Analysis Of Expression Profiles Research Articles

IRX-related homeobox gene MKX is a novel oncogene in acute myeloid leukemia.

Homeobox genes encode transcription factors which organize differentiation processes in all tissue types including the hematopoietic compartment. Recently, we have reported physiological expression of TALE-class homeobox gene IRX1 in early myelopoiesis restricted to the megakaryocyte-erythroid-progenitor stage and in early B-cell development to the pro-B-cell stage. In contrast, sister homeobox genes IRX2, IRX3 and IRX5 are aberrantly activated in the corresponding malignancies acute myeloid leukemia (AML) and B-cell progenitor acute lymphoid leukemia. Here, we examined the role of IRX-related homeobox gene MKX (also termed IRXL1 or mohawk) in normal and malignant hematopoiesis. Screening of public datasets revealed silent MKX in normal myelopoiesis and B-cell differentiation, and aberrant expression in subsets of AML and multiple myeloma (MM) cell lines and patients. To investigate its dysregulation and oncogenic function we used AML cell line OCI-AML3 as model which strongly expressed MKX at both RNA and protein levels. We found that IRX5, JUNB and NFkB activated MKX in this cell line, while downregulated GATA2 and STAT5 inhibited its expression. MKX downstream analysis was conducted by siRNA-mediated knockdown and RNA-sequencing in OCI-AML3, and by comparative expression profiling analysis of a public dataset from MM patients. Analysis of these data revealed activation of CCL2 which in turn promoted proliferation. Furthermore, MKX upregulated SESN3 and downregulated BCL2L11, which may together underlie decreased etoposide-induced apoptosis. Finally, myeloid differentiation genes CEBPD and GATA2 were respectively up- and downregulated by MKX. Taken together, our study identified MKX as novel aberrantly expressed homeobox gene in AML and MM, highlighting the function of IRX1 in normal myelopoiesis and B-cell development, and of IRX-related genes in corresponding malignancies. Our data merit further investigation of MKX and its deregulated target genes to serve as novel markers and/or potential therapeutic targets in AML patient subsets.

LncRNA Expression Profiles in C6 Ceramide Treatment Reveal lnc_025370 as a Promoter in Canine Mammary Carcinoma CHMp Cells Progression

Canine mammary carcinomas (CMCs) represent the most prevalent form of cancer in female dogs, characterized by a high incidence and mortality rate. C6 ceramide is recognized for its multifaceted anti-cancer properties, yet its specific influence on CMCs remains to be elucidated. Long noncoding RNAs (lncRNAs), now recognized as functional “dark matter” in precision oncology, are particularly intriguing, with 44% of canine lncRNAs exhibiting tissue-specific expression. In this study, we performed a thorough analysis of lncRNA expression profiles to uncover the mechanisms behind C6 ceramide’s anti-cancer activity in CHMp cells. Our findings reveal that C6 ceramide notably inhibits the proliferation of CHMp cells. RNA sequencing identified 4522 lncRNAs with expression changes following C6 ceramide treatment, of which 2936 were upregulated and 1586 were downregulated. Further investigation into Lnc_025370 showed that it is predominantly nuclear-localized and is significantly downregulated by C6 ceramide treatment. Functional studies discovered that overexpression of Lnc_025370 enhances the growth and metastatic capabilities of CHMp cells, which is associated with an increase in NRG1, and concurrently diminishes the anti-cancer effectiveness of C6 ceramide in vitro. Mouse xenograft models also showed that Lnc_025370 overexpression promotes tumor growth and Ki67 expression. Together, our results suggest that Lnc_025370 acts as a pivotal target mediator of C6 ceramide’s anti-cancer effects, facilitating the malignant progression of CHMp cells.

Charting the transcriptomic landscape of primary and metastatic cancers in relation to their origin and target normal tissues.

