhythmias in ischemic cardiomyopathy and establishes a prediction model with potential clinical utility for risk stratification and targeted therapies. Further investigation into the specific mechanisms underlying the identified molecular clusters may reveal new therapeutic targets for the management of arrhythmias in ischemic cardiomyopathy patients.