Changes In Transcriptomic Profiles Research Articles

Metabolome and Transcriptome Association Analysis Reveals the Link Between Pigmentation and Nutrition Utilization in the Juveniles of Sea Cucumber Holothuria leucospilota.

The sea cucumber Holothuria leucospilota is an economically and ecologically important tropical species. Following development into juveniles, H. leucospilota undergoes a color change from white to black, involving a pigmentation process for over a period of several months. In this study, a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Next-Generation sequencing (NGS) were employed to investigate the changes in metabolomic and transcriptomic profiles during pigmentation in H. leucospilota juveniles. The metabolomic analysis identified a total of 341 metabolites, of which 52 were found to be differentially regulated (P < 0.05 and VIP > 1), with 27 being upregulated in white individuals and 25 in black individuals. Additionally, 632 differentially expressed genes (DEGs) were identified, with 380 genes upregulated in white samples and 252 genes upregulated in black samples. Interestingly, the melanin content and tyrosinase transcript levels did not display significant differences between the two groups. Metabolomic data suggested the involvement of the linoleic acid metabolic pathway in pigmentation. Transcriptomic analysis, coupled with realtime PCR validation, revealed a decrease in the transcript levels of digestive enzymes like α-amylase, maltase-glucoamylase, and trehalase after the juveniles changed to black. Furthermore, the mRNA expressions of major yolk proteins showed a decline, indicating a shift in the accumulation of protein nutrient sources. Overall, our findings suggest that during the pigmentation process in H. leucospilota, no significant changes were observed in the classical melanin pathway, while notable alterations were observed in their nutritional status. This study provides valuable insights into the regulatory mechanisms of pigmentation in marine organisms.

Analysis of transcriptomic, lipidomic and phospho-kinase profiles reveals the effects of chlorogenic acid on 3T3-L1 preadipocytes differentiation

Chlorogenic acid has been widely reported to profoundly influence preadipocyte differentiation but without consensus being achieved. In the current study, 3 T3-L1 preadipocytes were differentiated using adipogenic cocktail with or without chlorogenic acid. The influences of chlorogenic acid on the mRNA, lipid and phospho-kinase profiles were evaluated using transcriptomic analysis, lipidomic analysis and phospho-kinase array assay. The obtained results showed that chlorogenic acid treatment during 3T3-L1 cells differentiation altered a series of genes transcription, resulted in obvious changes in the contents of various lipids, and inhibited phosphorylation of several adipogenesis-related kinases. Overall, the observed changes in transcriptomic, lipidomic and phospho-kinase profiles in the current study favor the findings that chlorogenic acid may inhibit the differentiation of 3T3-L1 preadipocytes. In conclusion, the findings from this study highlighted the therapeutic potential of chlorogenic acid for the treatment of obesity.

Sex-dependent differences in the genomic profile of lingual sensory neurons in naïve and tongue-tumor bearing mice

Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: (1) FACS sorting obtained higher number of neurons from female trigeminal ganglia (TG) compared to males; (2) Naïve female neurons innervating the tongue expressed immune cell markers such as Csf1R, C1qa and others, that weren’t expressed in males. This was validated by Immunohistochemistry. (3) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. (4) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, (5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.

Sink-source imbalance triggers delayed photosynthetic induction: Transcriptomic and physiological evidence.

Sink-source imbalance causes accumulation of nonstructural carbohydrates (NSCs) and photosynthetic downregulation. However, despite numerous studies, it remains unclear whether NSC accumulation or N deficiency more directly decreases steady-state maximum photosynthesis and photosynthetic induction, as well as underlying gene expression profiles. We evaluated the relationship between photosynthetic capacity and NSC accumulation induced by cold girdling, sucrose feeding, and low nitrogen treatment in Glycine max and Phaseolus vulgaris. In G. max, changes in transcriptome profiles were further investigated, focusing on the physiological processes of photosynthesis and NSC accumulation. NSC accumulation decreased the maximum photosynthetic capacity and delayed photosynthetic induction in both species. In G. max, such photosynthetic downregulation was explained by coordinated downregulation of photosynthetic genes involved in the Calvin cycle, Rubisco activase, photochemical reactions, and stomatal opening. Furthermore, sink-source imbalance may have triggered a change in the balance of sugar-phosphate translocators in chloroplast membranes, which may have promoted starch accumulation in chloroplasts. Our findings provide an overall picture of photosynthetic downregulation and NSC accumulation in G. max, demonstrating that photosynthetic downregulation is triggered by NSC accumulation and cannot be explained solely by N deficiency.

