Variation In Expression Research Articles

Single-cell sequencing reveals that AK5 inhibits apoptosis in AD oligodendrocytes by regulating the AMPK signaling pathway

BackgroundNeuroinflammation and abnormal energy metabolism have been shown to significantly contribute to the progression of Alzheimer’s disease (AD). Adenylate kinase 5 (AK5), an enzyme predominantly expressed in the brain regulates ATP metabolism, has an unclear role in energy metabolism and neuroinflammation in AD.MethodsThe AD datasets were derived from the GEO public database to analyze the expression levels of AK5 in AD and normal samples and to assess the relationship between AK5 expression and the clinical characteristics of AD patients. Functional enrichment analysis was employed to investigate the effects of changes in AK5 expression on energy metabolism and immunoinflammation in AD, as well as the underlying mechanisms. Moreover, the influence of AK5 expression variations on oligodendrocyte development was assessed, and the predicted outcomes were validated through cellular experiments.ResultsBioinformatic analysis revealed that AK5 was lowly expressed in AD olfactory lobe tissues, accompanied by increased immunoinflammation and apoptosis. Increased expression of AK5 was associated with the activation of AMPK signaling, enhanced oxidative phosphorylation, and overall stimulation of energy metabolism. In oligodendrocytes treated with Aβ1-42, overexpression of AK5 resulted in elevated levels of P-AMPK, SIRT1, and BCL-2 proteins, while reducing the levels of BAX, CASPASE-3, and NF-κB proteins. This modulation activated AMPK signaling, thereby inhibiting neuroinflammation and apoptosis. In contrast, low levels of AK5 expression during early differentiation triggered inflammatory responses and increased apoptosis in oligodendrocytes.ConclusionAK5 inhibits AD oligodendrocyte apoptosis by activating the AMPK signaling pathway.

Transcriptomic profiles of single-cell autophagy-related genes (ATGs) in lung diseases

Autophagy related genes (ATGs) play essential roles in maintaining cellular functions, although biological and pathological alterations of ATG phenotypes remain poorly understood. To address this knowledge gap, we utilized the single-cell sequencing technology to elucidate the transcriptomic atlas of ATGs in lung diseases, with a focus on lung epithelium and lymphocytes. This study conducted a comprehensive investigation into RNA profiles of ATGs in the lung tissues obtained from healthy subjects and patients with different lung diseases through single-cell RNA sequencing (scRNA-seq), including COVID-19 related acute lung damage, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), systemic sclerosis (SSC), and lung adenocarcinoma (LUAD). Our findings revealed significant variations of ATGs expression across lung epithelial cell subsets, e.g., over-expression of MAPK8 in basal cells, ATG10 in club cells, and BCL2 in a goblet cell subset. The changes of autophagy-related pathways varied between lung epithelial and lymphocyte subsets. We identified the disease-associated changes in ATG expression, including significant alterations in BCL2, BCL2L1, PRKCD, and PRKCQ in inflammatory lung diseases (COPD and IPF), and MAP2K7, MAPK3, and RHEB in lung cancer (LUAD), as compared to normal lung tissues. Key ligand-receptor pairs (e.g., CD6-ALCAM, CD99-CD99) and signaling pathways (e.g., APP, CD74) might serve as biomarkers for lung diseases. To evaluate ATGs responses to external challenges, we examined ATGs expression in different epithelial cell lines exposed to cigarette smoking extract (CSE), lysophosphatidylcholine (lysoPC), lipopolysaccharide (LPS), and cholesterol at various doses and durations. Notable changes were observed in CFLAR, EIF2S1, PPP2CA, and PPP2CB in A549 and H1299 against CSE and LPS. The heterogeneity of ATGs expression was dependent on cell subsets, pathologic conditions, and challenges, as well as varied among cellular phenotypes, functions, and behaviors, and the severity of lung diseases. In conclusion, our data might provide new insights into the roles of ATGs in epithelial biology and pulmonary disease pathogenesis, with implications for disease progression and prognosis.Graphical 1. The transcriptomic profiles of autophagy-related genes (ATGs) varied among pulmonary epithelial and lymphocyte subsets in lung diseases.2. ATGs play essential roles in the maintenance of pulmonary cell functions and intercellular interactions between lung epithelia and lymphocytes by reprogramming the metabolisms and the energy balance.3. ATGs can be the candidates of biology-specific biomarkers and therapeutic targets for pulmonary diseases.

