Oxidation-reduction process of Arabidopsis thaliana roots induced by bisphenol compounds based on RNA-seq analysis.

Bisphenol compounds (BPs) have various industrial uses and can enter the environment through various sources. To evaluate the ecotoxicity of BPs and identify potential gene candidates involved in the plant toxicity, Arabidopsis thaliana was exposed to bisphenol A (BPA), BPB, BPE, BPF, and BPS at 1, 3, 10 mg/L for a duration of 14 days, and their growth status were monitored. At day 14, roots and leaves were collected for internal BPs exposure concentration detection, RNA-seq (only roots), and morphological observations. As shown in the results, exposure to BPs significantly disturbed root elongation, exhibiting a trend of stimulation at low concentration and inhibition at high concentration. Additionally, BPs exhibited pronounced generation of reactive oxygen species, while none of the pollutants caused significant changes in root morphology. Internal exposure concentration analysis indicated that BPs tended to accumulate in the roots, with BPS exhibiting the highest level of accumulation. The results of RNA-seq indicated that the shared 211 differently expressed genes (DEGs) of these 5 exposure groups were enriched in defense response, generation of precursor metabolites, response to organic substance, response to oxygen-containing, response to hormone, oxidation-reduction process and so on. Regarding unique DEGs in each group, BPS was mainly associated with the redox pathway, BPB primarily influenced seed germination, and BPA, BPE and BPF were primarily involved in metabolic signaling pathways. Our results provide new insights for BPs induced adverse effects on Arabidopsis thaliana and suggest that the ecological risks associated with BPA alternatives cannot be ignored.

Perioperative systemic IL-6 and immune-adipose- metabolism transcription in tumour and tumour adjacent breast cancer.

Surgical resection is the primary treatment approach for patients with breast cancer. Despite optimal multimodal treatment, metastatic recurrence remains a risk. Surgery-mediated systemic inflammation and local tissue inflammation generate an immunosuppressive and wound-healing environment that may accelerate cancer recurrence and metastasis post-operatively. Investigating the impact of surgery on local and systemic inflammation may provide knowledge for improvement of patient prognosis and treatment opportunities. Systemic cytokines were quantified in the blood plasma of patients with breast cancer pre-operatively, early post-operatively, and late post-operatively. Early post-operative levels of IL-6 were significantly elevated in patients who underwent mastectomy compared with wide local excision. Post-operative IL-6 levels correlate with clinicopathological features (age and BMI). The transcriptomes of local matched tumour and normal tumour adjacent (normal) breast tissue, from patients with breast cancer, were analysed by RNA-Seq. Elevated gene expressions of IL6, ADIPOQ, FABP4, LPL, PPARG, and CD36 in normal tissue were associated with worse overall survival of patients with ER-positive breast cancer. In tissue with higher expression of IL6 and ADIPOQ, a higher abundance of M2-like macrophage gene expression was identified. This study revealed perioperative systemic dynamics of inflammatory mediators and identified local immune-adipose-metabolism gene expression in tumour-adjacent tissue associated with pro-tumour function.

Multiple congenital anomalies in two fetuses with glutathione-synthetase deficit (GSS).

Glutathione synthetase deficiency is a rare inborn metabolic disease usually caused by biallelic variants in GSS. Clinical severity varies from isolated hemolytic anemia, sometimes associated with chronic metabolic acidosis and 5-oxoprolinuria, to severe neurological phenotypes with neonatal lethality. Here we report on two fetal siblings from two pregnancies with glutathione synthetase deficiency exhibiting similar multiple congenital anomalies associating phocomelia, cleft palate, intra-uterine growth retardation, genito-urinary malformations, and congenital heart defect. Genome sequencing showed that both fetuses were compound heterozygous for two GSS variants: the previously reported pathogenic missense substitution NM_000178.4 c.800G>A p.(Arg267Gln), and a 2.4 kb intragenic deletion NC_000020.11:g.34944530_34946833del. RNA-seq on brain tissue revealed the out-of-frame deletion of the exon 3 and an almost monoallelic expression of the missense variant (88%), suggesting degradation of the deletion-harboring allele by nonsense-mediated mRNA decay. 5-oxoproline (pyroglutamic acid) levels in amniotic fluid were elevated, suggesting an alteration of the gamma-glutamyl cycle, and corroborating the pathogenicity of the two GSS variants. Only one case of glutathione synthetase deficiency with limb malformations has previously been reported, in a newborn homozygous for the c.800G>A variant. Thus, our data allow us to discuss a potential phenotypic extension of glutathione synthetase deficiency, with a possible involvement of the c.800G>A variant.