Transcriptomic and Proteomic Analyses of Myzus persicae Carrying Brassica Yellows Virus.

Viruses in the genus Polerovirus infect a wide range of crop plants and cause severe economic crop losses. BrYV belongs to the genus Polerovirus and is transmitted by Myzus persicae. However, the changes in transcriptome and proteome profiles of M. persicae during viral infection are unclear. Here, RNA-Seq and TMT-based quantitative proteomic analysis were performed to compare the differences between viruliferous and nonviruliferous aphids. In total, 1266 DEGs were identified at the level of transcription with 980 DEGs being upregulated and 286 downregulated in viruliferous aphids. At the protein level, among the 18 DEPs identified, the number of upregulated proteins in viruliferous aphids was twice that of the downregulated DEPs. Enrichment analysis indicated that these DEGs and DEPs were mainly involved in epidermal protein synthesis, phosphorylation, and various metabolic processes. Interestingly, the expressions of a number of cuticle proteins and tubulins were upregulated in viruliferous aphids. Taken together, our study revealed the complex regulatory network between BrYV and its vector M. persicae from the perspective of omics. These findings should be of great benefit to screening key factors involved in the process of virus circulation in aphids and provide new insights for BrYV prevention via vector control in the field.

Hepatic Global Transcriptomic Profiles of Holstein Cows According to Parity Reveal Age-Related Changes in Early Lactation.

Cows can live for over 20 years, but their productive lifespan averages only around 3 years after first calving. Liver dysfunction can reduce lifespan by increasing the risk of metabolic and infectious disease. This study investigated the changes in hepatic global transcriptomic profiles in early lactation Holstein cows in different lactations. Cows from five herds were grouped as primiparous (lactation number 1, PP, 534.7 ± 6.9 kg, n = 41), or multiparous with lactation numbers 2-3 (MP2-3, 634.5 ± 7.5 kg, n = 87) or 4-7 (MP4-7, 686.6 ± 11.4 kg, n = 40). Liver biopsies were collected at around 14 days after calving for RNA sequencing. Blood metabolites and milk yields were measured, and energy balance was calculated. There were extensive differences in hepatic gene expression between MP and PP cows, with 568 differentially expressed genes (DEGs) between MP2-3 and PP cows, and 719 DEGs between MP4-7 and PP cows, with downregulated DEGs predominating in MP cows. The differences between the two age groups of MP cows were moderate (82 DEGs). The gene expression differences suggested that MP cows had reduced immune functions compared with the PP cows. MP cows had increased gluconeogenesis but also evidence of impaired liver functionality. The MP cows had dysregulated protein synthesis and glycerophospholipid metabolism, and impaired genome and RNA stability and nutrient transport (22 differentially expressed solute carrier transporters). The genes associated with cell cycle arrest, apoptosis, and the production of antimicrobial peptides were upregulated. More surprisingly, evidence of hepatic inflammation leading to fibrosis was present in the primiparous cows as they started their first lactation. This study has therefore shown that the ageing process in the livers of dairy cows is accelerated by successive lactations and increasing milk yields. This was associated with evidence of metabolic and immune disorders together with hepatic dysfunction. These problems are likely to increase involuntary culling, thus reducing the average longevity in dairy herds.

Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

Transcriptome Sequence Analysis of Defense Response of Resistant and Susceptible Bottle Gourd to Powdery Mildew

Powdery mildew (PM) is the main disease that afflicts bottle gourd. Previous studies on PM mainly focused on its effects on pumpkin, melon, and other crops; however, the exact molecular mechanism of bottle gourd resistance to PM remains unclear. RNA sequencing (RNA-Seq) technology was used to investigate the dynamic changes in leaf transcriptome profiles between resistant and susceptible gourd at 12, 24, 48, and 72 h post-inoculation with powdery mildew. Compared with a susceptible variety (G3), the expression levels of the differentially expressed genes of phenylpropanoid biosynthesis, starch, and sucrose metabolism, and plant–pathogen interaction pathways in disease-resistant plants were upregulated. We propose that disease resistance and tolerance in bottle gourd are enhanced via several pathways, including the antioxidant system, phenylalanine biosynthesis, and cell wall cellulose synthesis. Our research will provide an important basis for further screening and breeding PM resistance in bottle gourd.

Systematic Large Fragment Deletions in the Genome of Synechococcus elongatus and the Consequent Changes in Transcriptomic Profiles.

Genome streamlining, as a natural process in the evolution of microbes, has become a common approach for generating ideal chassis cells for synthetic biology studies and industrial applications. However, systematic genome reduction remains a bottleneck in the generation of such chassis cells with cyanobacteria, due to very time-consuming genetic manipulations. Synechococcus elongatus PCC 7942, a unicellular cyanobacterium, is a candidate for systematic genome reduction, as its essential and nonessential genes have been experimentally identified. Here, we report that at least 20 of the 23 over 10 kb nonessential gene regions could be deleted and that stepwise deletions of these regions could be achieved. A septuple-deletion mutant (genome reduced by 3.8%) was generated, and the effects of genome reduction on the growth and genome-wide transcription were investigated. In the ancestral triple to sextuple mutants (b, c, d, e1), an increasingly large number of genes (up to 998) were upregulated relative to the wild type, while slightly fewer genes (831) were upregulated in the septuple mutant (f). In a different sextuple mutant (e2) derived from the quintuple mutant d, much fewer genes (232) were upregulated. Under the standard conditions in this study, the mutant e2 showed a higher growth rate than the wild type, e1 and f. Our results indicate that it is feasible to extensively reduce the genomes of cyanobacteria for generation of chassis cells and for experimental evolutionary studies.

Abstract 532: Deficiency of CCN3 in Smooth Muscle Cells Perturbs Vascular Homeostasis and Exacerbates Thoracic Aortic Dissection

Introduction: Thoracic aortic dissection (TAD) is a life-threatening aortic disease with a high mortality rate in the event of rupture. Key mechanisms underlying the development of TAD include extracellular matrix (ECM) degradation and vascular smooth muscle cell (SMC) reprograming. Cellular communication network factor 3 (CCN3), a matricellular protein highly expressed in SMC, exerts a myriad of context-dependent biological functions. However, its role in SMC homeostasis and in the development of TAD remains largely undefined. Methods: A single-cell sequencing (Sc-seq) dataset (GSE213740) from human aortic dissection patients was analyzed to determine the association between CCN3 expression and TAD. In newly weaned male CCN3-floxed mice harboring Myh11-driven inducible Cre, 4 consecutive tamoxifen injections induced CCN3 deletion in SMCs. Mice without Cre expression were subjected to the same procedures and were used as controls. After 1 week of washout, 4-week-old mice were given 0.5% BAPN (β-aminopropionitrile monofumarate) in their drinking water to induce TAD. Histologic and protein measurements were performed on TAD tissues from control and CCN3-SMC-KO mice. Sc-seq analysis was performed to recapitulate cell heterogeneities and transcriptome profile changes. The regulatory role of SMC-derived CCN3 in TGFβ signaling was evaluated by western blot and colocalization analyses. Results: Analysis of the human Sc-seq dataset revealed downregulation of CCN3 in aortas from patients with TAD. CCN3-positive SMCs have elevated ECM and elastin gene expression in comparison to CCN3-null SMCs. In vivo, concordant with increased mortality in CCN3-SMC-KO animals, SMC-specific deletion of CCN3 promotes BAPN-induced elastin fibers fragmentation and degradation. In vitro, SMC deficiency of CCN3 diminished TGFβ-induced elastogenesis. CCN3 binds to the TGFβ receptor on SMCs and augments TGFβ receptor phosphorylation, thereby activating Smads, upregulating SMC elastogenesis, and as a consequence, alleviates elastin fiber fragmentation and TAD development. Conclusion: CCN3 is a key determinate of SMC reprogramming and sustains vascular homeostasis. Our findings suggest a novel target for the prevention and treatment of TAD.