Advancements in single-cell RNA sequencing and spatial transcriptomics: transforming biomedical research

In recent years, significant advancements in biochemistry, materials science, engineering, and computer-aided testing have driven the development of high-throughput tools for profiling genetic information. Single-cell RNA sequencing (scRNA-seq) technologies have established themselves as key tools for dissecting genetic sequences at the level of single cells. These technologies reveal cellular diversity and allow for the exploration of cell states and transformations with exceptional resolution. Unlike bulk sequencing, which provides population-averaged data, scRNA-seq can detect cell subtypes or gene expression variations that would otherwise be overlooked. However, a key limitation of scRNA-seq is its inability to preserve spatial information about the RNA transcriptome, as the process requires tissue dissociation and cell isolation. Spatial transcriptomics is a pivotal advancement in medical biotechnology, facilitating the identification of molecules such as RNA in their original spatial context within tissue sections at the single-cell level. This capability offers a substantial advantage over traditional single-cell sequencing techniques. Spatial transcriptomics offers valuable insights into a wide range of biomedical fields, including neurology, embryology, cancer research, immunology, and histology. This review highlights single-cell sequencing approaches, recent technological developments, associated challenges, various techniques for expression data analysis, and their applications in disciplines such as cancer research, microbiology, neuroscience, reproductive biology, and immunology. It highlights the critical role of single-cell sequencing tools in characterizing the dynamic nature of individual cells.

Comparative transcriptomics reveal stage-dependent parasitic adaptations in the myxozoan Sphaerospora molnari

BackgroundParasitism as a life strategy has independently evolved multiple times within the eukaryotic tree of life. Each lineage has developed mechanisms to invade hosts, exploit resources, and ensure replication, but our knowledge of survival mechanisms in many parasitic taxa remain extremely limited. One such group is the Myxozoa, which are obligate, dixenous cnidarians. Evidence suggests that myxozoans evolved from free-living ancestors to endoparasites around 600 million years ago and are likely one of the first metazoan parasites on Earth. Some myxozoans pose significant threats to farmed and wild fish populations, negatively impacting aquaculture and fish stocks; one such example is Sphaerospora molnari, which forms spores in the gills of common carp (Cyprinus carpio), disrupting gill epithelia and causing somatic and respiratory failure. Sphaerospora molnari undergoes sequential development in different organs of its host, with large numbers of morphologically distinct stages occurring in the blood, liver, and gills of carp. We hypothesize that these parasite life-stages differ in regards to their host exploitation, pathogenicity, and host immune evasion strategies and mechanisms. We performed stage-specific transcriptomic profiling to identify differentially expressed key functional gene groups that relate to these functions and provide a fundamental understanding of the mechanisms S. molnari uses to optimize its parasitic lifestyle. We aimed to identify genes that are likely related to parasite pathogenicity and host cell exploitation mechanisms, and we hypothesize that genes unique to S. molnari might be indicative of evolutionary innovations and specific adaptations to host environments.ResultsWe used parasite isolation protocols and comparative transcriptomics to study early proliferative and spore-forming stages of S. molnari, unveiling variation in gene expression between each stage. We discovered several apparent innovations in the S. molnari transcriptome, including proteins that are likely to function in the uptake of previously unknown key nutrients, immune evasion factors that may contribute to long-term survival in hosts, and proteins that likely improve adhesion to host cells that may have arisen from horizontal gene transfer. Notably, we identified genes that are similar to known virulence factors in other parasitic organisms, particularly blood and intestinal parasites like Plasmodium, Trypanosoma, and Giardia. Many of these genes are absent in published cnidarian and myxozoan datasets and appear to be specific to S. molnari; they may therefore represent potential innovations enabling Sphaerospora to exploit the host’s blood system.ConclusionsIn order to address the threat posed by myxozoans to both cultured fish species and wild stocks, it is imperative to deepen our understanding of their genetics. Sphaerospora molnari offers an appealing model for stage-specific transcriptomic profiling and for identifying differentially expressed key functional gene groups related to parasite development. We identified genes that are thus far unique to S. molnari, which reveal their evolutionary novelty and likely role as adaptations to specific host niches. In addition, we describe the pathogenicity-associated genetic toolbox of S. molnari and discuss the implications of our discoveries for disease control by shedding light on specific targets for potential intervention strategies.