Sink strength, nutrient allocation, cannabinoid yield and associated transcript profiles vary in two drug-type Cannabis chemovars.

Cannabis sativa L. is one of the oldest domesticated crops. Hemp-type cultivars, which predominantly produce non-intoxicating cannabidiol (CBD), have been selected for their fast growth, seed, and fibre production, while drug-type chemovars were bred for high accumulation of tetrahydrocannabinol (THC). We investigated how the generation of CBD-dominant chemovars by introgression of hemp- into drug-type Cannabis impacted plant performance. The THC-dominant chemovar showed superior sink strength, higher flower biomass and demand-driven control of nutrient uptake. By contrast, the CBD-dominant chemovar hyperaccumulated phosphate in sink organs leading to reduced carbon and nitrogen assimilation in leaves, which limited flower biomass and cannabinoid yield. RNA-seq analyses determined organ- and chemovar-specific differences in expression of genes associated with nitrate and phosphate homeostasis as well as growth-regulating transcription factors that were correlated with measured traits. Among these were genes positively selected for during Cannabis domestication encoding an inhibitor of the phosphate starvation response SPX DOMAIN GENE3, nitrate reductase and two nitrate transporters. Altered nutrient sensing, acquisition or distribution are likely a consequence of adaption to growth on marginal, low-nutrient input lands in hemp. Our data provide evidence that such ancestral traits may become detrimental for female flower development and consequently overall CBD yield in protected cropping environments.

Predictive biomarkers for embryotoxicity: a machine learning approach to mitigating multicollinearity in RNA-Seq.

Multicollinearity, characterized by significant co-expression patterns among genes, often occurs in high-throughput expression data, potentially impacting the predictive model's reliability. This study examined multicollinearity among closely related genes, particularly in RNA-Seq data obtained from embryoid bodies (EB) exposed to 5-fluorouracil perturbation to identify genes associated with embryotoxicity. Six genes-Dppa5a, Gdf3, Zfp42, Meis1, Hoxa2, and Hoxb1-emerged as candidates based on domain knowledge and were validated using qPCR in EBs perturbed by 39 test substances. We conducted correlation studies and utilized the variance inflation factor (VIF) to examine the existence of multicollinearity among the genes. Recursive feature elimination with cross-validation (RFECV) ranked Zfp42 and Hoxb1 as the top two among the seven features considered, identifying them as potential early embryotoxicity assessment biomarkers. As a result, a t test assessing the statistical significance of this two-feature prediction model yielded a p value of 0.0044, confirming the successful reduction of redundancies and multicollinearity through RFECV. Our study presents a systematic methodology for using machine learning techniques in transcriptomics data analysis, enhancing the discovery of potential reporter gene candidates for embryotoxicity screening research, and improving the predictive model's predictive accuracy and feasibility while reducing financial and time constraints.

Transcriptome analysis of the aged SAMP8 mouse model of Alzheimer's disease reveals novel molecular targets of formononetin protection.

