Comprehensive Gene Expression Profiling Research Articles

RNA sequencing reveals molecular mechanisms of endometriosis lesion development in mice.

Understanding of molecular mechanisms contributing to the pathophysiology of endometriosis, and upstream drivers of lesion formation, remains limited. Using a C57Bl/6 mouse model in which decidualized endometrial tissue is injected subcutaneously in the abdomen of recipient mice, we generated a comprehensive profile of gene expression in decidualized endometrial tissue (n=4), and in endometriosis-like lesions at Day 7 (n=4) and Day 14 (n=4) of formation. High-throughput mRNA sequencing allowed identification of genes and pathways involved in the initiation and progression of endometriosis-like lesions. We observed distinct patterns of gene expression with substantial differences between the lesions and the decidualized endometrium that remained stable across the two lesion timepoints, and showed similarity to transcriptional changes implicated in human endometriosis lesion formation. Pathway enrichment analysis revealed several immune and inflammatory response-associated canonical pathways, multiple potential upstream regulators, and involvement of genes not previously implicated in endometriosis pathogenesis, including IRF2BP2 and ZBTB10, suggesting novel roles in disease progression. Collectively, the provided data will be a useful resource to inform research on the molecular mechanisms contributing to endometriosis-like lesion development in this mouse model.

Physiological and Transcriptomic Characterization of Rice Genotypes under Drought Stress

Drought is a primary abiotic stress that inhibits rice (Oryza sativa L.) growth and development, and during the reproductive stage it has a negative impact on the rice seed-setting rate. This research study examined two rice lines, La-96 (drought sensitive) and La-163 (drought resistant), for drought stress treatment (with soil moisture at 20% for 7 days) and control (normal irrigation and kept soil moisture ≥40%). To elucidate the photosynthesis and molecular mechanisms underlying drought tolerance in rice, leaf photosynthetic traits and transcriptome sequencing were used to compare differences between two contrasting recombinant inbred lines (RIL) during drought and subsequent recovery at the booting stage. The rice line La-96 showed a significant decrease in seed-setting rate after being treated for seven days’ drought stress (from 86.64% to 22.75%), while La-163 was slightly affected (from 89.04% to 79.33%). The photosynthetic activities of both lines significantly decreased under the drought treatment, and these traits of La-163 recovered to a comparable level with the control after three days of rewatering. The transcriptome of both lines in three treatments (the control, drought stress, and subsequent recovery) were tested, and a total of 16,051 genes were identified, among which 10,566 genes were differentially expressed in various treatments and rice lines. Comprehensive gene expression profiles revealed that the specifically identified DEGs were involved in the ribosome synthesis and the metabolic pathway of photosynthesis, starch, and sucrose metabolism. The DEGs that are activated and respond quickly, as seen during recovery in the tolerant rice line, may play essential roles in regulating subsequent growth and development. This study uncovered the molecular genetic pathways of drought tolerance and extended our understanding of the drought tolerance mechanisms and subsequent recovery regulation in rice.

Comprehensive genomic analysis and expression profiling of the cytochrome P450 genes during abiotic stress and flavonoid biosynthesis in potato (Solanum tuberosum)

The research focused on the cytochrome P450 (CYP) superfamily, a prominent enzyme family involved in plant metabolism. A total of 253 StCYP genes from the potato genome were investigated and characterized through various methods, including phylogentic analysis, physical and chemical characterization, chromosome localization, gene structure and conserved protein domain classification, gene duplication occurrences, collinearity and expression pattern analysis. The 253 StCYPs were classified into seven clans and 33 families, distributed unevenly across 12 chromosomes. Collinearity analysis revealed 28 pairs of orthologous genes between potato StCYPs and Arabidopsis AtCYPs. Moreover, the expression of the StCYPs in various tissue parts of doubled haploid (DM) potatoes under abiotic stress and exogenous hormone treatment was investigated by using RNA-seq data from the PGSC database. It was discovered that one StCYP gene likely contributes to drought stress response through the ABA-dependent pathway. Furthermore, RNA-seq analysis of pigmented tuber flesh from three potato clones at three developmental stages enabled the identification of StCYPs potentially implicated in the flavonoid biosynthesis in potato tubers. In particular, StCYP705A-like was further validated for its potential role in this process. Employing Gfanno software in conjunction with RNA-seq data, seven StCYP genes (Two StC4H, three StF3′H, one StFNS Ⅱ and one StF3′5′H) were identified as flavonoid biosynthetic pathway genes in potato. Additionally, five StCYPs in CYP 71 clan demonstrated a strong association with flavonoid biosynthesis. These findings lay a groundwork for a more comprehensive understanding of the StCYP gene family and further exploration of the functions of StCYP genes in potato abiotic stress tolerance and flavonoids biosynthesis.

