Transcriptomic Differences Research Articles

Differential transcriptome reprogramming induced by the soybean cyst nematode Type 0 and Type 1.2.5.7 during resistant and susceptible interactions.

Soybean cyst nematode (SCN, Heterodera glycines) is a serious root parasite of soybean (Glycine max) that induces extensive gene expression changes associated with pleiotropic biological activities in infected cells. However, the impacts of various SCN Hg Types on host transcriptome reprogramming remain largely unknown. Here, we developed and used two recombinant inbred lines (RIL-72 and RIL-137) to profile transcriptome reprogramming in the infection sites during the resistant and susceptible interactions with SCN Hg Type 1.2.5.7 and Type 0. SCN bioassays indicated that RIL-72 was susceptible to Type 1.2.5.7 but resistant to Type 0, whereas RIL-137 was resistant to both types. Comparative analysis of gene expression changes induced by Type 1.2.5.7 in the resistant and susceptible lines revealed distinct transcriptome regulation with a number of similarly and oppositely regulated genes. The expression levels of similarly regulated genes in the susceptible line appeared to be insufficient to mount an effective defense against SCN. The functional importance of oppositely regulated genes was confirmed using virus induced gene silencing and overexpression approaches. Further transcriptome comparisons revealed shared as well as Hg Type- and genotype-specific transcriptome reprogramming. Shared transcriptome responses were mediated through common SCN-responsive genes and conserved immune signaling, whereas genotype-specific responses were derived from genetic variability, metabolic and hormonal differences, and varied regulation of protein phosphorylation and ubiquitination. The conserved defense mechanisms together with genotype-specific responses would enable plants to trigger effective and tailored immune responses to various Hg types and adapt the defense response to their genetic backgrounds.

New insights into transcriptional and metabolic differences between pregnant males and embryos in seahorses

Seahorses exhibit a unique reproduction mode of male pregnancy. Successful gestation plays a crucial role in the seahorse reproduction process and the aquaculture industry. However, the substantial paternal-embryo differences in seahorses remain largely unexplored. In the present study, transcriptomic and metabolic differences between pregnant males and embryos during gestation were explored to investigate the roles of key genes and metabolites in pregnant males and embryos in seahorses. Higher levels of essential amino acids and polyunsaturated fatty acids in embryos compared to fathers may indicate their vital role in embryonic development. The high expression of immune-related genes (CCLs and CCRs) and downregulation of antioxidants in fathers may provide immune protection and reduce the immunological rejection of embryos during gestation. The transcript-metabolite correlation network during pregnancy further indicated the role of key genes and metabolites during embryonic eye (N-acetyl-L-histidine, PAX6, and FZT4) and tail (WNT5 and WNT8) development. The findings of the present study not only provide valuable data on differences between pregnant males and embryos in seahorse, but also lay foundation for future research on seahorse aquaculture.

Transcriptional responses in a mouse model of silicone wire embolization induced acute retinal artery ischemia and reperfusion.

Acute retinal ischemia and ischemia-reperfusion injury are the primary causes of retinal neural cell death and vision loss in retinal artery occlusion (RAO). The absence of an accurate mouse model for simulating the retinal ischemic process has hindered progress in developing neuroprotective agents for RAO. We developed a unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model using silicone wire embolization combined with carotid artery ligation. The survival of retinal ganglion cells and visual function were evaluated to determine the duration of ischemia. Immunofluorescence staining, optical coherence tomography, and haematoxylin and eosin staining were utilized to assess changes in major neural cell classes and retinal structure degeneration at two reperfusion durations. Transcriptomics was employed to investigate alterations in the pathological process of UPOAO following ischemia and reperfusion, highlighting transcriptomic differences between UPOAO and other retinal ischemia-reperfusion models. The UPOAO model successfully replicated the acute interruption of retinal blood supply observed in RAO. 60 min of Ischemia led to significant loss of major retinal neural cells and visual function impairment. Notable thinning of the inner retinal layer, especially the ganglion cell layer, was evident post-UPOAO. Temporal transcriptome analysis revealed various pathophysiological processes related to immune cell migration, oxidative stress, and immune inflammation during the non-reperfusion and reperfusion periods. A pronounced increase in microglia within the retina and peripheral leukocytes accessing the retina was observed during reperfusion periods. Comparison of differentially expressed genes (DEGs) between the UPOAO and high intraocular pressure models revealed specific enrichments in lipid and steroid metabolism-related genes in the UPOAO model. The UPOAO model emerges as a novel tool for screening pathogenic genes and promoting further therapeutic research in RAO.