Background: Senescence-accelerated mouse prone 8 (SAMP8) and age-matched SAMR1 mice are used to study the pathogenesis and therapeutics of Alzheimer's disease (AD); however, the molecular mechanisms are not completely understood. Objective: This study aimed to examine the effects of the 5-month administration of formononetin in SAMP8 mice and used RNA-seq to explore the molecular targets. Methods: SAMP8 mice were orally administered formononetin (0, 8, and 16 mg/kg) from 4 months of age, and age-matched SAMR1 mice were used as controls. Behavioral tests were performed in 9-month-old mice, followed by histopathologic analysis. Total RNA from the hippocampus was isolated and subjected to RNA-seq, RT-qPCR, and bioinformatics analysis. Results: The 9-month-old SAMP8 mice exhibited cognition deficits, evidenced by novel object recognition, open-field test, elevated plus maze, and passive avoidance. Nissl bodies in the cortex and hippocampus were decreased. Formononetin treatments ameliorated behavioral deficits and improved morphological changes, which were evidenced by Nissl and H&E staining. RNA-seq revealed distinct gene expression patterns between SAMP8 and SAMR1 mice. Differentially expressed genes in SAMP8 mice were attenuated or normalized by formononetin. Ingenuity pathway analysis (IPA) of canonical pathway and upstream regulators revealed increases in proinflammatory factors and immune dysfunction and decreases in NRF2 and SIRT-1 signaling pathways, leading to neuroinflammation. Formononetin treatment attenuated or reversed these molecular changes. The transcriptome of SAMP8 mice was correlated with transcriptomic profiles of other AD mouse models in the GEO database. Conclusion: Neuroinflammation and decreased antioxidant and SIRT-1 signaling contributed to cognitive deficits in aged SAMP8 mice, which are potential therapeutic targets of formononetin in combination with other therapies.

Transcriptomic network underlying physiological alterations in the stem of Myricaria laxiflora in response to waterlogging stress.

Myricaria laxiflora is an endangered shrub plant with remarkable tolerance to waterlogging stress, however, little attention has been paid to understanding the underlying mechanisms. Here, physiological and transcriptomic approaches were applied to uncover the physiological and molecular reconfigurations in the stem of M. laxiflora in response to waterlogging stress. The accumulation of the contents of H2O2 and malonaldehyde (MDA) alongside increased activities of enzymes for scavenging the reactive oxygen species (ROS) in the stem of M. laxiflora were observed under waterlogging stress. The principal component analysis (PCA) of transcriptomes from five different timepoints uncovered PC1 counted for 17.3 % of total variations and separated the treated and non-treated samples. A total of 8714 genes in the stem of M. laxiflora were identified as differentially expressed genes (DEGs) under waterlogging stress, which could be assigned into two different subgroups with distinct gene expression patterns and biological functions. The DEGs involved in glycolysis were generally upregulated, whereas opposite results were observed for nitrogen uptake and the assimilation pathway. The contents of abscisic acid (ABA) and jasmonic acid (JA) were sharply decreased alongside the decreased mRNA levels of the genes involved in corresponding synthesis pathways upon waterlogging stress. A network centered by eight key transcription factors has been constructed, which uncovered the inhibited cell division processes in the stem of M. laxiflora upon waterlogging stress. Taken together, the obtained results showed that glycolysis, nitrogen metabolism and meristem activities played an important role in the stem of M. laxiflora in response to waterlogging stress.

scRNA-Explorer: An End-user Online Tool for Single Cell RNA-seq Data Analysis Featuring Gene Correlation and Data Filtering.

In the majority of downstream analysis pipelines for single-cell RNA sequencing (scRNA-seq), techniques like dimensionality reduction and feature selection are employed to address the problem of high-dimensional nature of the data. These approaches involve mapping the data onto a lower-dimensional space, eliminating less informative genes, and pinpointing the most pertinent features. This process ultimately leads to a reduction in the number of dimensions used for downstream analysis, which in turn speeds up the computation of large-scale scRNA-seq data. Most approaches are directed to isolate from biological background the genes characterizing different cells and or the condition under study by establishing lists of differentially expressed or coexpressed genes. Herein, we present scRNA-Explorer an open-source online tool for simplified and rapid scRNA-seq analysis designed with the end user in mind. scRNA-Explorer utilizes: (i) Filtering out uninformative cells in an interactive manner via a web interface, (ii) Gene correlation analysis coupled with an extra step of evaluating the biological importance of these correlations, and (iii) Gene enrichment analysis of correlated genes in order to find gene implication in specific functions. We developed a pipeline to address the above problem. The scRNA-Explorer pipeline allows users to interrogate in an interactive manner scRNA-sequencing data sets to explore via gene expression correlations possible function(s) of a gene of interest. scRNA-Explorer can be accessed at https://bioinformatics.med.uoc.gr/shinyapps/app/scrnaexplorer.

Genome-wide identification of cannabinoid biosynthesis genes in non-drug type Cannabis (Cannabis sativa L.) cultivar.