COMPREHENSIVE MOLECULAR PROFILING OF UVEAL MELANOMA EVALUATED WITH GENE EXPRESSION PROFILING, PREFERENTIALLY EXPRESSED ANTIGEN IN MELANOMA EXPRESSION, AND NEXT-GENERATION SEQUENCING.

To determine the association between gene-expression profiling (GEP), next-generation sequencing (NGS), preferentially expressed antigen in melanoma (PRAME) features, and metastatic risk in patients with uveal melanoma (UM). A retrospective analysis of patients with UM treated by brachytherapy or enucleation by a single ocular oncologist was conducted from November 2020 and July 2022. Clinicopathologic features, patient outcomes, GEP classification, NGS, and PRAME results were recorded. Comprehensive GEP, PRAME, and NGS testing was performed on 135 UMs. The presence of eukaryotic translation initiation factor 1A, X-chromosomal and splicing factor 3B subunit 1 mutations was significantly associated with GEP class 1A and GEP class 1B, respectively. The presence of BRCA- associated protein-1 mutation was significantly associated with GEP class 2. The average largest basal diameter for tumors with eukaryotic translation initiation factor 1A, X-chromosomal mutations was significantly smaller than those with splicing factor 3B subunit 1 mutations and BRCA1-associated protein-1 mutations. Class 2 tumors metastasized sooner than GEP class 1 tumors. Tumors with splicing factor 3B subunit 1 and/or BRCA1-associated protein-1 mutations metastasized sooner compared with tumors that had either no driver mutation or no mutations at all. Tumors with splicing factor 3B subunit 1 did not have a significantly different time to metastasis compared with tumors with BRCA1-associated protein-1 (P value = 0.97). Forty tumors (30%) were PRAME positive, and the remaining 95 tumors (70%) were PRAME negative. Tumors with PRAME-positive status did not have a significantly different time to metastasis compared with tumors without PRAME-positive status (P value = 0.11). GEP, NGS, and PRAME expression analysis help determine different levels of metastatic risk in UM. Although other prognostic tests exist, the following study reports on the use of NGS for metastatic prognostication in UM. However, limitations of NGS exist, especially with small lesions that are technically difficult to biopsy.

Heterogeneity of hepatocellular carcinoma that responds differently to combination therapy with TACE and Sorafenib as determined by digital spatial gene expression profiling.

The combination of Sorafenib and transcatheter arterial chemoembolization (TACE) exhibits limited efficacy in the treatment of certain advanced hepatocellular carcinomas (HCC), and the molecular mechanisms underlying resistance to this combination remain unclear. This study aims to underscore the distinctive contribution of GeoMx DSP technology in elucidating the molecular intricacies of HCC resistance to the Sorafenib and TACE combination. Patients with advanced HCC during the waiting period before liver transplantation were classified into sensitive and resistant groups based on their response to Sorafenib and TACE combination therapy. Employing GeoMx DSP technology for comprehensive gene expression profiling, we identified pivotal molecular targets linked to resistance against combination therapy. The investigation scrutinized intra-tumoral and inter-individual variances, unveiling a spectrum of crucial molecular targets, such as PLG, PLVAP, immunoglobulin genes, ORM1, and NR4A1, among others. Additionally, we explored signaling pathways associated with treatment responsiveness, including the PPAR signaling pathway. Notably, we emphasized the significance of the immune microenvironment characterized by heightened SPP1 expression in HCC resistance to combination therapy. In the resistant group, SPP1+ tumor-associated macrophage (TAM) infiltration was notably pronounced (p = 0.037), while T-cell depletion showed a mitigated presence (p = 0.013). The study reveals intra- and inter-individual heterogeneity in HCC that is differentially responsive to the combination of Sorafenib and TACE, highlighting multiple key molecular targets associated with treatment resistance. The immune microenvironment is important, and in particular, SPP1+ TAM infiltration may play a key role. Meanwhile, the introduction of immunotherapy in patients resistant to combination therapy may lead to positive results.