Transcriptional responses in a mouse model of silicone wire embolization induced acute retinal artery ischemia and reperfusion

Acute retinal ischemia and ischemia-reperfusion injury are the primary causes of retinal neural cell death and vision loss in retinal artery occlusion (RAO). The absence of an accurate mouse model for simulating the retinal ischemic process has hindered progress in developing neuroprotective agents for RAO. We developed a unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model using silicone wire embolization combined with carotid artery ligation. The survival of retinal ganglion cells and visual function were evaluated to determine the duration of ischemia. Immunofluorescence staining, optical coherence tomography, and haematoxylin and eosin staining were utilized to assess changes in major neural cell classes and retinal structure degeneration at two reperfusion durations. Transcriptomics was employed to investigate alterations in the pathological process of UPOAO following ischemia and reperfusion, highlighting transcriptomic differences between UPOAO and other retinal ischemia-reperfusion models. The UPOAO model successfully replicated the acute interruption of retinal blood supply observed in RAO. 60 min of Ischemia led to significant loss of major retinal neural cells and visual function impairment. Notable thinning of the inner retinal layer, especially the ganglion cell layer, was evident post-UPOAO. Temporal transcriptome analysis revealed various pathophysiological processes related to immune cell migration, oxidative stress, and immune inflammation during the non-reperfusion and reperfusion periods. A pronounced increase in microglia within the retina and peripheral leukocytes accessing the retina was observed during reperfusion periods. Comparison of differentially expressed genes (DEGs) between the UPOAO and high intraocular pressure models revealed specific enrichments in lipid and steroid metabolism-related genes in the UPOAO model. The UPOAO model emerges as a novel tool for screening pathogenic genes and promoting further therapeutic research in RAO.

Cell and transcriptomic diversity of infrapatellar fat pad during knee osteoarthritis

ObjectivesIn this study, we employ a multiomic approach to identify major cell types and subsets, and their transcriptomic profiles within the infrapatellar fat pad (IFP), and to determine differences in...

Sex differences in the molecular profile of adult diffuse glioma are shaped by IDH status and tumor microenvironment.

Sex differences in adult diffuse glioma (ADG) are well-established clinically, yet the underlying molecular mechanisms remain inadequately understood. Here, we aim to reveal molecular features and cellular compositions unique to each sex in ADG to comprehend the role of sex in disease etiology. We quantified sex differences in transcriptome of ADG using multiple independent glioma patient datasets. Next, we delved into the single-cell landscape to examine sex differences in gene expression and cellular composition. To explore how sex influences disease progression, we analyzed paired samples from primary and recurrent ADG cases, aiming to identify sex-specific differences in molecular and cellular features. Our analysis revealed that mutations in isocitrate dehydrogenase (IDH) genes and the tumor microenvironment emerged as primary influencers of sex-differential molecular enrichments. In IDHwt tumors, genes in neuronal signaling pathway are found to be enriched in male tumors, while genes in hypoxia and inflammatory response pathways are enriched in female tumors. This pattern was reversed in IDHmut gliomas. We hypothesized that these distinctions could be attributed to heterogeneous cellular composition between sexes. Using single-cell data, we observed distinctive patterns of sex differences in cell states, cell composition and cell-cell interaction in IDHwt and IDHmut tumors separately. Further, by comparing molecular changes in paired primary and recurrent ADG samples, we identified sex-specific differences in molecular characteristics and cellular compositions of recurrent tumors. Our results provide a comprehensive multi-level characterization of sex differences in ADG, such findings provide novel insights into glioma disease progression in each sex.