Metastasis is a leading cause of cancer-related deaths, yet understanding how metastatic tumors adapt from their origin to their target tissues remains a fundamental challenge. To address this, we assessed whether primary and metastatic tumors more closely resemble their tissues of origin or target tissues in terms of gene expression. We analyzed expression profiles from multiple cancer types and normal tissues, including single-cell and bulk RNA sequencing data from both paired and unpaired patient cohorts. Primary tumors were overall more transcriptomically similar to their tissues of origin, while metastases shifted toward their target tissues. However, pathway-level analysis highlighted critical metabolic and immune transcriptomic changes toward target tissues during metastasis in both primary and metastatic tumors. In addition, primary tumors exhibited higher activity in cancer hallmarks such as "Activating Invasion and Metastasis" when compared to metastases. This comprehensive analysis provides a transcriptome-wide view of the processes through which cancer tumors adapt to their metastatic environments before and after metastasis.

Biopsy vitrification: New tool for endometrial tissue cryopreservation for research applications

Patient-derived endometrial biopsies serve as a crucial source for molecular studies, highlighting the necessity for tissue cryopreservation methods that preserve cell viability and tissue morphology with minimal to no impact. The passive slow freezing (PSF) protocol has demonstrated efficacy for cryopreserving endometrial biopsies, allowing for the subsequent isolation of viable epithelial and stromal cells. Vitrification (VT) enables the avoidance of ice crystal formation and could therefore potentially prevent mechanical injury to tissues. In this study, PSF and VT techniques were applied to endometrial biopsies, and the effects of cryopreservation on tissue samples were evaluated using traditional histology. In addition, transmission electron microscopy (TEM), gene expression profiling analyses, the viability of endometrial cells, and the ability to form epithelial organoids were compared between PSF and VT endometrial biopsies in a subset of samples. The histology and TEM studies demonstrated relatively mild cellular and sub-cellular damage in both cryopreservation protocols which did not affect tissue functionality and the formation of the organoids. Additionally, the cryopreservation methodology did not affect the gene expression profile of the 68 endometrial-receptivity associated genes studied. In conclusion, our findings indicate that although current cryopreservation methodologies need further improvements, they still allow us to achieve acceptable cell viability and functionality, showing promising potential for facilitating the utilization of cryopreserved endometrial tissue samples for research purposes.

The expression profile analysis and functional prediction of lncRNAs in peripheral blood mononuclear cells in maintenance hemodialysis patients developing heart failure

Heart failure (HF) is the leading cause of death in patients with maintenance hemodialysis (MHD). Biomarkers has an important guiding role in the early diagnosis, risk stratification, and prognostic assessment of HF. Increasing studies have indicated that long non-coding RNAs (lncRNAs) have played an indispensable role in the regulatory network of HF. This study was aiming to explore the expression profiles of lncRNAs in patients treated with MHD developing heart failure. Peripheral blood mononuclear cells were isolated from 4 hemodialysis patients with reduced ejection fraction (HFrEF) and 4 hemodialysis patients with preserved ejection fraction (HFpEF), respectively. The expression profile analysis of lncRNAs was performed by using Illumina Novaseq 6000 sequencer. Quantitative real time polymerase chain reaction (qRT-PCR) was used to verify the expression of representative differentially expressed lncRNAs. Based on lncRNA-miRNA-mRNA-KEGG network analysis, the potential role of candidate lncRNAs and their association with the severity of HF were further evaluated. In total, 1,429 differentially expressed lncRNAs were found between patients with HFrEF and patients with HFpEF, of which 613 were up-regulated and 816 were down-regulated (P < 0.05). Five candidate lncRNAs were screened out by a series of bioinformatic analyses. After being compared with miRBase, ENST00000561762, one of the 5 candidates, was considered the most likely lncRNA to be serving as a precursor for miRNA. Nine predicted target genes were found by further lncRNA-miRNA-mRNA-KEGG network analysis, and among which ITGB5 was enriched in the actin dynamics signaling pathway. In another cohort of hemodialysis patients, the expression of lncRNA ENST00000561762 was verified by qRT-PCR. Further analysis revealed that there was a strong correlation between left ventricular ejection fraction and ENST00000561762, proBNP, and 6-minute walk distance, respectively. LncRNAs expression profile was remarkably different in hemodialysis patients with HFrEF compared to those with HFpEF. Among which, lncRNA ENST00000561762 was considered as a promising biomarker for patients with HFrEF as it was predicted to be a miRNA precursor to regulate the actin dynamics signaling pathway.