‘What happened to the un-omitted subjects?’

Abstract This research examines variations of subject expression in spoken Korean. While much focus has been put on the omission of subjects in Korean as a pro-drop language, what is less explored is under what conditions choices are made into different forms when subjects are expressed, which in Korean includes not only pronouns but also kinship terms, professional titles, personal names and other lexical forms. In this paper, I test the distribution of subject expression in spoken Korean corpora for first and second person subjects and analyse the discursive aspects of the referential choices for the expressed subjects. The results confirm that first and second person subjects are expressed with varied referential forms and that relative age between discourse participants becomes a significant factor in the referential choice. This study also remarks on the importance of socio-cultural and pragmatic interpretation in the discussion of subject expression in Korean.

A Head-Specific Transcriptomic Study Reveals Key Regulatory Pathways for Winter Diapause in the Mosquito Culex pipiens.

The primary vector of the West Nile virus, Culex pipiens, undergoes reproductive dormancy during the adverse winter season. While our current understanding has mainly focused on cellular signals and phenotypic shifts occurring at a global scale during diapause, information on tissue-specific transcriptomic changes remains limited. This knowledge gap is a major challenge in interpreting the regulatory mechanisms at the tissue level. To address this, the present work utilized RNA-seq technology to investigate the transcriptional changes in the head that house the brain and crucial endocrinal organs such as corpora allata. We obtained RNA samples from the heads of diapausing and nondiapausing female mosquitoes at two specific time intervals, ZT0 and ZT16, and then subjected them to sequencing. Our results revealed differences in differentially expressed genes between diapause and non-diapause at ZT0 and ZT16, highlighting the phenotypic and diel variations in gene expression. We also selected twelve genes associated with the diapause phenotype and examined the transcript abundance at six different time points over 24 h. qRT-PCR analysis showed similar up- and downregulation of transcripts between the diapause and nondiapause phenotypes thus validating the results of RNA-seq. In summary, our study identified new genes with phenotypic and diel differentiation in their expression, potentially linking photoperiod to seasonal reproductive dormancy in insects. The newly presented information will significantly advance our understanding of head-specific genes crucial for insect diapause.

Localization of Lesions in Autoimmune Blistering Diseases Is Independent of Site-Specific Target Antigen Expression

Autoimmune blistering diseases (AIBDs) involve autoantibodies targeting proteins in the epidermal/epithelial desmosome (pemphigus) or basement membrane zone (pemphigoid). Despite widespread antigen distribution, lesions exhibit a scattered involvement pattern. This study maps the frequency/severity of AIBD lesions on various body parts and investigates whether differential antigen expression contributes to specific predilection sites. We analyzed affected sites presenting blisters/erosions, erythematous/urticarial lesions, and mucosal lesions in bullous pemphigoid (BP-cohort 1, n = 65; BP-cohort 2, n = 119), pemphigus vulgaris (PV, n = 67), and pemphigus foliaceus (PF, n = 20) patients. To assess antigen expression, we conducted indirect immunofluorescence (IF) staining of 11 AIBD antigens from 13 anatomical sites of 10 body donors without AIBD. In BP, blisters/erosions and erythematous/urticarial lesions predominantly affected arms and legs, while PV/PF patients exhibited frequent involvement of buccal mucosa and back, respectively. IF staining identified significant regional differences in BP180, BP230, and integrin β4 expression, although these variations did not correlate with a higher lesion frequency/severity. Other antigens showed consistent expression across all regions. Our findings suggest that predilection sites for BP and PV/PF are largely unaffected by regional variations in antigen expression but may be influenced by factors like microbiota, mechanical stress, sunlight exposure, local immunity, or genetics.

Expression profile analysis of estrous-phased ovarian tissue of high and low prolific lines of inbred Swiss albino mice.