BACKGROUND: Cannabis sativa cultivars can be classified as marijuana or hemp, depending on its amount of the psychoactive cannabinoid Δ9-tetrahydrocannabinolic acid (THCA). Hemp Cheungsam is a non-drug type Cannabis sativa that is characterized by low THCA content. However, the transcripts and expression profile of cannabinoid biosynthesis pathway genes of hemp Cheungsam have not been investigated. METHODS: RNA-sequencing (RNA-seq) was performed on three different tissue types (flower, leaf, and stem) of hemp Cheungsam to understand their transcriptomes. The expression of cannabinoid biosynthesis pathway genes was further analyzed in each tissue type. Multiple sequence alignment and conserved domain analyses were used to investigate the homologs of cannbinoid biosynthesis genes. RESULTS: We found that the cannabinoid biosynthesis pathway was mainly expressed in the flowers of hemp Cheungsam, similar to other Cannabis cultivars. However, expression of cannabidiolic acid (CBDA) synthase was much higher than THCA synthase and cannabichromenic acid (CBCA) synthase, suggesting that the transcription profile favors CBDA biosynthesis. Sequence analysis of cannabinoid biosynthesis pathway genes suggested the identity of orthologs in hemp Cheungsam. CONCLUSIONS: Cannabinoid biosynthesis in hemp Cheungsam mostly occurs in the flowers, compared to other plant organs. While CBDA synthase expression is high, THCA and CBCA synthase expression is considerably low, indicating lesser THCA biosynthesis and thus low THCA content. Sequence analysis of key genes (CBDA, THCA, and CBCA synthases) of the cannabinoid biosynthetic pathway indicates that orthologs are present in hemp Cheungsam.

Construction and validation of a TAMRGs prognostic signature for gliomas by integrated analysis of scRNA and bulk RNA sequencing data.

BACKGROUND: This study aimed to construct and validate a prognostic model based on tumor associated macrophage-related genes (TAMRGs) by integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) data. METHODS: The scRNA-seq data of three inhouse glioma tissues were used to identify the tumor-associated macrophages (TAMs) marker genes, the DEGs from the The Cancer Genome Atlas (TCGA) - Genotype-Tissue Expression (GTEx) dataset were used to further select TAMs marker genes. Subsequently, a TAMRG-score was constructed by Least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analysis in the TCGA dataset and validated in the Chinese Glioma Genome Atlas (CGGA) dataset. RESULTS: We identified 186 TAMs marker genes, and a total of 6 optimal prognostic genes including CKS2, LITAF, CTSB, TWISTNB, PPIF and G0S2 were selected to construct a TAMRG-score. The high TAMRG-score was significantly associated with worse prognosis (log-rank test, P<0.001). Moreover, the TAMRG-score outperformed the other three models with AUC of 0.808. Immune cell infiltration, TME scores, immune checkpoints, TMB and drug susceptibility were significantly different between TAMRG-score groups. In addition, a nomogram were constructed by combing the TAMRG-score and clinical information (Age, Grade, IDH mutation and 1p19q codeletion) to predict the survival of glioma patients with AUC of 0.909 for 1-year survival. CONCLUSION: The high TAMRG-score group was associated with a poor prognosis. A nomogram by incorporating TMARG-score could precisely predict glioma survival, and provide evidence for personalized treatment of glioma.

Genome-wide analysis reveals transcriptional and translational changes during diapause of the Asian corn borer (Ostrinia furnacalis).

BACKGROUND: Diapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB). RESULTS: Our results unveil genes classified as "forwarded", "exclusive", "intensified", or "buffered" during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process. CONCLUSIONS: In summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.

Effect of High Glucose on Embryological Development of Zebrafish, Brachyodanio, Rerio through Wnt Pathway.