Characterization of lipases revealed tissue-specific triacylglycerol hydrolytic activity in Spodoptera frugiperda.

Lipids perform a diverse and unique array of functions in insects. Lipases are key enzymes in lipid metabolism, and their metabolic products are crucial for development and reproduction of insects. Here, a total of 110 lipase genes were identified in the genome of Spodoptera frugiperda. Cluster analysis indicated that neutral lipases constitute the majority of lipases. Tissue expression profile analysis displayed that most lipase genes were highly expressed in the larval gut of S. frugiperda. Some lipases exhibited a diet-specific expression pattern, which implied their roles in host adaptation. Key domain analysis proved that none of the neutral lipases highly expressed in the gut has an integrated lid domain, while most lipases highly expressed in the fat body contained both the integrated lid domain and β9 loop, indicating the activity loss of neutral lipases in guts. The assay of triacylglycerol (TAG) hydrolytic activity confirmed that the gut had the lowest activity when compared to that of fat body and epidermis. Interestingly, the opposite TAG hydrolytic activity trends across mating were observed between adult males and females, implying that lipase played different roles in the reproduction of both sexes. In conclusion, neutral lipases lost TAG hydrolytic activity in S. frugiperda guts, but retained the activity in fat body. Neutral lipases would play vital roles in many physiological processes in insects, especially in insect reproduction, which provides palpable targets for novel insecticide development to control insect population growth.

Integrated analysis of miRNA and mRNA expression profiles associated with wooden breast myopathy in broiler chickens

Wooden breast (WB) myopathy has raised a worldwide concern among broiler industry during the past decade. Despite progress in understanding its etiology from transcriptional regulation, post-transcriptional mechanisms including the regulation of microRNAs (miRNAs) remain largely unknown. In the current study, we described an integrative analysis between mRNA and miRNA expression profiles of pectoralis major muscle from normal and WB myopathic broilers. A total of 1983 differentially expressed mRNAs (DEmRNAs) and 155 DEmiRNAs were identified in WB. We screened crucial biological processes and core DEmRNAs enriched in functional pathways, and established the protein-protein interaction network. DEmiRNAs and negatively correlated DEmRNAs regulatory networks were constructed, including 44 exist DEmiRNAs and 478 DEmRNAs, forming 772 miRNA-mRNA pairs. Upregulated DEmiRNAs including gga-miR-21-3p, gga-miR-460a-5p and gga-miR-6631-5p, as well as downregulated DEmiRNAs including gga-miR-182-5p, gga-miR-183 and gga-miR-96-5p were identified as hub miRNAs. Meanwhile, functional enrichment analysis indicated that upregulated DEmRNAs in the network were enriched in biological processes of response to stimulus, inflammatory response, extracellular matrix organization, whereas downregulated DEmRNAs were enriched in carbohydrate, amino acid and nucleotide metabolic processes. Collectively, our integrative miRNA and mRNA analysis highlighted candidate miRNAs and mRNAs, as well as potential miRNA-mRNA regulatory mechanisms involved in WB myopathy in broiler chicken.

The cell adhesion molecule CD44 acts as a modulator of 5-HT7 receptor functions

BackgroundHomo- and heteromerization of G protein-coupled receptors (GPCRs) plays an important role in the regulation of receptor functions. Recently, we demonstrated an interaction between the serotonin receptor 7 (5-HT7R), a class A GPCR, and the cell adhesion molecule CD44. However, the functional consequences of this interaction on 5-HT7R-mediated signaling remained enigmatic.MethodsUsing a quantitative FRET (Förster resonance energy transfer) approach, we determined the affinities for the formation of homo- and heteromeric complexes of 5-HT7R and CD44. The impact of heteromerization on 5-HT7R-mediated cAMP signaling was assessed using a cAMP responsive luciferase assay and a FRET-based cAMP biosensor under basal conditions as well as upon pharmacological modulation of the 5-HT7R and/or CD44 with specific ligands. We also investigated receptor-mediated G protein activation using BRET (bioluminescence resonance energy transfer)-based biosensors in both, homo- and heteromeric conditions. Finally, we analyzed expression profiles for 5-HT7R and CD44 in the brain during development.ResultsWe found that homo- and heteromerization of the 5-HT7R and CD44 occur at similar extent. Functionally, heteromerization increased 5-HT7R-mediated cAMP production under basal conditions. In contrast, agonist-mediated cAMP production was decreased in the presence of CD44. Mechanistically, this might be explained by increased Gαs and decreased GαoB activation by 5-HT7R/CD44 heteromers. Unexpectedly, treatment of the heteromeric complex with the CD44 ligand hyaluronic acid boosted constitutive 5-HT7R-mediated cAMP signaling and receptor-mediated transcription, suggesting the existence of a transactivation mechanism.ConclusionsInteraction with the hyaluronan receptor CD44 modulates both the constitutive activity of 5-HT7R as well as its agonist-mediated signaling. Heteromerization also results in the transactivation of 5-HT7R-mediated signaling via CD44 ligand.