Litter size in mice is an important fitness and economic feature that is controlled by several genes and influenced by non-genetic factors too. High positive selection pressure in each generation for Litter size at birth (LSB), resulted in the development of high and low prolific lines of inbred Swiss albino mice (SAM). Despite uniform management conditions, these lines showed variability in LSB across the generation. Variation in estrous-phased ovarian gene expression between high (LSB ≥ 12) and low prolific lines (LSB ≤ 3) of F4 inbred SAM, was explored using RNA-Seq.Estrous phase assessment was done using vaginal cytology. A total of 870 differentially expressed genes (DEGs) were identified; among which, 287 genes were significantly up-regulated while 583 genes were down-regulated in HLS as compared to the LLS group. DEGs were assigned to 166 Gene Ontology (GO) terms and KEGG pathways. In HLS, the significantly up-regulated DEGs were involved in ovarian cell-cell signaling, regulation of biological activity and ovarian metabolic-associated pathways. Most down-regulated DEGs were expressed in immune-related pathways, indicating that immunological dampening is associated with a high ovulation rate and higher level of progesterone concentration leading to physiological changes responsible for higher fecundity. The present study, based on bulk RNA-seq analysis reflects the aggregate gene expression of the whole ovarian tissue, and reveals 24 DEGs that could be used as candidates for litter size attributes in future polymorphism and functional studies to gain further insights into the mechanisms underlying litter size variations in animals.

Intraspecific gene regulation in cis- and trans.

Changes in gene expression underlie much of evolution and occur via either cis-acting mutations, which lie near the affected gene and act in a context specific manner, or trans-acting mutations, which may be far from the affected gene and act through diffusible molecules such as transcription factors. A commonly held view is that most expression variation within species is controlled in trans- while expression differences between species are largely controlled in cis-. Here, we summarize recent intraspecific gene regulation studies and find, contrary to this widely held view, that many studies in diverse taxa have revealed a large role for cis-acting mutations underlying expression variation within species. A review of the existing literature also shows that preparations using whole organisms rather than individual tissues may be biased towards identifying trans-regulation. Moreover, we note several examples of predominantly cis-acting regulation in recently diverged populations adapted to different environments. We highlight the challenges of drawing general conclusions from comparisons among studies that use different methodologies and we offer suggestions for studies that will address outstanding questions concerning the evolution of gene regulation.

Epigenetic Mechanisms Driving Human Evolutionary Changes

A crucial aspect of our comprehension of human evolution is epigenetics, the study of heritable modifications in gene function that do not include modifications in the DNA sequence. This intriguing field of study emphasises on how reversible and environmental-sensitive mechanisms, such as histone, DNA methylation, and non-coding RNA activity, control gene expression and support phenotypic variation. These techniques allow animals to adapt to changing environments without experiencing permanent genetic changes, providing a flexible basis for evolutionary processes. Understanding human evolution now heavily relies on epigenetics. Since animals can adapt to changing environments without enduring permanent genetic changes, evolutionary processes have a flexible basis. Epigenetic modifications have closely linked characteristics such as immune system function and brain development throughout human evolution. Epigenetic patterns have been impacted by environmental factors like diet, illness, and climate, which has allowed organisms to flourish and procreate in a variety of ecological niches. With an emphasis on their function in human evolution, this study explores the molecular mechanisms underlying epigenetic control and how epigenetics allows humans to display amazing phenotypic plasticity. The ramifications of epigenetic studies in anthropology are also examined, with a focus on how modern humans differ from their hominid ancestors and how epigenetics influences cultural and social practices. This review further highlights the revolutionary potential of epigenetics in deciphering the intricacies of human evolution by fusing knowledge from genetics, anthropology, and environmental studies.