Gestational diabetes mellitus (GDM) is a worldwide pregnancy complication. Gestational diabetes can significantly impact fetus development. However, the effects of high glucose on embryological development post-fertilization are yet to be researched. Danio rerio embryos are a great model for studying embryonic development. In this study, the effects on embryological (morphological and genetic) development were examined in the presence of a high-glucose environment that mimics the developing fetus in pregnant women with GDM. Fertilized zebrafish embryos were treated with normal media and high glucose for 5 days from 3 h post-fertilization (hpf) to 96 hpf, respectively, as control and experimental groups. Morphological changes are recorded with microscope images. Hatch rate and heart rate are compared between groups at set time points. RNA-Seq is performed to examine the gene changes in the experimental group. Glucose delayed the zebrafish embryo development by slowing the hatch rate by about 24 h. The brain, heart, and tail started showing smaller morphology in the glucose group compared to the control group at 24 hpf. Heart rate was faster in the glucose group compared to the control group on days 2 and 3 with a statistically significant difference. Among the zebrafish whole genome, the significantly changed genes were 556 upregulated genes and 1118 downregulated genes, respectively, in the high-glucose group. The metabolic and Wnt pathways are altered under high-glucose conditions. These conditions contribute to significant physiological differences that may provide insight into the functionality of post-embryological development.

Antennal Transcriptome Screening and Identification of Chemosensory Proteins in the Double-Spine European Spruce Bark Beetle, Ips duplicatus (Coleoptera: Scolytinae).

The northern bark beetle, Ips duplicatus, is an emerging economic pest, reportedly infesting various species of spruce (Picea spp.), pine (Pinus spp.), and larch (Larix spp.) in Central Europe. Recent climate changes and inconsistent forest management practices have led to the rapid spread of this species, leaving the current monitoring strategies inefficient. As understanding the molecular components of pheromone detection is key to developing novel control strategies, we generated antennal transcriptomes from males and females of this species and annotated the chemosensory proteins. We identified putative candidates for 69 odorant receptors (ORs), 50 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 27 odorant-binding proteins (OBPs), including a tetramer-OBP, 9 chemosensory proteins (CSPs), and 6 sensory neuron membrane proteins (SNMPs). However, no sex-specific chemosensory genes were detected. The phylogenetic analysis revealed conserved orthology in bark beetle chemosensory proteins, especially with a major forest pest and co-habitant, Ips typographus. Recent large-scale functional studies in I. typographus chemoreceptors add greater significance to the orthologous sequences reported here. Nevertheless, identifying chemosensory genes in I. duplicatus is valuable to understanding the chemosensory system and its evolution in bark beetles (Coleoptera) and, generally, insects.

Upregulation of CKS2 in immunosuppressive cells is associated with metastasis and poor prognosis in prostate cancer: a single-cell RNA-sequencing analysis.

Background: Metastasis worsens prostate cancer (PCa) prognosis, with the immunosuppressive microenvironment playing a key role in bone metastasis. This study aimed to investigate how an immunosuppressive environment promotes PCa metastasis and worsens prognosis of patients with PCa. Methods: Candidate oncogenes were identified through analysis of the Gene Expression Omnibus (GEO) database. A prognostic model was developed for the purpose of identifying target genes. A single-cell RNA sequencing data from GEO database was used to analyze the localization of target genes in the tumor microenvironment. A pan-cancer analysis was conducted to study the cancer-causing potential of target genes across different types of tumors. Results: Fifty-one genes were found to be differentially expressed in bone metastasis compared to non-metastatic PCa, with CKS2 identified as the most significant gene associated with poor prognosis. CKS2 was shown to be linked to an immunosuppressive microenvironment and osteoclastic bone metastases, as shown by its negative correlation with immune cell infiltration and osteoblast-related gene expression. Moreover, CKS2 was found in immunosuppressive cells and was linked to bone metastasis in PCa. It was also overexpressed in different types of tumors, making it as an oncogenic gene. Conclusions: This research offers a new perspective on the potential utility of CKS2 as a therapeutic target for the prevention of metastatic PCa.

Identifying epithelial-mesenchymal transition-related genes as prognostic biomarkers and therapeutic targets of hepatocellular carcinoma by integrated analysis of single-cell and bulk-RNA sequencing data.