8302 A Novel Tool to Map Estrogen Responsiveness Within Estrogen Sensitive Hypothalamic Neurons in Mice

Abstract Disclosure: A.L. Cara: None. A.L. Hall: None. K.K. Soma: None. J. van Veen: None. S.M. Correa: None. Identification of hormone sensitive cells has been traditionally evaluated by hormone receptor expression. Receptor positive cells can be labeled via expression of mRNA transcript via in situ hybridization, presence of protein via immunohistology, or via conditionally-dependent Cre recombinase driven reporter expression. However, these methods used to label and manipulate cells only indicate which cells are capable of sensing hormones, and not necessarily which cells are responding to a hormonal signal in a specific physiologic or behavioral context. Furthermore, broad labeling of all receptor positive cells may not accurately reflect the heterogeneous nature of certain cell populations, particularly in the brain. The hypothalamus is a brain region with well characterized hormone receptor expression, including estrogen receptors, such as ERα (encoded by the Esr1 gene). Estrogens have a broad array of physiologic and behavioral functions which are mediated through hypothalamic nuclei, including thermoregulation, metabolism, and reproduction. Neuronal populations mediating these functions have been characterized, targeted, and manipulated based on ERα expression, but the dynamic response to estrogens in these highly heterogeneous populations has yet to be uncovered. We developed a custom reporter virus that fluorescently labels estrogen-sensitive and responsive cells. The virus contains a flip-excision switch (FLEX) to drive Cre-dependent expression of a FusionRed fluorescent reporter, eight tandem estrogen response elements (ERE), and a destabilized enhanced green fluorescent protein (EGFP) with a 2-hour half-life. The Cre-dependent expression of FusionRed identifies all Esr1-Cre cells at the time of transduction, while the ERE-EGFP identifies a subset of Esr1-Cre+ cells that are actively responding to estrogens via nuclear receptor signaling. We stereotactically injected the ERE-EGFP virus to distinct hypothalamic nuclei of Esr1-Cre female mice. Following transduction, mice were sacrificed during different stages of the estrous cycle. Spatial analysis of ERE-EGFP expression revealed dynamic changes across the estrous cycle, with responses differing based on hypothalamic nucleus. Reporter expression dynamics were compared to estrogen levels measured by LC-MS/MS in ovary, serum, and microdissected brain tissue. A separate group of mice were ovariectomized and given estradiol benzoate (EB) or vehicle control, and neurons were flow sorted for bulk RNA sequencing. We discovered transcriptomic differences in FusionRed+ cells with high or low ERE-EGFP expression, including upregulation of estrogen target genes (e.g., Pgr, encoding progesterone receptor) with EB treatment in ERE-EGFP+ cells. In summary, this new viral reporter reveals divergent responses to estrogens across subsets of estrogen sensitive neurons, brain regions, and hormonal states. Presentation: 6/2/2024

Pancreatic cancer tumor organoids exhibit subtype-specific differences in metabolic profiles

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease characterized by complex metabolic rewiring that enables growth in changing nutrient availability and oxygen conditions. Transcriptome-based prognostic PDAC tumor subtypes, known as ‘basal-like’ and ‘classical’ subtypes are associated with differences in metabolic gene expression including genes involved in glycolysis. Tumor subtype-specific metabolism phenotypes may provide new targets for treatment development in PDAC, but their functional relevance has not been fully elucidated. We aimed to investigate differences in metabolic profiles and transcriptomes in tumor models derived from patients with basal-like and classical tumors.MethodsPatient-derived organoids (PDOs) were established from tumor biopsies collected from patients with metastatic PDAC, including three PDOs from basal-like and five PDOs from classical tumors. Metabolic analyses included assessment of differences in metabolic activity using Seahorse Glycolysis and Mito Stress tests and 13C-glucose metabolites tracing analysis. In order to investigate the influence of mitochondrial pyruvate transport on metabolic differences, PDOs were treated with the mitochondrial pyruvate carrier 1 (MPC1) inhibitor UK-5099. Prognostic relevance of MPC1 was determined using a tumor tissue microarray (TMA) in resectable, and proteomics profiling in metastatic PDAC datasets. Whole genome and transcriptome sequencing, differential gene expression and gene set enrichment analyses were performed in PDOs.ResultsMetastatic PDAC PDOs showed subtype-specific differences in glycolysis and oxidative phosphorylation (OXPHOS). Basal-like tumor-derived PDOs had a lower baseline extracellular acidification rate, but higher glycolytic reserves and oxygen consumption rate (OCR) than classical tumor-derived PDOs. OCR difference was eliminated following treatment with UK-5099. In the 13C-glucose metabolites tracing experiment, a basal-like tumor PDO showed lower fractions of some M + 2 metabolites but higher sensitivity to UK-5099 mediated reduction in M + 2 metabolites than a classical tumor PDO. Protein level analyses revealed lower MPC1 protein levels in basal-like PDAC cases and association of low MPC1 levels with clinicopathologic parameters of tumor aggressiveness in PDAC. PDO differential gene expression analyses identified additional subtype-specific cellular pathways and potential disease outcome biomarkers.ConclusionsOur findings point to distinct metabolic profiles in PDAC subtypes with basal-like tumor PDOs showing higher OXPHOS and sensitivity to MPC1 inhibition. Subtypes-specific metabolic vulnerabilities may be exploited for selective therapeutic targeting.