Abstract P006: Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model

Abstract Purpose/Objective: Radiopharmaceutical therapy (RPT) enables the systemic delivery of an inherently heterogeneous dose of radiation. The RPT dose heterogeneity is created by a tumor’s non-uniform perfusion and the variation in RPT target expression. Radiation therapy is known to immunomodulate the tumor microenvironment (TME) in a dose-dependent manner. The hypothesis is that RPT with a heterogeneous distribution, delivering a low to high dose gradient in a single TME, will activate multiple dose dependent immune effects that are more effective in promoting priming of adaptive immunity compared to a more homogenous distribution of RPT. Methods: RPT was administered using PNT6555, a fibroblast activation protein alpha targeting agent, and uptake was confirmed with B16 murine melanoma. The B16 cell line is engineered to overexpress mouse fibroblast activation protein alpha (mFAP). The engineered B16 was implanted either alone to form a tumor with 100% mFAP expression (B16-mFAP+) or in a mixture with the parental cell line to create a heterogeneous expression of mFAP (mixed B16-mFAP+/B16-mFAP-). Utilizing the SPECT/CT data and ex vivo biodistribution, image-based dosimetry for 177Lu-PNT6555 was performed using the Monte-Carlo-based RAPID platform. C57BL/6 mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- tumors were treated with non-radiolabeled PNT6555, 5, 10, 20 or 40 Gy on day 0. Tumors were harvested on day 7 post-treatment. RT-qPCR was performed to evaluate the type I interferon response and tumor immune susceptibility markers. Mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- received a mean tumor dose of 5 Gy 177Lu-PNT6555. Mice were monitored for tumor growth and overall survival. In a separate cohort, tumors and tumor draining lymph nodes (TDLN) were harvested at days 4, 7, and 12 post-injection. Flow cytometry was performed on the tumor to evaluate immune infiltration and TDLN to evaluate dendritic cell and B cell activation. Statistical significance was determined by a one-way ANOVA (flow) or Kaplan-Meier with log-rank testing (therapy). Results: Optimal upregulation of immune-related genes by 177Lu-PNT6555 occurred at 2.5 Gy for Icam, 10 Gy for Ifnβ1, and 20 Gy for H2-K1 (murine gene coding MHCI). A heterogeneous delivery of 5 Gy to mice bearing mixed B16-mFAP+/mFAP- resulted in upregulation of all three genes in a single TME. For the therapy study, a heterogeneous distribution of 5 Gy 177Lu-PNT6555 significantly increased overall survival compared to a homogenous distribution. Flow cytometry revealed a significant increase in dendritic cell activation by CD80 and MHCII expression in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Additionally, there was a significant increase in infiltration of CD4+ cells, CD8+ cells, NK cells and B cells as well as preservation of CD8+ cell in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Conclusions: These preclinical studies demonstrate the capacity of a heterogeneous dose of RPT to enhance the immunomodulatory effect of RPT and improve overall survival in a monotherapy setting. Citation Format: Maya E. Takashima, Ohyun Kwon, Shin Hye Ahn, Anthony Belanger, Won Jong Jin, Charlotte Sawicki, Paul A. Clark, Tessa Chen, Ria Kumari, Bryan Bednarz, Zachary S. Morris. Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P006

An involvement of a new zinc finger protein PbrZFP719 into pear self-incompatibility reaction.

This study indicated that the CCHC-type zinc finger protein PbrZFP719 involves into self-incompatibility by affecting the levels of reactive oxygen species and cellulose content at the tips of pollen tubes. S-RNase-based self-incompatibility (SI) facilitates cross-pollination and prevents self-pollination, which in turn increases the costs associated with artificial pollination in fruit crops. Self S-RNase exerts its inhibitory effects on pollen tube growth by altering cell structures and components, including reactive oxygen species (ROS) level and cellulose content. Presently, only a limited number of genes have been implicated in the gametophytic SI. In this study, the CCHC-type zinc finger proteins (ZFP), PbrZFP719, was found to be more highly expressed in pollen grains and pollen tubes than other ZFPs. Experimental over-expression of PbrZFP719 via pollen magnetofection and its knockdown using antisense oligonucleotides demonstrated that PbrZFP719 positively mediates pollen tube growth in pear. Further analyses revealed that variations in PbrZFP719 expression correlate with the changes in ROS levels and cellulose content at the tips of pollen tubes. Notably, PbrZFP719 expression was reduced in pollen tubes treated with self S-RNase. These results suggest that self S-RNase can inhibit pollen tube growth by decreasing ROS levels and cellulose content through the downregulation of PbrZFP719 expression. The information provide insights into a novel mechanism by which self S-RNase inhibits pollen tube growth during SI reaction and offers a refined approach for gene over-expression in pollen tube.