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

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

Genome-Wide Identification and Expression Profiling Analysis of the Mitochondrial Calcium Uniporter Gene Family Under Abiotic Stresses in Medicago sativa.

The mitochondrial calcium uniporters (MCUs) are a family of calcium unidirectional transporters important for cytoplasmic Ca2+ signals. Though the MCU proteins in several plant species have been investigated, genome-wide analysis of MCUs in alfalfa is lacking. Here, via genome-wide analysis, a total of 5, 20, and 6 MCU genes were identified in three different alfalfa cultivars, namely Medicago truncatula Jemalong A17, Medicago sativa XinJiangDaYe, and M. sativa Zhongmu No. 1, respectively. They were further phylogenetically classified into three subfamilies. Most MCU genes have only one intron, and gene duplication events of MCU genes were observed within each alfalfa accession and between different accessions. All alfalfa MCU proteins contained a highly conserved MCU domain and 10 conserved motifs, featuring two transmembrane domains and a DI/VME motif. According to the tissue expression data of M. sativa Zhongmu No. 1, MsMCU6.2 was the most abundant transcript with the highest expression in the leaf, and MsMCU5 and MsMCU1.2 showed higher expression levels in the stem than other tissues. We analyzed the expression profiles of five MCU genes (MsMCU1.1/1.2/5/6.1/6.2) under salt, drought, and cold stresses via qRT-PCR assays. All five MCU genes were induced by drought stress, except MsMCU5, whose expression was up-regulated by salt stress, while cold stress slightly altered MsMCU expression. Nine potential interacting proteins and three miRNAs targeting MtMCUs were predicted. These results provide detailed knowledge of alfalfa MCU genes and suggest their potential functions in abiotic stress response.

Inulae Flos is a potential herbal medicine to treat glioma: a study based on gene expression profile analysis, network pharmacology and molecular docking

Inulae Flos is a promising herbal medicine; however, its underlying mechanism in treating glioma remains unclear. This study aimed to elucidate the potential active ingredients and mechanisms of Inulae Flos in glioma treatment. Differentially expressed genes (DEGs) associated with gliomas were identified by analyzing the GSE186057 dataset. The bioactive components of Inulae Flos were searched in the Traditional Chinese Medicine Systems Pharmacology database, and related targets were obtained from the Swiss Target Prediction database. A protein-protein interaction (PPI) network was constructed using the STRING database. Next, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) of these common targets were analyzed using the DAVID database. Finally, AutoDock Vina was used for molecular docking analysis. The tumor-suppressive properties of the crude extract of Inulae Flos (XFH) and britanin were then evaluated with in vitro and in vivo assays. Nineteen active ingredients were obtained from Inulae Flos, and 20 DEGs were identified as the targets of Inulae Flos in glioma treatment. Britanin, luteolin and 1-O-Acetylbritannilactone were identified as the key ingredients. PIK3R1, CDK2, CCNB1, MAPK1, MAPK8, CCNA2, and AR were considered to be the hub target genes of Inulae Flos in glioma treatment. Good binding affinity was observed between the key components and hub targets. In addition, in vitro studies showed that XFH and britanin, a key component of Inulae Flos, could inhibit the growth, migration and invasion glioma cell lines U87 and U138, and repress the tumorigenesis of U138 cells in nude mice. Inulae Flos is promising for glioma treatment, and it exerts its anti-tumor activities via multiple components, targets and pathways.