Background: Hepatocellular carcinoma (HCC) remains one of the most lethal cancers globally. Patients with advanced HCC tend to have poor prognoses and shortened survival. Recently, data from bulk RNA sequencing have been employed to discover prognostic markers for various cancers. However, they fall short in precisely identifying core molecular and cellular activities within tumor cells. In our present study, we combined bulk-RNA sequencing (bulk RNA-seq) data with single-cell RNA sequencing (scRNA-seq) to develop a prognostic model for HCC. The goal of our research is to uncover new biomarkers and enhance the accuracy of HCC prognosis prediction. Methods: Integrating single-cell sequencing data with transcriptomics were used to identify epithelial-mesenchymal transition (EMT)-related genes (ERGs) implicated in HCC progression and their clinical significance was elucidated. Utilizing marker genes derived from core cells and ERGs, we constructed a prognostic model using univariate Cox analysis, exploring a multitude of algorithmic combinations, and further refining it through multivariate Cox analysis. Additionally, we conducted an in-depth investigation into the disparities in clinicopathological features, immune microenvironment composition, immune checkpoint expression, and chemotherapeutic drug sensitivity profiles between high- and low-risk patient cohorts. Results: We developed a prognostic model predicated on the expression profiles of eight signature genes, namely HSP90AA1, CIRBP, CCR7, S100A9, ADAM17, ENG, PGF, and INPP4B, aiming at predicting overall survival (OS) outcomes. Notably, patients classified with high-risk scores exhibited a propensity towards diminished OS rates, heightened frequencies of stage III-IV disease, increased tumor mutational burden (TMB), augmented immune cell infiltration, and diminished responsiveness to immunotherapeutic interventions. Conclusions: This study presented a novel prognostic model for predicting the survival of HCC patients by integrating scRNA-seq and bulk RNA-seq data. The risk score emerges as a promising independent prognostic factor, showing a correlation with the immune microenvironment and clinicopathological features. It provided new clinical tools for predicting prognosis and aided future research into the pathogenesis of HCC.

Innovative aspects and applications of single cell technology for different diseases.

Recent developments in single-cell technologies have provided valuable insights from cancer genomics to complex microbial communities. Single-cell technologies including the RNA-seq, next-generation sequencing (NGS), epigenomics, genomics, and transcriptomics can be used to uncover the single cell nature and molecular characterization of individual cells. These technologies also reveal the cellular transition states, evolutionary relationships between genes, the complex structure of single-cell populations, cell-to-cell interaction leading to biological discoveries and more reliable than traditional bulk technologies. These technologies are becoming the first choice for the early detection of inflammatory biomarkers affecting the proliferation and progression of tumor cells in the tumor microenvironment and improving the clinical efficacy of patients undergoing immunotherapy. These technologies also hold a central position in the detection of checkpoint inhibitors and thus determining the signaling pathways evoked by tumor invasion. This review addressed the emerging approaches of single cell-based technologies in cancer immunotherapies and different human diseases at cellular and molecular levels and the emerging role of sequencing technologies leading to drug discovery. Advancements in these technologies paved for discovering novel diagnostic markers for better understanding the pathological and biochemical mechanisms also for controlling the rate of different diseases.

Elucidation of mechanisms underlying active oxygen burst in Citrus sinensis after Diaporthe citri infection using transcriptome analysis.

Introduction: Reactive oxygen species (ROS) generation is a common disease defense mechanism in plants. However, it is unclear whether Citrus host activates defense response against Diaporthe citri causing citrus melanose disease by producing ROS, and the underlying molecular mechanisms are unknown. Methods: DAB staining and RNA-Seq technology were used to compare the active oxygen burst and differential gene expression, respectively, in uninfected and infected Citrus sinensis leaves at different time points during D. citri infection in vivo. The functions of CsRBOH (a significant DEG) were confirmed in N. benthamiana through the Agrobacterium-mediated transient expression system. Results: DAB staining indicated that C. sinensis initiated defense against D. citri infection within 24 h by generating ROS. Illumina sequencing revealed 25,557 expressed genes of C. sinensis. The most upregulated DEGs (n = 1,570) were identified 72 h after fungal inoculation (sample denoted as CD72). In the CD72 vs. Cs (samples at 0 h after fungal inoculation) comparison, the KEGG pathway category with the highest number of genes (n = 62) and most significant enrichment was Protein processing in endoplasmic reticulum, followed by Glutathione metabolism and MAPK signaling pathway-plant. GO analysis revealed that the DEGs of CD72 vs. Cs related to active oxygen burst and chitin recognition were significantly grouped into the regulation of biological processes and molecular functions, with GO terms including response to ROS, response to fungus, and oxidoreductase activity. Remarkably, CsRBOH was significantly enriched in the GO and KEGG analyses, and its expression pattern in qRT-PCR and DAB staining results were consistent. Among the 63 ROS-related DEGs, HSP genes and genes associated with the peroxidase family were highly significant as revealed by protein-protein interaction networks. Furthermore, ROS accumulation, cell death, and upregulation of defense-related genes were observed in N. benthamiana leaves with CsRBOH expressed through the Agrobacterium-mediated transient expression system. Conclusion: Our findings suggested that C. sinensis activates CsRBOH and ROS-related genes, leading to ROS accumulation to resist the invasion by D. citri. This study laid the foundation for future research on molecular mechanisms and breeding of C. sinensis cultivars resistant to citrus melanose.