Transcriptome Characterization and Gene Changes Induced by Fusarium solani in Sweetpotato Roots.

Sweetpotato (Ipomoea batatas) is an important root crop that is infected by Fusarium solani in both seedling and root stages, causing irregular black or brown disease spots and root rot and canker. This study aims to use RNA sequencing technology to investigate the dynamic changes in root transcriptome profiles between control check and roots at 6 h, 24 h, 3 days, and 5 days post-inoculation (hpi/dpi) with F. solani. The results showed that the defense reaction of sweetpotato could be divided into an early step (6 and 24 hpi) without symptoms and a late step to respond to F. solani infection (3 and 5 dpi). The differentially expressed genes (DEGs) in response to F. solani infection were enriched in the cellular component, biological process, and molecular function, with more DEGs in the biological process and molecular function than in the cellular component. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the main pathways were metabolic pathways, the biosynthesis of secondary metabolites, and carbon metabolism. More downregulated genes were identified than upregulated genes in the plant-pathogen interaction and transcription factors, which might be related to the degree of host resistance to F. solani. The findings of this study provide an important basis to further characterize the complex mechanisms of sweetpotato resistance against biotic stress and identify new candidate genes for increasing the resistance of sweetpotato.

Rapid changes in transcriptomic profile and mitochondrial function in human soleus muscle after three-day dry immersion.

We aimed to explore the effect of the 3-day dry immersion - a model of physical unloading - on mitochondrial function, transcriptomic and proteomic profiles in a slow-twitch m. soleus of six healthy females. We registered that a marked reduction (25-34%) in the ADP-stimulated respiration in permeabilized muscle fibers was not accompanied by a decrease in the content of mitochondrial enzymes (mass spectrometry-based quantitative proteomics), hence it is related to the disruption in regulation of respiration. We detected a widespread change in the transcriptomic profile (RNA-seq) upon dry immersion. Down-regulated mRNAs were strongly associated with mitochondrial function, as well as with lipid metabolism, glycolysis, insulin signaling, and various transporters. Despite the substantial transcriptomic response, we found no effect on the content of highly abundant proteins (sarcomeric, mitochondrial, chaperon and extracellular matrix-related, etc.) that may be explained by long half-life of these proteins. We suggest that during short-term disuse the content of some regulatory (and usually low abundant) proteins such as cytokines, receptors, transporters, transcription regulators is largely determined by its mRNA concentration. These mRNAs revealed in our work may serve as putative targets for the future studies aimed at developing the approaches for the prevention of muscle deconditioning-induced by disuse.

Changes in life history parameters and transcriptome profile of Serangium japonicum associated with feeding on natural prey (Bemisia tabaci) and alternate host (Corcyra cephalonica eggs)