CNSC-08. PROGNOSTIC BIOMARKER DISCOVERY THROUGH PROTEOMIC ANALYSIS OF GLIOBLASTOMA MULTIFORME

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor known for its high recurrence rates and dismal survival outcomes. Despite advancements in surgical and therapeutic strategies, the prognosis for GBM patients remains poor. This study applies proteomic analysis to postoperative GBM tumor specimens to identify molecular markers that could enhance prognosis prediction. Through the analysis of protein expression profiles before and after tumor recurrence, we identified significant alterations in protein pathways via Gene Set Enrichment Analysis (GSEA), highlighting potential biomarkers for disease outcome prediction. METHODS We employed Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH-MS) proteomics to analyze protein samples from both primary and recurrent GBM patients. Gene Set Enrichment Analysis (GSEA) was utilized to compare protein expression profiles and discern significantly altered pathways. RESULTS Analysis revealed 778 differentially expressed proteins (DEPs), with notable alterations in pathways such as glycolysis, interleukin signaling, and MAPK signaling. Proteins such as TOLLIP, ENO1, and ITGA2B emerged as potential prognostic markers. Validation through an online tumor prognostic analysis platform (ToPP), integrated with The Cancer Genome Atlas (TCGA) data, facilitated the development of a prognostic risk score model. This model effectively categorized patients into high and low-risk groups based on their protein expression profiles, enhancing the prediction of patient outcomes. The proteins and pathways identified offer new insights into the molecular dynamics of GBM postoperatively. The association between protein expression profiles and patient prognosis underscores the potential of these markers in developing customized therapeutic strategies. Additionally, the prognostic risk score model serves as a valuable tool for improving prognostic accuracy and could guide personalized treatment plans. CONCLUSION Proteomic profiling of GBM tumors significantly contributes to uncovering the molecular mechanisms driving this aggressive cancer and facilitates the development of dependable prognostic tools. These findings not only affirm the critical role of proteomics in cancer research but also advance personalized medicine strategies in GBM management.

TMOD-17. FOXR2-ACTIVATED CNS NEUROBLASTOMA ORIGINATES FROM THE MEDIAL GANGLIONIC EMINENCE LINEAGE

Abstract FOXR2-activated CNS neuroblastoma is a rare subtype of pediatric brain tumor characterized by the elevated expression of the transcription factor FOXR2 due to genomic rearrangement. However, the precise pathogenic mechanisms, including the cell type of origin and the function of FOXR2, are not fully understood. An expression profile analysis of patient tumors showed increased expression of the marker genes associated with the Medial Ganglionic Eminence (MGE) in the ventral forebrain such as NKX2.1 and SOX6. Based on this result, we hypothesized that FOXR2-activated CNS neuroblastoma originates from the MGE lineage. To test this hypothesis, we overexpressed FOXR2 in human embryonic stem cell-derived MGE progenitors and, as a control cell type, cortical progenitors. Our data showed that FOXR2 overexpression resulted in a significantly increased proliferation in the MGE progenitors but not in the cortical progenitors, indicating a cell-type-specific effect of FOXR2. When injected into the brain of immunodeficient mice, FOXR2-expressing MGE progenitors formed tumors that molecularly resemble patient tumors, while FOXR2-expressing cortical progenitors did not. To further dissect the underlying molecular mechanisms, we conducted an RNA-seq experiment and found that FOXR2 overexpression activated the MEK/ERK signaling pathway through a suppression of the endogenous RAS inhibitor DIRAS3. Moreover, the FDA-approved MEK inhibitor trametinib significantly suppressed the proliferation and induced cell death in FOXR2-expressing MGE progenitors in vitro with the IC50 value of 30 nM. Collectively, our study has revealed the cell type of origin of FOXR2-activated CNS neuroblastoma and identified the MEK/ERK pathway as a critical downstream of FOXR2 that can be targeted therapeutically.