Comparative analysis of transcriptomics of Fasciola hepatica at different developmental stages.

This study explored the transcriptome differences in Fasciola hepatica at different developmental stages and identified functional genes related to growth and development during juvenile stages. DNBSEQ eukaryotic strand-specific transcriptome resequencing technology was used to sequence the transcriptomes of Fasciola hepatica eggs, juveniles, and adults. Additionally, the genes that were highly expressed during the juvenile stage were validated using qRT-PCR. The Q20 values of all three phases of sequencing were above 98%, and the Q30 values were above 94%. The differentially expressed genes (DEGs) in pairwise comparisons were analyzed by GO functional classification and the KEGG pathway database. Many immune-, growth-, and development-related pathways were found, which might be related to cell proliferation, development, and host immune evasion by Fasciola hepatica. In addition, five DEGs with high expression levels during the juvenile stage were identified: Cathepsin B, Glutathione S-transferase mu, heat shock protein 67B2, Kunitz-CH, and Legumain. Validation analyses revealed that these genes play key roles in maintaining normal growth, development, and immunological processes in liver Fasciola hepatica. RNA-seq was used to analyze the biological characteristics of the DEGs at different developmental stages concerning GO functional classification and KEGG metabolic pathways. Five DEGs with high expression during the juvenile stage were identified. These genes are related to the growth, development, and immune function of Fasciola hepatica, which provides a theoretical basis for subsequent research on the proteomics of Fasciola hepatica and the screening of candidate genes for early diagnosis.

The P2Y6 receptor as a potential keystone in essential hypertension.

Essential hypertension (HT) is a highly prevalent cardiovascular disease of unclear physiopathology. Pharmacological studies suggest that purinergic P2Y6 receptors (P2ry6) play important roles in cardiovascular function and may contribute to angiotensin II (AgtII) pathophysiological effects. Here, we tested the hypothesis that functional coupling between P2ry6 and AgtII receptors mediates altered vascular reactivity in HT. For this, a multipronged approach was implemented using mesenteric vascular smooth muscle cells (VSMCs) and arteries from BPN (Blood Pressure Normal) and BPH (Blood Pressure High) mice. Differential transcriptome profiling of mesenteric artery VSMCs identified P2ry6 purinergic receptor mRNA as one of the top upregulated transcripts in BPH. P2Y receptor activation elicited distinct vascular responses in mesenteric arteries from BPN and BPH mice. Accordingly, 10 µM UTP produced a contraction close to half-maximal activation in BPH arteries but no response in BPN vessels. AgtII-induced contraction was also higher in BPH mice despite having lower AgtII receptor type-1 (Agtr1) expression and was sensitive to P2ry6 modulators. Proximity Ligation Assay (PLA) and super-resolution microscopy (SRM) showed closer localization of Agtr1 and P2ry6 at/near the membrane of BPH mice. This proximal association was reduced in BPN mice, suggesting a functional role for Agtr1-P2ry6 complexes in the hypertensive phenotype. Intriguingly, BPN mice were resistant to AgtII-induced HT and showed reduced P2ry6 expression in VSMCs. Altogether, results suggest that increased functional coupling between P2ry6 and Agtr1 may contribute to enhanced vascular reactivity during HT. In this regard, blocking P2ry6 could be a potential pharmacological strategy to treat HT.