BackgroundThe mass production of natural predators with prolonged shelf life is a prerequisite for their field application as pest control agents. The traditional methods used for the mass production of Serangium japonicum rely heavily on the consistent supply of natural prey. This study explains the effects of B. tabaci (natural prey) and C. cephalonica eggs (alternative food) on life history and transcriptome profile of S. japanicum.MethodsThis study compares the effects of B. tabaci (natural prey) and C. cephalonica eggs (alternative food) on biology, reproduction, and predatory efficacy, and transcriptome profile of S. japanicum.ResultsThis study revealed that S. japonicum was able to successfully complete its life cycle while feeding on B. tabaci (natural prey) and C. cephalonica eggs (alternative food). The C. cephalonica eggs fed S. japonicum individuals had longer developmental period and lower fecundity as compared to those feeding on whitefly but the survival rates (3rd instar nymphs, 4th instar nymphs and pupae) and predatory efficacy of C. cephalonica eggs fed S. japonicum individuals were significantly similar to to those feeding on whitefly.Transcriptome analysis showed that when faced with dietary changes, S. japanicum could successfully feed on C. cephalonica eggs by regulating genes related to nutrient transport, metabolism, and detoxification. Moreover, S. japanicum degraded excess cellular components through ribosomal autophagy and apoptosis, which provided sufficient materials and energy for survival and basic metabolism.ConclusionCorcyra cephalonica eggs can be used as an alternate host for the predator, Serangium japonicum, as the survival rates and predatory efficacy of the predator are similar to those feeding on the natural host (B.tabaci). When faced with dietary changes, S. japanicum could successfully feed on C. cephalonica eggs as revealed by upregulation of genes related to nutrient transport, metabolism, and detoxification. These findings are of great significance for studying the functional evolution of S. japonicum in response to dietary changes.

Abstract PD4-06: PD4-06 Obesity-associated changes in transcriptomic profile and immune landscape of primary breast cancer revealed by bulk and single-cell gene expression data

Abstract Background: Breast cancer (BC) is one of the cancer types recognized as an obesity-associated disease. Current understandings of molecular mechanisms underlying the BC-obesity connection however largely came from experimental models while systematic investigation of the impact of obesity on BC biology in large patient series is still lacking. The purpose of this study is to discover changes in the transcriptomic profile of primary BC according to patients’ body mass index (BMI). Data and Methods: Bulk and single-cell gene expression data from treatment-naïve primary breast tumors from non-underweight patients were retrieved from the MINDACT trial (NCT00433589; N = 1481) and the pre-treatment cohort of the BioKey trial (NCT03197389, N = 36), respectively. Three categories were considered for BMI: lean, overweight and obese. The main analyses focused on the invasive carcinoma of no special type (NST) estrogen receptor-positive/HER2-negative (ER+/HER2-, N_bulk = 735, N_single-cell(sc) = 10) and NST ER-/HER2- (N_bulk = 118, N_sc = 15) subgroups. The bulk expression data was subjected to differential gene expression analyses according to BMI which was adjusted for menopausal status and tumor grade, then followed by gene set enrichment analyses. Clustering and cluster annotation were performed on the single-cell profiling data before differentially expressed genes according to BMI were identified for each of the present cell types. Results: Obesity-associated differences in the transcriptomic profile of breast tumors, which were subtle but potentially indicative of a biological relationship, were revealed by the bulk data. In both investigated subgroups, tumors from obese patients were shown to be enriched in cell cycle hallmarks. In ER-/HER2- tumors, adiposity further increased MYC signaling. We also observed different obesity-associated changes according to the ER status. Among ER+/HER2- tumors, those from obese patients were enriched in hallmarks related to inflammatory response compared to those from lean patients. In contrast, these hallmarks appeared to be enriched in the ER-/HER2- tumors from lean patients. Our investigation of the single-cell data further revealed shifts in the cell composition of tumor tissue and cell type-specific transcriptomic differences according to BMI which were more pronounced than those detected from the bulk data. ER+/HER2- tumors from obese patients have a higher frequency of immunosuppressive and pro-tumoral cell subpopulations such as dendritic cells (DC) enriched in immunoregulatory molecules (p = .03), LYVE1+ macrophages (p = .02) and myofibroblasts (p = .03) than those from lean patients. Overexpression of Cyclin D1 and CD24 was found in cancer cells in ER+/HER2- tumors from obese patients. A reduction in anti-tumor immune responses was evident with downregulation of multiple interferons in CD8+ and CD4+ T cells as well as B cells. We observed in the ER-/HER2- subgroup increased infiltration of plasmacytoid DC (p = .01), CCL2+ macrophages (p = .01) in tumors from obese versus lean patients, while fibroblasts showed an opposite tendency. Additionally, significant obesity-associated downregulation of major histocompatibility complex (MHC) molecules class I in cancer cells and MHC class II molecules in B cells could be suggestive of deficient antigen presentation and activation of cytotoxic and helper T cells. Conclusion: We highlighted the impact of obesity on the remodeling of tumor and tumor microenvironment which might generally lead to a suppression of anti-tumor immune responses, albeit potentially via diverse axes according to the ER status. Although investigation on a larger cohort is warranted, our current results suggest that obesity-associated transcriptomic changes in BC could be highly cell type-specific, hence we recommend single-cell approaches in addition to spatial multi-omics analysis to further elucidate the interplay between obesity and BC. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Marion Maetens, Karen Van Baelen, Maxim De Schepper, Coralie Poncet, Mauro Delorenzi, Marjanka K. Schmid, Emiel Rutgers, Laura Van ’t Veer, Martine Piccart, Fatima Cardoso, Giuseppe Viale, Ayse Bassez, Hanne Vos, Patrick Neven, Ines Nevelsteen, Kevin Punie, Hans Wildiers, Giuseppe Floris, Diether Lambrechts, Ann Smeets, Elia Biganzoli, François Richard, Christine Desmedt. PD4-06 Obesity-associated changes in transcriptomic profile and immune landscape of primary breast cancer revealed by bulk and single-cell gene expression data [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-06.