A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.

Assessment of long-term stored specimens in the Siriraj Hospital colorectal cancer biobank for RNA sequencing and profiling

Abstract Objectives Biobanks play an important role in advancing cancer research, yet concerns persist regarding the molecular integrity of long-term stored samples. This study assessed fresh frozen (FF) tissues and formalin-fixed paraffin-embedded (FFPE) tissues from the Siriraj Hospital colorectal cancer (CRC) biobank collected during two distinct periods (2011–2012 and 2020–2021). Methods In 2022, FF and FFPE primary cancer tissues from 75 CRC patients were evaluated. RNA sequencing (RNA-Seq) analyzed comprehensive gene expression profiles in FF tissues preserved at −80 °C, while nCounter profiling elucidated cancer-specific RNA transcripts in FFPE tissues stored at ambient temperature. Comparative analyses were conducted between specimens from 2011 to 2012 and 2020–2021. Results The FF tissues stored for approximately 10.5 years were well-suited for RNA-Seq compared to the intact tissues preserved for 1.5 years. Despite consistencies in RNA quantity, RNA integrity, amount of sequencing reads, and CRC gene signature, gene enrichment analysis revealed the decreased ribosome biogenesis, spliceosome and antifolate resistance pathways in the 2011–2012 group. Moreover, the FFPE tissues also showed no alteration in RNA quantity between the two periods, and the nCounter profiling demonstrated comparable CRC-specific gene counts in spite of the significant reduction of raw counts in the 2011–2012 group. Conclusions We report that FF tissues from CRC patients, stored for 10 years, are viable for whole transcriptome RNA-Seq, despite altered pathways such as ribosome biogenesis, spliceosome, and antifolate resistance. Moreover, 10-year-stored FFPE CRC tissues remain suitable for specific RNA profiling using the nCounter pan-cancer panel, despite a significant reduction in raw counts. These findings underscore the enduring contribution of biobanks to molecular research, highlighting their value a decade post-collection.

Cryptic Resilience: Decoding Molecular Networks in Pearl Millet for Enhanced Heat Stress

Pearl millet (<em>Pennisetum glaucum</em>), a vital cereal crop renowned for its drought tolerance, is a cornerstone for smallholder farmers in arid and semi-arid regions, ranking as the fifth most significant cereal globally. Despite its resilience, the molecular mechanisms underlying its tolerance to heat stress remained elusive. To address this knowledge gap, we subjected ten-day-old pearl millet seedlings to an unprecedented temperature of 50°C for 60 seconds. Subsequent next-generation RNA sequencing aimed to unravel differential gene expression in heat-stressed seedlings compared to control conditions. Our analysis revealed a remarkable 29.8% differential expression in the genome sequence in response to heat stress. Heat-stressed pearl millet leaves exhibited differential expression in 11,483 genes, with fold changes ranging from 2 to 18.6 compared to the control group. Of these, 3,612 genes displayed upregulation, while 7,871 genes exhibited downregulation. These genes play roles in diverse biological processes involving crucial enzymes such as aminoacyl-tRNA synthetases, ligases, methyltransferases, oxidoreductases, and DNA-directed RNA polymerases. The Photosystem II Type I Chlorophyll-a/b-binding protein and heat shock proteins displayed the most significant fold changes in heat-stressed leaves. Moreover, various transcription factor families, including bHLH, ERF, NAC, WRKY, MYB-related, C2H2, bZIP, MYB, FAR1, and B3, vital in controlling pearl millet's response to heat stress, were linked to over 100 differentially expressed genes. The dataset generated through this research, shedding light on the molecular processes enabling pearl millet to withstand heat, holds immense value given the crop's role in food security and resilience to extreme weather. In the context of climate change and global warming, this knowledge lays the foundation for further studies on metabolic engineering and selecting crops resilient to high temperatures. Our transcriptomics approach provides comprehensive gene expression profiles of heat-stressed plants. It elucidates pearl millet's response to heat stress, offering a crucial resource for future investigations into crop adaptation strategies.