Genes and proteins expression profile of 2D vs 3D cancer models: a comparative analysis for better tumor insights.

When juxtaposed with 2D cell culture models, multicellular tumor spheroids demonstrate a capacity to faithfully replicate certain features inherent to solid tumors. These include spatial architecture, physiological responses, the release of soluble mediators, patterns of gene expression, and mechanisms of drug resistance. The morphological and behavioural similarities between 3D-cultured cells and cells within tumor masses highlight the potential of these models in studying cancer biology and drug responses. The liquid overlay method, hanging drop technique, and ultra-low adhesion plates are among the various methods for generating tumor spheroids, each with its advantages and applications. Gene expression studies, employing advanced methods such as microarrays, suppression subtractive hybridization, qRT-PCR, and mass-spectrometry-based proteomics revealed distinct expression patterns in 3D spheroids compared to 2D cultures, uncovering upregulation and downregulation of genes associated with tumor development, metastasis, and drug resistance. Protein expression studies identified alterations in key signaling pathways, metabolic characteristics, and phosphorylation levels, highlighting the impact of 3D culture on cellular responses. This study explores genes and proteins expression variations in various cancer cell lines cultivated in 3D spheroids, shedding light on the complexity of interactions in a more tumor-mimicking environment. The fusion of these analytical approaches not only advances scientific understanding but also holds promise for the development of more effective cancer treatments.

MicroRNA-197-3p Transfection: Variations in Cardiomyocyte Gene Expression with Anaesthetics Drugs in a Model of Hypoxia/Reperfusion

MicroRNA-197-3p Transfection: Variations in Cardiomyocyte Gene Expression with Anaesthetics Drugs in a Model of Hypoxia/Reperfusion

Mapping the topography of spatial gene expression with interpretable deep learning.

Spatially resolved transcriptomics technologies provide high-throughput measurements of gene expression in a tissue slice, but the sparsity of these data complicates analysis of spatial gene expression patterns. We address this issue by deriving a topographic map of a tissue slice-analogous to a map of elevation in a landscape-using a quantity called the isodepth. Contours of constant isodepths enclose domains with distinct cell type composition, while gradients of the isodepth indicate spatial directions of maximum change in expression. We develop GASTON (gradient analysis of spatial transcriptomics organization with neural networks), an unsupervised and interpretable deep learning algorithm that simultaneously learns the isodepth, spatial gradients and piecewise linear expression functions that model both continuous gradients and discontinuous variation in gene expression. We show that GASTON accurately identifies spatial domains and marker genes across several tissues, gradients of neuronal differentiation and firing in the brain, and gradients of metabolism and immune activity in the tumor microenvironment.

Computational analysis of musical elements across twenty-two European countries

Abstract This work examines the similarities and differences between twenty-two European countries by using the computational model Information Dynamics of Music (IDyOM) to analyze various musical elements in folk songs, children’s folk songs, and children’s songs. The examination of the (dis)similarities between 22 European countries tests two hypotheses. First, it examines whether there are significant differences in the use of musical elements between European countries that are considered to have a common musical style. Secondly, it explores whether the musical elements used in the representative music of a particular country are more similar in countries with similar cultural, political, historical and economic backgrounds and geographical proximity. The results of the research, which compared the three genres across 22 European countries, revealed significant differences that highlight the unique ways in which these genres manifest themselves and how musical elements are integrated into the musical structure, suggesting that European countries do not possess a single musical style. Furthermore, some geographically distant countries have exhibited similarities, while other geographically close countries showed dissimilarities. This implies that either there is no shared musical foundation across different countries, or that the unique variations in musical expression within certain countries have had a significant influence on the overall population.