Abstract P6-14-12: Evaluation of changes in sequencing quality and transcriptomic profiles with increasing post-mortem interval: results from an optimization experiment within the UPTIDER tissue donation program

Abstract Background. Postmortem tissue donation programs can importantly enhance sample access for translational research on metastatic disease. However, this post-mortem setting poses logistical and technical challenges in terms of preserving nucleic acid quality and in particular RNA. Here we present the results of an experiment within our breast cancer tissue donation program UPTIDER (NCT04531696), aiming at assessing RNA degradation rates and expression profile changes in function of tissue type and sample-specific postmortem interval (ssPMI). Patients &amp; Methods. For 7 patients, bulk RNA sequencing was performed using the Lexogen protocol on fresh frozen samples from healthy or tumour tissues taken repeatedly (at 1.5h time intervals) during the autopsy. ssPMI was defined as the time between the death of the patient and the freezing of the sample. Quality threshold was set at 0.5 million (M) of assigned reads (AR). Other quality metrics included number of expressed genes, evolution of proliferation-, hypoxia-, stromal- and immune-related transcriptional signatures (PMID:18698033, 20087356) with increasing ssPMI. Associations between quality metrics and ssPMI were assessed by linear regressions for longitudinal data, with quality metrics as dependent variable, time as independent variable and accounting for the clustering of the data by patient and organ using the generalized estimating equation method. Three nested models - with constant, linear and non-linear relationship - were compared using ANOVA testing strategy. Non-linearity was rendered by a restrict cubic spline with three knots. All tests were performed by the Wald test on regression coefficients. Results. Ninety samples (67 healthy, 23 tumour) were analyzed. Median ssPMI was 7.50 hours (range: 3.07-11.12). Most (87%) samples passed quality thresholds, with median AR being 1.70M (interquartile range: [0.70M-3.57M]). No association was found between quality metrics and time in healthy samples. In tumor samples, regarding sequencing quality, negative associations with increasing time were found for AR and for number of expressed genes with an average decay of 242308 reads per hour (95 confidence interval (95CI): [94415.62-390200.50], p-value=.001) and 251 genes per hour (95CI: [17.30-485], p-value=.035), respectively. At the transcriptomic level, potential subtle changes were observed regarding immune (e.g. STAT1 signature: -0.02, 95CI: [-0.05;0.00]) and hypoxia-related signatures (e.g. PGAM1 signature: -0.03, 95CI [-0.05;-0.01]), while no effect of time was seen for the proliferation and stromal-related signatures. Sample size precluded organ specific analyses. Conclusion. A decrease in the number of AR and number of expressed genes with increasing ssPMI was found in tumor samples leading to subtle changes in few transcriptional programs. Healthy samples showed stable quality metrics over time possibly explained by a lower cell activity in healthy as compared to tumour cells. Knowledge derived from this study will be integrated in the upcoming transcriptomic analyses of the samples collected within UPTIDER. Citation Format: François Richard, Tatjana Geukens, Giuseppe Marano, Wouter Van Den Bogaert, Maxim De Schepper, Marion Maetens, Amena Mahdami, Karen Van Baelen, Ha-Linh Nguyen, Anirudh Pabba, Sophia Leduc, Edoardo Isnaldi, Maysam Hajipirloo, Imane Bachir, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Evaluation of changes in sequencing quality and transcriptomic profiles with increasing post-mortem interval: results from an optimization experiment within the UPTIDER tissue donation program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-12.