CNSC-58. BIOPROCESSING OF VIABLE SURGICAL PEDIATRIC BRAIN TUMOR SPECIMENS FOR GENOME-GUIDED PERSONALIZED DRUG TESTING

Abstract Novel treatment approaches are urgently needed for pediatric central nervous system (CNS) tumors as they are the leading cause of cancer-related deaths in children. A lack of research materials impedes progress towards developing such therapies. Although various brain cancer biobanks exist, these rarely store viable patient-derived tissue and cells for live cell analyses, including cell fate assays and testing for drug sensitivities. We propose that a biorepository of viable cell dissociates and tissue broadly representing CNS tumor entities overcomes these limitations. From a combination of molecular diagnoses and histopathologic assessment of over 120 samples collected, we generated a biorepository of roughly 35 distinct entities of pediatric CNS tumors in our Stanford neuro-bioprocessing cohort. Based on the Central Brain Tumor Registry of the United States, the proportion of subtypes represented in our cohort match that of the general population with exceptions. We established a standardized bioprocessing pipeline that can be used as a template for tissue collection and model development in the broader disease context and for multiple downstream applications. Our emphasis on cryostorage of viable cells and tissue facilitates ad hoc live cell analyses. In addition to gaining a better understanding of tumor pathophysiology, we attempted a rapid bed-to-bench-to-bedside approach using comparative RNA expression profile analyses in an individual patient’s pilocytic astrocytoma. Novel drug targets were identified by analyzing RNA transcripts that are highly expressed (outliers) compared with a compendium of 12,747 brain and non-brain tumor samples. We validated outliers as drug targets using acute cell isolates, and identified a novel target in recurrent low-grade glioma. These studies illustrate that biobanking of viable patient-derived material enables developing personalized therapies with the goal of improving patient outcomes. As demonstrated here, patient-derived acute cell isolates facilitate identifying drug vulnerability, creating an opportunity for more personalized treatment of patients with CNS tumors.

Comprehensive analysis of mRNA and miRNA differential expression profiles in the hypothalamus-pituitary-gonadal axis in laying and broodiness period of Wanxi white geese

The hypothalamus-pituitary-gonadal (HPG) axis is an important neuroendocrine regulatory center involved in egg-laying process in poultry. However, its mechanism of regulating broodiness behavior and laying performance in geese remains unclear. This study explored the molecular mechanism by which the HPG axis regulates brooding behavior in Wanxi white geese (WWG). The hypothalamus, pituitary, and ovarian tissues of Wanxi white geese were collected at laying and brooding periods for transcriptome sequencing analysis. A total of 240 (BH vs. LH), 319 (BP vs. LP), and 445 (BO vs. LO) differentially expressed genes, and 56 (BH vs. LH), 82 (BP vs. LP), and 48 (BO vs. LO) differentially expressed miRNAs were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis showed that differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were significantly enriched in hormone level regulation, cell communication, calcium signaling pathway, GnRH signaling pathway, MAPK signaling pathway, Wnt signaling pathway, and other processes. Six DEGs and four DEMs were randomly selected for real-time fluorescence quantitative reverse transcription PCR (RT-qPCR). The results showed that the transcriptome sequencing data were accurate and reliable. In addition, 22 potential hub miRNAs were screened. Dual luciferase reporter assays confirmed the targeting relationship between miR-144-y and DIO3. The results showed that the miRNAs mainly regulated the laying performance and brooding behavior of WWG by mediating the expression of target genes. In this study, we systematically elucidated the mechanisms by which the HPG axis regulates the broodiness behavior and laying performance of WWG at the post-transcriptional level. Several miRNAs and mRNAs associated with the reproductive performance of WWG were identified, providing a crucial reference for the subsequent use of gene editing technologies to breed new varieties and advance the development of WWG breeding industry.