Cross-species comparison of airway epithelium transcriptomics

Studies of lung transcriptomics across species are essential for understanding the complex biology and disease mechanisms of this vital organ. Single-cell RNA sequencing (scRNA-seq) has emerged as a key tool for understanding cell dynamics across various species. However, comprehensive cross-species comparisons are limited. Therefore, the aims of this study was to investigate the transcriptomic similarities and differences in lung cells across four species—humans, monkeys, mice, and rats—in healthy and asthma conditions using scRNA-seq. The results revealed significant transcriptomic similarities between monkeys and humans and significant cross-species conservation of cell-specific marker genes, transcription factors (TFs), and biological pathways. Additionally, we explored sex differences, identifying distinct sex-specific expression patterns that may influence disease susceptibility. These insights refine our understanding of the mechanism underlying airway cell biology across species and have important implications for studying lung diseases, particularly the mechanisms of mucus clearance in asthma.

Transcriptomic Analysis of the Hepatopancreas in the Sex-Related Size Differences of Macrobrachium nipponense.

Macrobrachium nipponense, a commercially popular crustacean species within the Chinese context, is recognized for its exceptional nutritional composition and palatability. There are significant differences in growth between male and female M. nipponense. Herein, transcriptomics was used to determine the hepatopancreas transcriptome differences between sex-related size differences in M. nipponense. We identified 974 differentially expressed genes (DEGs) between the SHE (female) and BHE (male) groups, which were validated using RT-qPCR. The genes encoding matrix metalloproteinase-9 (MM9), Ribosome-binding protein 1 (RBP1), Aly/REF export factor 2, and hematological and neurological expressed 1 (HN1) may play a role in modulating the sex-related size differences observed in M. nipponense. Clusters of orthologous groups and gene ontology functional analysis demonstrated that the DEGs for sex-related size in M.nipponense were associated with various biological functions. The Kyoto Encyclopedia of Genes and Genomes pathways analysis demonstrated that upregulated DEGs were mainly enriched in lysine biosynthesis, tryptophan metabolism, and lysine degradation pathways, whereas the downregulated DEGs were mainly enriched in ascorbate and aldarate metabolism, retinol metabolism, and drug metabolism-cytochrome P450 pathways. The results indicated the molecular mechanism underlying the sex-related size differences and identified key genes. This data will be invaluable to support explanations of individual differences between male and female prawns.

Integrated cytological and transcriptomic analyses provide new insights into restoration of pollen viability in synthetic allotetraploid Brassica carinata.

Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata.

Single-cell RNA-sequencing reveals a unique landscape of the tumor microenvironment in obesity-associated breast cancer

As two diseases with rapidly increasing incidence, the molecular linkages between obesity and breast cancer (BC) are intriguing. Overall, obesity may be a negative prognostic factor for BC. Single-cell RNA-sequencing (scRNA-seq) was performed on tumor tissues from 6 obese and non-obese BC patients. With 48,033 cells analyzed, we found heterogeneous tumor epithelium and microenvironment in these obese and lean BC patients. Interestingly, the obesity-associated epithelial cells exhibited specific expression signatures which linked tumor growth and hormone metabolism in BC. Notably, one population of obesity-specific macrophage up-regulated the nuclear receptor subfamily 1 group H member 3 (NR1H3), which acted a transcription factor and regulated FABP4 expression through its interaction with the DNA of SREBP1, and further increased the proliferation of tumor cells in BC. Using single-cell signatures, our study illustrate cell diversity and transcriptomic differences in tumors from obese and non-obese BC patients, and sheds light on potential molecular link between lipid metabolism and BC.

Using a combination of bulk and single cell RNA sequencing to identify transcriptomic differences in metastatic vs non-metastatic phaeochromoctyomas and paragangliomas

Using a combination of bulk and single cell RNA sequencing to identify transcriptomic differences in metastatic vs non-metastatic phaeochromoctyomas and paragangliomas

Abstract A062: Neurotropic fibroblast population increases following exposure to chemotherapy in pancreatic adenocarcinoma