Chronic ethanol exposure induces mitochondrial dysfunction and alters gene expression and metabolism in human cardiac spheroids.

Chronic alcohol consumption in adults can induce various cardiac toxicities such as arrhythmias, cardiomyopathy, and heart failure. Prenatal alcohol exposure can increase the risk of developing congenital heart defectsamongoffspring. Understandingthe molecular mechanisms underlying long-term alcohol exposure-induced cardiotoxicity canhelpguidethedevelopmentof therapeutic strategies. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) were engineered into cardiac spheroids and treated with clinically relevant concentrations of ethanol (17 and 50 mM) for 5 weeks. The cells were then analyzed for changes in mitochondrial features, transcriptomic and metabolomic profiles, and integrated omics outcomes. Following chronic ethanol treatment of hiPSC-CMs, a decrease in mitochondrial membrane potential and respiration and changes in expression of mitochondrial function-related genes were observed. RNA-sequencing analysis revealed changes in various metabolic processes, heart development, response to hypoxia, and extracellular matrix-related activities. Metabolomic analysis revealed dysregulation of energy metabolism and increased metabolites associated with theupregulation of inflammation. Integrated omics analysis further identified functional subclusters and revealed potentially affected pathways associated with cardiac toxicities. Chronic ethanol treatment of hiPSC-CMs resulted in overall decreased mitochondrial function, increased glycolysis, disrupted fatty acid oxidation, and impaired cardiac structural development.

Metabolomic and transcriptomic response to imatinib treatment of gastrointestinal stromal tumour in xenograft-bearing mice

BackgroundAlthough imatinib is a well-established first-line drug for treating a vast majority of gastrointestinal stromal tumours (GIST), GISTs acquire secondary resistance during therapy. Multi-omics approaches provide an integrated perspective to empower the development of personalised therapies through a better understanding of functional biology underlying the disease and molecular-driven selection of the best-targeted individualised therapy. In this study, we applied integrative metabolomic and transcriptomic analyses to elucidate tumour biochemical processes affected by imatinib treatment. Materials and methodsA GIST xenograft mouse model was used in the study, including 10 mice treated with imatinib and 10 non-treated controls. Metabolites in tumour extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). RNA sequencing was also performed on the samples subset (n=6). ResultsMetabolomic analysis revealed 21 differentiating metabolites, whereas next-generation RNA sequencing data analysis resulted in 531 differentially expressed genes. Imatinib significantly changed the profile of metabolites associated mainly with purine and pyrimidine metabolism, butanoate metabolism, as well as alanine, aspartate, and glutamate metabolism. The related changes in transcriptomic profiles included genes involved in kinase activity and immune responses, as well as supported its impact on the purine biosynthesis pathway. ConclusionsOur multi-omics study confirmed previously known pathways involved in imatinib anticancer activity as well as correlated imatinib-relevant downregulation of expression of purine biosynthesis pathway genes with the reduction of respectful metabolites. Furthermore, considering the importance of the purine biosynthesis pathway for cancer proliferation, we identified a potentially novel mechanism for the anti-tumour activity of imatinib. Based on the results, we hypothesise metabolic modulations aiming at the reduction in purine and pyrimidine pool may ensure higher imatinib efficacy or re-sensitise imatinib-resistant tumours.

Fear Conditioning Leads to Enduring Alterations in RNA Transcripts in Hippocampal Neuropil that are Dependent on EphB2 Forward Signaling.