P0004 Transcriptomic profiling as a tool for prediction of endoscopic inflammation in quiescent Crohn's Disease patients

Abstract Background Biological agents have revolutionized therapy in Crohn's disease (CD), yet ~50% of patients do not respond / lose response to therapy. Therefore, patient-specific therapies are in critical need. Preliminary studies have demonstrated that transcriptomic analysis of fecal aspirates from the colon are associated with patients' inflammatory status1,2. CORE (Capsule & Omics for pRedicting Exacerbation of Crohn’s disease) is a highly defined prospective cohort of clinically non-active CD patients undergoing clinical and endoscopic serial evaluations at Sheba Medical Center (MC). Scarce data exists as to the association of small intestinal biopsies and fecal aspirates from the colon with small bowel inflammation status in quiescent CD. Methods As part of an ongoing prospective cohort study, a biobank of lower-GI biopsies and aspirates (e.g. fluids suctioned from the colon) from CD patients was established at Sheba MC between 2023-2024, along with comprehensive clinical, endoscopic and demographic data. Following RNA extraction and bulk RNA deep sequencing, transcriptomic analysis was performed for all biopsies and fecal aspirated and correlated with clinical and endoscopic data. Results 40 fecal aspirate samples and 50 biopsies were obtained and analyzed from 56 CD patients (median age: 32 years, 73% males). 63% had ileal disease, 5% had colonic disease and 32% had ileo-colonic disease. 48% were treated with Adalidumab, 7% with Infliximab, 16% with Vedolizumab, 12% with Ustekinumab and 17% were not treated with biologics. All patients were in clinical remission at time of analysis. 57% had endoscopically active disease per colonoscopy and 46% per capsule endoscopy. Figure 1 demonstrates the distinct spatial distribution of samples, demonstrating that gene expression variation is significantly associated with small bowel inflammation. 639 genes were significantly differentially expressed between fecal washes of patients with inflamed and non-inflamed small bowel (padj < 0.01). Hierarchical clustering dendrograms show distinct expression patterns that effectively separate inflamed from non-inflamed samples, underlying again a strong transcriptional signature associated with inflammation (Figure 2). The Silhouette score of 0.332, on a scale ranging from -1 to 1, demonstrates moderate clustering separation, validating our analytical approach. Conclusion Colonic fecal-aspirates yield a reliable transcriptomic profile, predicting small bowel inflammation, based on a panel of 639 genes, in quiescent CD. Further studies will enable us to build a PCR based model for prediction of small bowel inflammation in quiescent Crohn's disease.

OP03 Mapping the observable IBDverse: Using massive-scale single-cell RNA sequencing of gut and blood samples to nominate genes, cell types and pathways driving IBD susceptibility

Abstract Background Genome-wide association studies (GWAS) have identified 370 loci associated with inflammatory bowel disease (IBD) susceptibility1. The majority of these are driven by non-coding variants, thought to dysregulate the expression of a nearby gene. The dysregulated disease effector gene, and the operative cell-type(s) is often unknown. Previous efforts to nominate effector genes have relied on identifying expression quantitative trait loci (eQTL) and expression-linked genes (eGenes). However, gene expression is often derived from healthy individuals, a single, non-primary site, and measured using bulk RNA sequencing. This likely misses cell-, site-, or disease-specific genetic effects, and has nominated disease effector genes for only a minority of IBD loci. Methods To better characterise the universe of IBD effector genes, we created IBDverse - the largest collection of single-cell RNAseq data from the terminal ileum (TI, nCD=119, nHealthy=243), rectum (nHealthy=275) and blood (nCD=95) - a total of >2.4 million high quality transcriptomes from 732 samples across 396 individuals. After grouping cells into different cell types, we mapped genetic variants associated with gene expression changes. Disease effector genes were nominated by identifying expression-linked genetic effects likely driven by the same causal variant as an IBD GWAS signal, as determined by colocalisation analysis2. Results Across all tissues, we identify 102 transcriptionally distinct cell types (Fig 1), and genetic effects associated with the expression of >17,000 genes. For 507 genes, the genetic association for their expression was strongly associated with genetic effects for IBD susceptibility, suggesting them to be drivers of disease risk. Remarkably, this doubles the previous best efforts to nominate disease effector genes3, doing so at >52% of IBD susceptibility loci. As well as their nomination, this study also helps contextualise disease effector expression variation. Disease effector genes were detectable in only a single tissue or cell annotation in >40% of cases, highlighting the need for high resolution data obtained across multiple tissues. We also find the first evidence for upregulation of RASGRP1, a gene previously correlated with exacerbated colitis in mice4, to drive Crohn’s disease susceptibility. As this is identified in a subset of tissue-resident memory T cells, and only from the TI, this also suggests this cell type and tissue as governing this effect. Conclusion This study serves as a step change in the understanding of cellular heterogeneity of these tissues, provides a compendium of genetic regulatory effects across constituent cell types, and quantifies the relevance of these to IBD susceptibility. References Liu et al., (2023). Nat. Genetics. ‘Genetic architecture of the inflammatory bowel diseases across East Asian and European ancestries’ Giambartolemi et al., (2014) PLOS Genetics. ‘Bayesian test for colocalisation between pairs of genetic association studies using summary statistics’ Panousis et al., (2024) Nat. Comms (in press). Wang et al., (2022) Nat. Comms. ‘RasGRP1 promotes the acute inflammatory response and restricts inflammation-associated cancer cell growth’