Donor regulatory T cells rapidly adapt to recipient tissues to control murine acute graft-versus-host disease

The adoptive transfer of regulatory T cells is a promising strategy to prevent graft-versus-host disease after allogeneic bone marrow transplantation. Here, we use a major histocompatibility complex-mismatched mouse model to follow the fate of in vitro expanded donor regulatory T cells upon migration to target organs. Employing comprehensive gene expression and repertoire profiling, we show that they retain their suppressive function and plasticity after transfer. Upon entering non-lymphoid tissues, donor regulatory T cells acquire organ-specific gene expression profiles resembling tissue-resident cells and activate hallmark suppressive and cytotoxic pathways, most evidently in the colon, when co-transplanted with graft-versus-host disease-inducing conventional T cells. Dominant T cell receptor clonotypes overlap between organs and across recipients and their relative abundance correlates with protection efficacy. Thus, this study reveals donor regulatory T cell selection and adaptation mechanisms in target organs and highlights protective features of Treg to guide the development of improved graft-versus-host disease prevention strategies.

Decreased histone expression in chronic rhinosinusitis with nasal polyps.

Histones have been associated with human diseases. However, the implication of extranuclear histone proteins and their potential mechanism in the pathophysiology of chronic rhinosinusitis (CRS) have not been thoroughly investigated. This study was designed to evaluate the role of histones in patients with CRS by comparing histone expression between patients and controls. Nasal polyp (NP) tissues were obtained, and their comprehensive gene expression profiles were investigated by microarray analysis. Differences in expression were verified by reverse transcriptase polymerase chain reaction and immunohistochemical staining. Cell culture and flow cytometry were used to evaluate the role of histones in the pathogenesis of polyps. Significant differences in the microarray analysis were observed between the patient and control groups (P < 0.01). It was found by flow cytometry that the histone (H2BK) can promote cell apoptosis in NPs. Our results indicate that reduced expression of H2BK may contribute to the imbalance process of cell proliferation and apoptosis in CRS with NP.

Hippocampal Lactate-Infusion Enhances Spatial Memory Correlated with Monocarboxylate Transporter 2 and Lactylation.

Lactate has emerged as a key player in regulating neural functions and cognitive processes. Beyond its function as an energy substrate and signal molecule, recent research has revealed lactate to serve as an epigenetic regulator in the brain. However, the molecular mechanisms by which lactate regulates spatial memory and its role in the prevention of cognitive disorders remain unclear. Herein, we injected L-lactate (10 μmol/kg/d for 6 d) into the mouse's hippocampus, followed by the Morris water maze (MWM) test and molecular analyses. Improved spatial memory performances were observed in mice injected with lactate. Besides, lactate upregulated the expression of synaptic proteins post-synaptic density 95 (PSD95), synaptophysin (SYP), and growth associated protein 43 (GAP43) in hippocampal tissues and HT22 cells, suggesting a potential role in synaptic transmission and memory formation. The facilitative role of monocarboxylate transporter 2 (MCT2), a neuron-specific lactate transporter, in this process was confirmed, as MCT2 antagonists attenuated the lactate-induced upregulation of synaptic proteins. Moreover, lactate induced protein lactylation, a post-translational modification, which could be suppressed by MCT2 inhibition. RNA sequencing of lactated-injected hippocampal tissues revealed a comprehensive gene expression profile influenced by lactate, with significant changes in genes associated with transcriptional progress. These data demonstrate that hippocampal lactate injection enhances spatial memory in mice, potentially through the upregulation of synaptic proteins and induction of protein lactylation, with MCT2 playing a crucial role in these processes. Our findings shed light on the multi-faceted role of lactate in neural function and memory regulation, opening new avenues for therapeutic interventions targeting cognitive disorders.