Genome-wide identification and functional analysis of CYP450 genes in eggplant (Solanum melongena L.) with a focus on anthocyanin accumlation

BackgroundIn plants, Cytochrome P450 (CYP450) constitutes the largest family of metabolic enzymes and plays a crucial role in various physiological processes, including plant growth and development. Eggplants are well-known for their peel’s high concentration of anthocyanin compounds that provide significant health benefits to humans. The accumulation of anthocyanins in eggplant peels is an important process during growth and development. Therefore, it is essential to identify the CYP450 genes in eggplant (SmCYPs) and analyze their expression profiles during the period of anthocyanin accumulation in the peel.ResultsA total of 180 SmCYPs were identified in the eggplant genome and classified into eight subfamilies based on phylogenetic analysis. These SmCYPs exhibited highly conserved gene structure (exon/intron) and protein motifs, especially within their respective subgroup. Sixteen pairs of genes with collinearity were identified through gene duplication analysis. Promoter cis-acting element analysis revealed that SmCYPs are involved in various responses, including growth and development, stress responsiveness, and light responsiveness. Transcriptome data analysis revealed that all SmCYPs were expressed in various eggplant tissues, such as roots, stems, leaves, flowers, and fruits; with diverse expression patterns among members. The expression patterns of SmCYPs in eggplant peel also exhibited diversity during different stages of anthocyanin accumulation. qRT-PCR analysis demonstrated similar expression patterns for 15 selected SmCYPs as observed in the transcriptome data. Metabolomics analysis further suggested that SmCYPs are involved in the biosynthesis of secondary metabolites and metabolic pathways related to flavonoid and flavone/flavonol biosynthesis. Notably, three specific SmCYPs (SmCYP73A1/75A/98A1) play a significant role in flavonoid biosynthesis, particularly in anthocyanin synthesis in eggplant.ConclusionGenome-wide identification, phylogenetic analysis, expression profile analysis, and exploration of metabolic pathways related to SmCYPs provide valuable insights into the roles of these genes in anthocyanin accumulation in various tissues and organs, including eggplant peel. The findings from this study lay a foundation for the functional analysis of SmCYPs involvement in anthocyanin accumulation in eggplant peel, providing a molecular basis for breeders to cultivate novel varieties of eggplants with high levels of anthocyanin content.

Expression Profiling Analysis of the SWEET Gene Family in In Vitro Pitaya Under Low-Temperature Stress and Study of Its Cold Resistance Mechanism.

Pitaya (Hylocereus undatus) fruit is an attractive, nutrient-rich tropical fruit with commercial value. However, low-temperature stress severely affects the yield and quality of pitaya. The relevant mechanisms involved in the response of pitaya to low-temperature stress remain unclear. To study whether the SWEET gene family mediates the response of H. undatus to low-temperature stress and the related mechanisms, we performed genome-wide identification of the SWEET gene family in pitaya, and we used 'Baiyulong' tissue-cultured plantlets as material in the present study. We identified 28 members of the SWEET gene family from the H. undatus genome and divided these family members into four groups. Members of this gene family presented some differences in the sequences of introns and exons, but the gene structure, especially the motifs, presented relatively conserved characteristics. The promoter regions of most HuSWEETs have multiple stress- or hormone-related cis-elements. Three duplicated gene pairs were identified, including one tandem duplication gene and two fragment duplication gene pairs. The results revealed that the SWEET genes may regulate the transport and distribution of soluble sugars in plants; indirectly regulate the enzyme activities of CAT, POD, and T-SOD through its expression products; and are involved in the response of pitaya to low-temperature stress and play vital roles in this process. After ABA and MeJA treatment, the expression of HuSWEETs changed significantly, and the cold stress was also alleviated. This study elucidated the molecular mechanism and physiological changes in the SWEET gene in sugar metabolism and distribution of pitaya when it experiences low-temperature stress and provided a theoretical basis for cold-resistant pitaya variety breeding.

Multiple NADPH-cytochrome P450 reductases from Lycoris radiata involved in Amaryllidaceae alkaloids biosynthesis.