JrMYB44 is required for the accumulation of polyphenols and contributes to drought tolerance in Juglans regia

Juglans regia, an important economic tree species, is planted all over the world, and drought is one of the crucial factors limiting its growth and development. The various polyphenol content in walnut plants constitutes one of the material bases for the differences in stress resistance among various germplasms. However, the molecular mechanism underlying stress response mediated by polyphenol -dependent pathways remains unclear. v-Myb avian myeloblastosis viral oncogene homolog (MYB) protein of transcription factors play important regulatory roles in the process of plant stress responses. Previously, we identified JrMYB44 could be involved in osmotic stress response in walnut. In this study, we confirmed that the drought resistance of four walnut cultivars (‘Chandler’, ‘Xiangling’, ‘Xilin2’ and ‘Xifu1’) is positively correlated with the accumulation of polyphenols. The content and component changes of polyphenols in JrMYB44 overexpression (OE) and suppression (SE) lines in both walnut and Arabidopsis thaliana demonstrated that JrMYB44 positively regulated polyphenols accumulation. The variation of JrMYB44 expression and polyphenol levels under drought treatment indicated significant correlation between JrMYB44-induced drought tolerance and polyphenol accumulation, which was involved in reactive oxidative species (ROS) balance. The differentially expressed genes (DEGs) between OE and WT implied that JrMYB44 could positively activate downstream genes to participate in the drought stress response. Yeast one-hybrid (Y1H), transient GUS expression assay and dual-luciferase reporter assay (DLR) confirmed that JrMYB44 could recognize downstream JrWRKY7 and JrDREB2A, two transcription factors previously reported to be involved in drought response. Meanwhile, it was confirmed by Y2H, GST-pull down and luciferase complementation imaging assay (LCI) that JrMYB44 could interact with JrMYC2 and JrDof1, another two previously reported potential drought response regulators. Collectively, these results indicated that JrMYB44 could activate JrWRKY7, JrDREB2A and interact with JrMYC2 and JrDof1 to promote walnut polyphenol accumulation and improve drought resistance in a ROS dependent manner.

Comparison of Vegetable Waste Byproducts of Selected Cultivars of Foeniculum vulgare Mill. by an Integrated LC-(HR)MS and 1H-NMR-Based Metabolomics Approach.

The metabolome of plants is influenced by various factors, especially environmental, as the season in which they are grown. So, distinct varieties of the identical plant might show an increase or decrease in metabolites. The diversity of content of primary and secondary metabolites can also determine the variation in their biological properties. Due to the current occurrence of various fennel varieties, the crop can now be grown for the entire year. This work used an integrated approach of LC/MS and NMR analysis to characterize the metabolome of fennel waste of different varieties by multivariate statistical analysis. The extracts were investigated by NMR and LC/MS analysis to focus attention on the primary and secondary metabolites. Both LC-HRMS and NMR data were analyzed by principal component analysis (PCA). The 1H-NMR analysis led to the identification of 15 primary metabolites, such as amino acids, carbohydrates, and organic acid derivatives. The secondary metabolites identified by LC/MS analysis mainly belong to the phenolic, lipid, and fatty acid compounds classes. This integrated approach guarantees a precise and complete overview of the variations in the metabolic expression of the fennel varieties grown in different seasons.