Abstract Introduction Pancreatic adenocarcinoma (PDA) will be the second leading cause of cancer related death by 2030 and patient response to chemotherapy is poor with a strong likelihood of cancer recurrence. While past studies have investigated pre- and post-chemotherapy patient samples, there is a paucity of studies looking at fresh, patient matched samples which allow us to observe interpatient heterogeneity in chemotherapy response. Methods Here, we study the effects of chemotherapy on five different patients using sc-RNA sequencing of matched treatment naïve and post treatment tissue biopsies to compare the effects of chemotherapy, not only on the tumor cells, but also on the tumor microenvironment – a key contributor to tumor survival and proliferation. We utilized the Seurat v4 pipeline and CellChat v2 to observe transcriptomic differences in epithelial cells, myeloid cells, t-Cells, and fibroblasts and their corresponding putative interactions in pre- and post- treatment states. Results Unsurprisingly, we found insufficient tumor epithelial cells post-treatment to perform further analysis on due to the intended cytotoxic effects of chemotherapy. Therefore, we turned our focus to the tumor microenvironment, where we had sufficient numbers of stromal and immune cells, to investigate the effects of chemotherapy. We identified that fibroblasts changed the most consistently across all patients in response to chemotherapy. The fibroblasts clustered into three distinct groups: an immunomodulatory group, a myofibroblastic and neurotropic group, as well as a mixed group expressing genes from all three categories. Interestingly, the neurotropic fibroblast signature was universally enriched after chemotherapy in all five patients, despite having a small and clinically heterogeneous cohort. Conclusions Through an analysis of each fibroblast group in the treatment naïve and post treatment categories, we observed that a specific set of fibroblasts become less myofibroblastic and increasingly neurotropic following treatment, leading us to posit that fibroblasts altered by exposure to chemotherapy may potentially contribute to a ‘pro-tumor’ environment that supports recurrence. To test this in vitro, we treated patient derived fibroblasts with Gemcitabine and used RTPCR to analyze their relative changes in gene expression. We specifically probed for genes that were definitive of myofibroblasts and neurotropic fibroblasts and saw that post-chemotherapy exposure, neurotropic genes such as NEGR1 and NFIA were upregulated. This validated our scRNA conclusions that fibroblasts become increasingly neurotropic following treatment and then differentially interact with their surrounding environment. In future studies, we will further analyze the role these neurotropic fibroblasts play in chemoresponse and tumor recurrence. Matched samples are critical to studies of chemotherapy effects on PDA due to the fact that they allow us to deconvolute one aspect of PDA heterogenity - interpatient heterogeneity - which is a large limitation in studies of this disease. Citation Format: Aylin Z Henstridge, Padma Kadiyala, Ahmed M Elhossiny, Jianhua Liu, Vaibhav Sahai, Nicole Peterson, Jorge Machicado, Richard Kwon, Allison Schulman, Erik Wamsteker, George Philips, Martin Fernandez-Zapico, Mark Truty, Costas Lyssiotis, Timothy Frankel, Filip Bednar, Marina Pasca di Magliano, Eileen S Carpenter. Neurotropic fibroblast population increases following exposure to chemotherapy in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A062.

Transcriptomic Differences by RNA Sequencing for Evaluation of New Method for Long-Time In Vitro Culture of Cryopreserved Testicular Tissue for Oncologic Patients.

Earlier studies have established that culturing human ovarian tissue in a 3D system with a small amount of soluble Matrigel (a basement membrane protein) for 7 days in vitro increased gene fusion and alternative splicing events, cellular functions, and potentially impacted gene expression. However, this method was not suitable for in vitro culture of human testicular tissue. To test a new method for long-time in vitro culture of testicular fragments, thawed with two different regimes, with evaluation of transcriptomic differences by RNA sequencing. Testicular tissue samples were collected, cryopreserved (frozen and thawed), and evaluated immediately after thawing and following one week of in vitro culture. Before in vitro culture, tissue fragments were encapsulated in fibrin. Four experimental groups were formed. Group 1: tissue quickly thawed (in boiling water at 100 °C) and immediately evaluated. Group 2: tissue quickly thawed (in boiling water at 100 °C) and evaluated after one week of in vitro culture. Group 3: tissue slowly thawed (by a physiological temperature 37 °C) and immediately evaluated. Group 4: tissue slowly thawed (by a physiological temperature 37 °C) and evaluated after one week of in vitro culture. There are the fewest differentially expressed genes in the comparison between Group 2 and Group 4. In this comparison, significantly up-regulated genes included C4B_2, LOC107987373, and GJA4, while significantly down-regulated genes included SULT1A4, FBLN2, and CCN2. Differential genes in cells of Group 2 were mainly enriched in KEGG: regulation of actin cytoskeleton, lysosome, proteoglycans in cancer, TGF-beta signaling pathway, focal adhesion, and endocytosis. These Group 2- genes were mainly enriched in GO: spermatogenesis, cilium movement, collagen fibril organization, cell differentiation, meiotic cell cycle, and flagellated spermatozoa motility. Encapsulation of testicular tissue in fibrin and long-time in vitro culture with constant stirring in a large volume of culture medium can reduce the impact of thawing methods on cryopreserved testicular tissue.