Abstract 7501: Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system

Abstract Background: Targeting rare cell types such as circulating tumor cells (CTCs), considered to be metastatic precursors in cancer progression, may provide long-term clinical benefits by enhancing patient outcomes. The study of CTCs comes with technical challenges due to their heterogeneity and low cell numbers, leading to difficulties in genomic characterization. This study aims to overcome these technical challenges by developing a comprehensive gene expression profiling workflow using qPCR and RNA sequencing (RNA-Seq) on rare cells enriched with Genesis System with Celselect SlidesTM. Methods: To mimic CTCs in blood, we created a rare cell model sample by spiking A549 non-small cell lung cancer cells at concentrations of 50, 100, 500 and 1000 cells/ml in whole blood from healthy donors. The Genesis System, which can capture rare cells of 8µm-30µm, isolated 83.73% of spiked A549 cells in whole blood. Using the same blood samples without A549 spiked in, we enriched cells using the Genesis and it was used as a baseline for gene expression controls. High quality RNA was extracted from each of the enriched samples and used for both qPCR and RNA-Seq to detect the gene expression signature of the non-small cell lung cancer cells. Results: Our analyses revealed significant differential expression of cancer-related genes (p &amp;lt; 0.05) in all A549-spiked samples when compared to the blood-only controls. Notably, in the 50 cells/ml dilution, RNA-Seq analysis identified over 3,000 differentially expressed genes, encompassing protein-coding genes, long non-coding RNAs, and small non-coding RNAs. Among these, 759 genes were consistently present across all enriched samples, suggesting a common expression pattern. To investigate the potential utility of these genes as non-small cell lung cancer biomarkers, we constructed a custom qPCR array comprising 38 of these genes and pre-amplified their targets using 50pg of RNA from the enriched cell samples. Notably, six genes from this panel (MET, TOP2A, NF1, SPP1, GAPDH, and ANXA5) exhibited significant differential expression (+/- 4-fold, p&amp;lt;0.05) in the enriched samples. MET and TOP2A, in particular, are well-known biomarkers for non-small cell lung cancer. Conclusion: Our liquid biopsy workflow demonstrates the recovery of high-quality RNA from enriched rare cells, including CTCs. It enables comprehensive profiling of the tumor transcriptome, encompassing RNAs of various sizes, including miRNAs. Our approach has the potential to facilitate the identification of patient-specific biomarkers, enabling swift monitoring using preamplification and qPCR in advancement of targeted oncology therapy and research. Citation Format: Srikanth Perike, Angelica Olcott, Cissy Jiang, Nish Kumar, Nathan Knapp, Jennifer Placek, Candice Cox, Yoon-Tae Kang, Linda Lingelbach, Elizabeth Dreskin. Gene expression profiling of rare cells captured from liquid biopsy using the genesis cell isolation system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7501.