Alterations in mRNA transcription have been associated with changes in brain functions. We wanted to examine if fear conditioning causes long-term changes in transcriptome profiles in the basolateral amygdala (BLA) and hippocampus using RNA-Seq and laser microdissection microscopy. We further aimed to uncover whether these changes are involved in memory formation by monitoring their levels in EphB2lacZ/lacZ mice, which lack EphB2 forward signaling and can form short-term fear conditioning memory but not long-term fear conditioning memory. We found transcriptome signatures unique to each brain region that arecomprise ofspecific cellular pathways. We also revealed that fear conditioning leads to alterations in mRNAs levels 24h after training in hippocampal neuropil, but not in hippocampal cell layers or BLA. The two main groups of altered mRNAs encode proteins involved in neuronal transmission, neuronal morphogenesis and neuronal development and the vast majority are known to be enriched in neurons. None of these mRNAs levels were altered by fear conditioning in EphB2lacZ/lacZ mice, which were also impaired in long-term fear memory. We show here that fear conditioning leads to an enduring alteration in mRNAs levels in hippocampal neuropil that is dependent on processes mediated by EphB2 that are needed for long-term memory formation.

IPSC-Derived MSCs Are a Distinct Entity of MSCs with Higher Therapeutic Potential than Their Donor-Matched Parental MSCs

Mesenchymal stromal cells derived from induced pluripotent stem cells (iMSCs) have been proposed as alternative sources of primary MSCs with various advantages for cell therapeutic trials. However, precise evaluation of the differences between iMSCs and primary MSCs is lacking due to individual variations in the donor cells, which obscure direct comparisons between the two. In this study, we generated donor-matched iMSCs from individual bone marrow-derived MSCs and directly compared their cell-autonomous and paracrine therapeutic effects. We found that the transition from primary MSCs to iMSCs is accompanied by a functional shift towards higher proliferative activity, with variations in differentiation potential in a donor cell-dependent manner. The transition from MSCs to iMSCs was associated with common changes in transcriptomic and proteomic profiles beyond the variations of their individual donors, revealing expression patterns unique for the iMSCs. These iMSC-specific patterns were characterized by a shift in cell fate towards a pericyte-like state and enhanced secretion of paracrine cytokine/growth factors. Accordingly, iMSCs exhibited higher support for the self-renewing expansion of primitive hematopoietic progenitors and more potent immune suppression of allogenic immune responses than MSCs. Our study suggests that iMSCs represent a separate entity of MSCs with unique therapeutic potential distinct from their parental MSCs, but points to the need for iMSC characterization in the individual basis.

Gene transcripts responsive to drought stress identified in Citrus macrophylla bark tissue transcriptome have a modified response in plants infected by Citrus tristeza virus

Citrus macrophylla Wester (CM) has valuable agronomic characteristics such as the ability to grow in saline soils, although with low tolerance to prolonged drought stress (DS) . To understand the mechanisms that characterize CM response to water scarcity, this study compared transcriptome profile changes in CM stem tissue when exposed to DS and identified a total of 2745 differentially expressed transcripts (DETs, fold change > 2), of which 631 were up-regulated and 2114 were down-regulated. DETs up-regulated by DS were enriched in pathways such as the redox and osmotic system or soluble carbohydrates and in transcripts for low molecular weight proteins such as late embryogenesis abundant protein (LEA). Down-regulated transcripts were mainly assigned to photosynthesis, transport, phenylpropanoids, calcium dependent kinases, brassinosteroids and other hormones including salicylic acid and abscisic acid. To assess the interplay between DS and Citrus tristeza virus (CTV) infection, twelve genes were profiled by quantitative Real-Time PCR (qPCR) analysis in control and CTV-infected CM plants, with or without DS. The twelve analyzed transcripts were significantly correlated ( r = 0.82, p < 0.001) with the RNA-Seq results and gave insight into the responses of CM to drought and/or to infection with CTV . Transcriptome results unveiled highly responsive genes to DS in stem tissue, which may be candidates for genetic selection of high drought tolerant plants of CM.