Role of TOMM34 on NF-κB activation-related hyperinflammation in severely ill patients with COVID-19 and influenza

Highly pathogenic respiratory RNA viruses such as SARS-CoV-2 and its associated syndrome COVID-19 pose a tremendous threat to the global public health. Innate immune responses to SARS-CoV-2 depend mainly upon the NF-κB-mediated inflammation. Identifying unknown host factors driving the NF-κB activation and inflammation is crucial for the development of immune intervention strategies. Published single-cell RNA sequencing (scRNA-seq) data was used to analyze the differential transcriptome profiles of bronchoalveolar lavage (BAL) cells between healthy individuals (n=27) and patients with severe COVID-19 (n=21), as well as the differential transcriptome profiles of peripheral blood mononuclear cells (PBMCs) between healthy individuals (n=22) and severely ill patients with COVID-19 (n=45) or influenza (n=16). Loss-of-function and gain-of-function assays were performed in diverse viruses-infected cells and male mice models to identify the role of TOMM34 in antiviral innate immunity. TOMM34, together with a list of genes encoding pro-inflammatory cytokines and antiviral immune proteins, was transcriptionally upregulated in circulating monocytes, lung epithelium and innate immune cells from individuals with severe COVID-19 or influenza. Deficiency of TOMM34/Tomm34 significantly impaired the type I interferon responses and NF-κB-mediated inflammation in various human/murine cell lines, murine bone marrow-derived macrophages (BMDMs) and invivo. Mechanistically, TOMM34 recruits TRAF6 to facilitate the K63-linked polyubiquitination of NEMO upon viral infection, thus promoting the downstream NF-κB activation. In this study, viral induction of TOMM34 is positively correlated with the hyperinflammation in severely ill patients with COVID-19 and influenza. Our findings also highlight the physiological role of TOMM34 in the innate antiviral signallings. A full list of funding sources can be found in the acknowledgements section.

Transcriptome profiles of the skin associated with the color of the black and white coat of Angora goats

The coat color of mammals, determined by the distribution of melanin, particularly eumelanin and pheomelanin, reflects intricate genetic and molecular processes. However, our understanding of the relationship between coat color, gene expression, and polymorphisms in goats remains nascent. This study investigates transcriptomic differences between black and white Angora goats across three distinct hair growth phases. Skin tissue samples from both colored and white Angora goats were analyzed using mRNA expression profiling. Three skin samples were taken from each goat as biological duplicates at every stage of hair follicle growth (September, January, and March). In total, 36 samples were analyzed in this study, including samples from two Angora goat varieties, three developmental stages (three biological replicates), and two technical replicates for RNA sequencing. Significant differences in gene expression were observed between black and white goats at each growth phase, particularly in genes associated with the melanogenesis pathway. Specifically, several pigmentation genes were identified solely in black goats, indicating phase-specific and breed-specific regulation. Noteworthy genes, such as SLC2A1, STAR, and SLC7A5, exhibited differential expression patterns across growth phases in black goats, further highlighting the complexity of melanogenesis regulation. This is the first study to use mRNA expression profiling of skin tissues to analyze coat color differences between black and white coated Angora goats at the anagen, catagen, and telogen stages. The identification of phase-specific and black goat-specific pigmentation genes provides valuable insights into the complex mechanisms governing coat color formation.

Foliar Application of Abscisic Acid Alleviates Cadmium Stress by Modulating Differential Physiological and Transcriptome Response in Leaves, Stems, and Roots of Mung Bean Seedlings

Foliar Application of Abscisic Acid Alleviates Cadmium Stress by Modulating Differential Physiological and Transcriptome Response in Leaves, Stems, and Roots of Mung Bean Seedlings