The RNA sequencing results revealed the expression of different genes and signaling pathways during chemotherapy resistance in peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) is a subtype of non-Hodgkin's lymphoma that occurs primarily at extranodal sites and is commonly treated using chemotherapy and radiotherapy. PTCL is more malignant than other lymphoid tumors, resulting in a poor prognosis.The 5-year recurrence rate remains high, and there is a lack of standard treatment for patients with relapse-resistant disease. However, the molecular mechanisms underlying the resistance of peripheral T-cell lymphoma cells to chemotherapeutic drugs, as well as identifying strategies to overcome drug resistance remains unclear. In this study, we aimed to identify pivotal genes and signaling pathways associated with chemotherapy resistance in PTCL.MethodsIn this study, a total of 5 healthy controls and 7 clinical patients were enrolled; 4 patients were classified as chemotherapy sensitive, and 3 patients were classified as chemotherapy resistant. Peripheral blood samples were collected from each participant, and total RNA was extracted from the white blood cells. RNA sequencing was conducted on the Illumina HiSeq platform to obtain comprehensive gene expression profiles. Subsequently, the expression patterns of the DEGs associated with the most enriched signaling pathways, with a special focus on cancer-related genes, were validated using quantitative real-time polymerase chain reaction (qRT–PCR) in peripheral TCL patients.ResultsRNA sequencing (RNA-seq) analysis revealed 4063 differentially expressed genes (DEGs) in peripheral T-cell lymphoma specimens from patients with chemotherapy resistance, of which 1128 were upregulated and 2935 were downregulated. Subsequent quantitative gene expression analysis confirmed a differential expression pattern in all the libraries, with 9 downregulated genes and 10 upregulated genes validated through quantitative real-time PCR in 6 clinical specimens from patients with chemotherapy resistance. KEGG pathway analysis revealed significant alterations in several pathways, with 6 downregulated pathways and 9 upregulated pathways enriched in the DEGs. Notably, the TNF signaling pathway, which is extensively regulated, was among the pathways that exhibited significant changes. These findings suggest that DEGs and the TNF signaling pathway may play crucial roles in chemotherapy resistance in peripheral T-cell lymphoma.ConclusionOur study revealed that the expression of specific genes, including TNFRSF1B, TRADD2, and MAP3K7, may play an important role in chemotherapy resistance in peripheral T-cell lymphoma. Moreover, we identified the downregulation of the TNF signaling pathway, a crucial pathway involved in cell survival, death, and differentiation, as a potential contributor to the development of chemotherapy resistance in peripheral T-cell lymphoma. These findings provide valuable insights into the molecular mechanisms underlying chemotherapy resistance and highlight potential targets for overcoming treatment resistance in this challenging disease.

C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

Recent studies have indicated the involvement of neutrophil-mediated inflammatory responses in the process leading to intracranial aneurysm (IA) rupture. Receptors mediating neutrophil recruitment could thus be therapeutic targets of unruptured IAs. In this study, complement C5a receptor 1 (C5AR1) was picked up as a candidate that may cause neutrophil-dependent inflammation in IA lesions from comprehensive gene expression profile data acquired from rat and human samples. The induction of C5AR1 in IA lesions was confirmed by immunohistochemistry; the up-regulations of C5AR1/C5ar1 stemmed from infiltrated neutrophils, which physiologically express C5AR1/C5ar1, and adventitial fibroblasts that induce C5AR1/C5ar1 in human/rat IA lesions. In in vitro experiments using NIH/3T3, a mouse fibroblast-like cell line, induction of C5ar1 was demonstrated by starvation or pharmacological inhibition of mTOR signaling by Torin1. Immunohistochemistry and an experiment in a cell-free system using recombinant C5 protein and recombinant Plasmin indicated that the ligand of C5AR1, C5a, could be produced through the enzymatic digestion by Plasmin in IA lesions. In conclusion, we have identified a potential contribution of the C5a–C5AR1 axis to neutrophil infiltration as well as inflammatory responses in inflammatory cells and fibroblasts of IA lesions. This cascade may become a therapeutic target to prevent the rupture of IAs.

Genome Identification and Expression Profiling of the PIN-Formed Gene Family in Phoebe bournei under Abiotic Stresses.

PIN-formed (PIN) proteins-specific transcription factors that are widely distributed in plants-play a pivotal role in regulating polar auxin transport, thus influencing plant growth, development, and abiotic stress responses. Although the identification and functional validation of PIN genes have been extensively explored in various plant species, their understanding in woody plants-particularly the endangered species Phoebe bournei (Hemsl.) Yang-remains limited. P. bournei is an economically significant tree species that is endemic to southern China. For this study, we employed bioinformatics approaches to screen and identify 13 members of the PIN gene family in P. bournei. Through a phylogenetic analysis, we classified these genes into five sub-families: A, B, C, D, and E. Furthermore, we conducted a comprehensive analysis of the physicochemical properties, three-dimensional structures, conserved motifs, and gene structures of the PbPIN proteins. Our results demonstrate that all PbPIN genes consist of exons and introns, albeit with variations in their number and length, highlighting the conservation and evolutionary changes in PbPIN genes. The results of our collinearity analysis indicate that the expansion of the PbPIN gene family primarily occurred through segmental duplication. Additionally, by predicting cis-acting elements in their promoters, we inferred the potential involvement of PbPIN genes in plant hormone and abiotic stress responses. To investigate their expression patterns, we conducted a comprehensive expression profiling of PbPIN genes in different tissues. Notably, we observed differential expression levels of PbPINs across the various tissues. Moreover, we examined the expression profiles of five representative PbPIN genes under abiotic stress conditions, including heat, cold, salt, and drought stress. These experiments preliminarily verified their responsiveness and functional roles in mediating responses to abiotic stress. In summary, this study systematically analyzes the expression patterns of PIN genes and their response to abiotic stresses in P. bournei using whole-genome data. Our findings provide novel insights and valuable information for stress tolerance regulation in P. bournei. Moreover, the study offers significant contributions towards unraveling the functional characteristics of the PIN gene family.