Ferulic Acid Methyl Ester Attenuates Cerebral Ischemia-Reperfusion Injury in Rats by Modulating PI3K/HIF-1α/VEGF Signaling Pathway.

Background: Cerebral ischaemia-reperfusion injury (CIRI) could worsen the inflammatory response and oxidative stress in brain tissue. According to previous studies, ferulic acid methyl ester (FAME), as the extract with the strongest comprehensive activity in the traditional Chinese medicine Huang Hua oil dot herb, has significant anti-oxidative stress and neuroprotective functions, and can effectively alleviate CIRI, but its mechanism of action is still unclear. Methods: Firstly, the pharmacological effects of FAME were investigated by in vitro oxidative stress and inflammatory experiments. Secondly, evaluate the therapeutic effects of FAME in the treatment of CIRI by brain histopathological staining and cerebral infarct area by replicating the in vivo MACO model. Thirdly, RNA-Seq and network pharmacology were utilized to predict the possible targets and mechanisms of FAME for CIRI at the molecular level. Finally, the expression of key target proteins, as well as the key regulatory relationships were verified by molecular docking visualization, Western Blotting and immunohistochemistry. Results: The results of in vitro experiments concluded that FAME could significantly reduce the content of TNF-α, IL-1ß and ROS, inhibiting COX-2 and iNOS protein expression in cells(p<0.01). FAME was demonstrated to have anti-oxidative stress and anti-inflammatory effects. The results of in vivo experiments showed that after the administration of FAME, the area of cerebral infarction in rats with CIRI was reduced, the content of Bcl-2 and VEGF was increased(p<0.05). Network pharmacology and RNA-Seq showed that the alleviation of CIRI by FAME may be through PI3K-AKT and HIF-1 signaling pathway. Enhanced expression of HIF-1α, VEGF, p-PI3K, p-AKT proteins in the brain tissues of rats in the FAME group was verified by molecular docking and Western Blotting. Conclusion: FAME possesses significant anti-inflammatory and anti-oxidative stress activities and alleviates CIRI through the PI3K/HIF-1α/VEGF signaling pathway.

Single-cell RNA sequencing reveals microenvironmental infiltration in non-small cell lung cancer with different responses to immunotherapy.

BACKGROUND: Immunotherapy represents a groundbreaking and monumental achievement in the field of cancer therapy, marking a significant advancement in fighting against this devastating disease. Lung cancer has showed consistent clinical improvements in response to immunotherapy treatments, yet, it is undeniable that challenges such as limited response rates acquire resistance, and the unclear fundamental mechanisms were inevitable problems. METHODS: The cellular composition was defined and distinguished through single-cell RNA sequencing (scRNA-seq) analysis of MPR (major pathologic response) and NMPR (non-major pathologic response) samples in GSE207422, including four primary MPR samples and eight primary NMPR samples. RESULTS: We found obvious difference in CD8+ T cell population between MPR and NMPR samples, with high expression of TYMS, RRM2, and BIRC5 in NPMR samples. Meanwhile, the proportion of macrophages and tumor epithelial cells infiltration increased in the NMPR samples. We discovered biomarkers (ACTN4, ATF3, BRD2, CDKN1A, and CHMP4B) in epithelial cells which were potentially represented worse outcomes. CONCLUSIONS: By exploring the difference of tumor microenvironment (TME) in samples with different corresponding degrees of neoadjuvant immunotherapy, this research introduces a number of novel biomarkers for predicting the response of treatment and a theoretical basis for overcoming immunotherapy resistance.