Amaryllidaceae alkaloids (AAs), such as galanthamine and lycorine, are natural products of Lycoris radiata possessing various pharmacological activities including anti-acetylcholinesterase, anti-inflammatory, and antitumour activities. Elucidating the biosynthesis of these special AAs is crucial for understanding their production and potential modification for improved clinical application, of which cytochrome P450 enzymes catalyse the formation of key alkaloid skeletons and subsequent modification processes, with the NAPDH cytochrome P450 reductases (CPRs) serving as essential redox partners. This study identified three CPRs, LrCPR1, LrCPR2, and LrCPR3, encoding 700, 697 and 695 amino acids, respectively, which belong to Class II CPRs. The LrCPRs reduced cytochrome c and ferricyanide in an NADPH-dependent manner, and their activities all followed the typical Michaelis-Menten curve. In yeast, the co-expression of LrCPRs and CYP96T6 produced the galantamine-like alkaloid namely N-demethylnarwedine, suggesting that they support the catalytic activity of CYP96T6. Quantitative analysis of the transcriptional expression profiles showed that LrCPRs were expressed in all the examined tissues of L. radiata, and their gene expression patterns are consistent with other genes that may be involved in the biosynthetic pathway of AAs, including cinnamate 4-hydroxylase and phenylalanine ammonia-lyase. Our study firstly provides the functional characterization of LrCPRs in L. radiata, which will contribute to the discovery of biosynthetic pathways and heterologous production of AAs.

Immune Microenvironment Alterations and Identification of Key Diagnostic Biomarkers in Chronic Kidney Disease Using Integrated Bioinformatics and Machine Learning.

Chronic kidney disease (CKD) involves complex immune dysregulation and altered gene expression profiles. This study investigates immune cell infiltration, differential gene expression, and pathway enrichment in CKD patients to identify key diagnostic biomarkers through machine learning methods. We assessed immune cell infiltration and immune microenvironment scores using the xCell algorithm. Differentially expressed genes (DEGs) were identified using the limma package. Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed to evaluate pathway enrichment. Machine learning techniques (LASSO and Random Forest) pinpointed diagnostic genes. A nomogram model was constructed and validated for diagnostic prediction. Spearman correlation explored associations between key genes and immune cell recruitment. The CKD group exhibited significantly altered immune cell infiltration and increased immune microenvironment scores compared to the normal group. We identified 2335 DEGs, including 124 differentially expressed immune-related genes. GSEA highlighted significant enrichment of inflammatory and immune pathways in the high immune score (HIS) subgroup, while GSVA indicated upregulation of immune responses and metabolic processes in HIS. Machine learning identified four key diagnostic genes: RGS1, IL4I1, NR4A3, and SOCS3. Validation in an independent dataset (GSE96804) and clinical samples confirmed their diagnostic potential. The nomogram model integrating these genes demonstrated high predictive accuracy. Spearman correlation revealed positive associations between the key genes and various immune cells, indicating their roles in immune modulation and CKD pathogenesis. This study provides a comprehensive analysis of immune alterations and gene expression profiles in CKD. The identified diagnostic genes and the constructed nomogram model offer potent tools for CKD diagnosis. The immunomodulatory roles of RGS1, IL4I1, NR4A3, and SOCS3 warrant further investigation as potential therapeutic targets in CKD.

Gene expression profile analysis of subregions of the adult female reproductive tract in the brown anole, Anolis sagrei.

The morphological diversity and functional role of the organs of the female reproductive system across tetrapods (limbed vertebrates) are relatively poorly understood. Though some features are morphologically similar, species-specific modification makes comparisons between species and inference about evolutionary origins challenging. In combination with the study of morphological changes, studying differences in gene expression in the adult reproductive system in diverse species can clarify the function of each organ. Here, we use the brown anole, Anolis sagrei, to study gene expression differences within the reproductive tract of the adult female. We generated gene expression profiles of four biological replicates of the three regions of the female reproductive tract, the infundibulum, glandular uterus, and nonglandular uterus by RNA-sequencing. We aligned read to the recently published Anolis sagrei genome and identified significantly differentially expressed genes between the regions using DEseq2. Each organ expressed approximately 14600 genes and comparison of gene expression profiles between organs revealed between 367-883 differentially expressed genes. We identify shared and region-specific transcriptional signatures for the three regions and compare gene expression in the brown anole reproductive tract to known gene expression patterns in other tetrapods. We find that genes in the Hox cluster have an anterior-posterior, colinear expression pattern as has been described in mammals. We also define a secretome for the glandular uterus. These data provide fundamental information for functional studies of the reproductive tract organs in the brown anole as well as an important phylogenetic anchor for comparative study of the evolution of the female reproductive tract.