Advances in genomics and genome editing for improving strawberry (Fragaria ×ananassa).

The cultivated strawberry, Fragaria ×ananassa, is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations and precision nucleotide substitutions in the target genome, revolutionizing functional genomics and crop improvement. Genome editing is beginning to gain traction in the more challenging polyploid crops, including allo-octoploid strawberry. The release of high-quality reference genomes and comprehensive subgenome-specific genotyping and gene expression profiling data in octoploid strawberry will lead to a surge in trait discovery and modification by using CRISPR/Cas. Genome editing has already been successfully applied for modification of several strawberry genes, including anthocyanin content, fruit firmness and tolerance to post-harvest disease. However, reports on many other important breeding characteristics associated with fruit quality and production are still lacking, indicating a need for streamlined genome editing approaches and tools in Fragaria ×ananassa. In this review, we present an overview of the latest advancements in knowledge and breeding efforts involving CRISPR/Cas genome editing for the enhancement of strawberry varieties. Furthermore, we explore potential applications of this technology for improving other Rosaceous plant species.

SGCAST: symmetric graph convolutional auto-encoder for scalable and accurate study of spatial transcriptomics.

Recent advances in spatial transcriptomics (ST) have enabled comprehensive profiling of gene expression with spatial information in the context of the tissue microenvironment. However, with the improvements in the resolution and scale of ST data, deciphering spatial domains precisely while ensuring efficiency and scalability is still challenging. Here, we develop SGCAST, an efficient auto-encoder framework to identify spatial domains. SGCAST adopts a symmetric graph convolutional auto-encoder to learn aggregated latent embeddings via integrating the gene expression similarity and the proximity of the spatial spots. This framework in SGCAST enables a mini-batch training strategy, which makes SGCAST memory-efficient and scalable to high-resolution spatial transcriptomic data with a large number of spots. SGCAST improves the overall accuracy of spatial domain identification on benchmarking data. We also validated the performance of SGCAST on ST datasets at various scales across multiple platforms. Our study illustrates the superior capacity of SGCAST on analyzing spatial transcriptomic data.

KnockTF 2.0: a comprehensive gene expression profile database with knockdown/knockout of transcription (co-)factors in multiple species.

Transcription factors (TFs), transcription co-factors (TcoFs) and their target genes perform essential functions in diseases and biological processes. KnockTF 2.0 (http://www.licpathway.net/KnockTF/index.html) aims to provide comprehensive gene expression profile datasets before/after T(co)F knockdown/knockout across multiple tissue/cell types of different species. Compared with KnockTF 1.0, KnockTF 2.0 has the following improvements: (i) Newly added T(co)F knockdown/knockout datasets in mice, Arabidopsis thalianaand Zea mays and also an expanded scale of datasets in humans. Currently, KnockTF 2.0 stores 1468 manually curated RNA-seq and microarray datasets associated with 612 TFs and 172 TcoFs disrupted by different knockdown/knockout techniques, which are 2.5 times larger than those of KnockTF 1.0. (ii) Newly added (epi)genetic annotations for T(co)F target genes in humans and mice, such as super-enhancers, common SNPs, methylation sitesand chromatin interactions. (iii) Newly embedded and updated search and analysis tools, including T(co)F Enrichment (GSEA), Pathway Downstream Analysisand Search by Target Gene (BLAST). KnockTF 2.0 is a comprehensive update of KnockTF 1.0, which provides more T(co)F knockdown/knockout datasets and (epi)genetic annotations across multiple species than KnockTF 1.0. KnockTF 2.0 facilitates not only the identification of functional T(co)Fs and target genes but also the investigation of their roles in the physiological and pathological processes.

Comprehensive Gene Expression Profiling Analysis of Adipose Tissue in Male Individuals from Fat- and Thin-Tailed Sheep Breeds.

It has been shown that tail fat content varies significantly among sheep breeds and plays a significant role in meat quality. Recently, significant efforts have been made to understand the physiological, biochemical, and genomic regulation of fat deposition in sheep tails in order to unravel the mechanisms underlying energy storage and adipose tissue lipid metabolism. RNA-seq has enabled us to provide a high-resolution snapshot of differential gene expression between fat- and thin-tailed sheep breeds. Therefore, three RNA-seq datasets were meta-analyzed for the current work to elucidate the transcriptome profile differences between them. Specifically, we identified hub genes, performed gene ontology (GO) analysis, carried out enrichment analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and validated hub genes using machine learning algorithms. This approach revealed a total of 136 meta-genes, 39 of which were not significant in any of the individual studies, indicating the higher statistical power of the meta-analysis. Furthermore, the results derived from the use of machine learning revealed POSTN, K35, SETD4, USP29, ANKRD37, RTN2, PRG4, and LRRC4C as substantial genes that were assigned a higher weight (0.7) than other meta-genes. Among the decision tree models, the Random Forest ones surpassed the others in adipose tissue predictive power fat deposition in fat- and thin-tailed breeds (accuracy > 0.85%). In this regard, combining meta-analyses and machine learning approaches allowed for the identification of three important genes (POSTN, K35, SETD4) related to lipid metabolism, and our findings could help animal breeding strategies optimize fat-tailed breeds' tail sizes.

Abstract 18810: Mitochondria-Rich Extracellular Vesicles From Stem Cell-Derived Cardiomyocytes Restore Bioenergetic Metabolism of Ischemic Myocardium in a Porcine Chronic Heart Failure Model

Introduction: The disruption of mitochondrial energy metabolism underlies the pathophysiology of ischemic heart failure. We previously demonstrated that intramyocardial injection of mitochondria-rich extracellular vesicles (M-EVs) derived from iPSC-derived cardiomyocytes (iCMs) transfers mitochondria and non-mitochondrial biologics into recipient cardiomyocytes. This process stimulates mitochondrial biogenesis and enhances functional capacities in murine and porcine acute myocardial infarction (MI) models. However, the therapeutic potential and underlying mechanism in chronic heart failure models remain to be assessed. Method: M-EVs were isolated from the conditioned medium of human iCMs using differential centrifugation. A total of 11 Yorkshire pigs underwent myocardial ischemia for one hour via balloon occlusion of the left anterior descending artery (Week-0). At week-4, 1.0 x 10 11 M-EVs or PBS control were administered transendocardially to the peri-infarct region (PIR) using a percutaneous catheter delivery system (Biocardia, Inc, CA). Cardiac functions and scar size were analyzed by cardiac MRI with gadolinium contrast at week-4 (pre-injection) and -8 (post-injection). Myocardial tissue samples were collected from the PIR and remote non-ischemic areas at week-8. Proteomic analysis and single-nucleus RNA sequencing were performed to investigate comprehensive protein and gene expression profiles of the myocardium. Results: Left ventricular (LV) ejection fractions were significantly decreased at Week-4 (32.7±5.4%). M-EV treatment significantly improved absolute LVEF from week-4 to -8 in the treatment vs. non-treatment groups, +3.15% vs. -4.99%, respectively, (p&lt;0.0001). No significant difference was observed in the scar size. Multiomic analysis of proteomes and single-nucleus transcriptomes suggested restoration of dysregulated cardiometabolic protein profile and mitochondrial energy metabolism in the myocardium. Conclusion: M-EV-mediated transfer of mitochondria and enriched proteins augmented LV dysfunction in the pig chronic MI model. M-EV therapy may represent a novel biologic approach that directly targets cardiac metabolism by restoring mitochondrial function and cellular bioenergetics.