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.

Olfr2-positive macrophages originate from monocytes proliferate in situ and present a pro-inflammatory foamy-like phenotype.

AIMS: Olfactory receptor 2 (Olfr2) has been identified in a minimum of 30% of vascular macrophages, and its depletion was shown to reduce atherosclerosis progression. Mononuclear phagocytes, including monocytes and macrophages within the vessel wall, are major players in atherosclerosis. Single-cell RNA sequencing studies revealed that atherosclerotic artery walls encompass several monocytes and vascular macrophages, defining at least nine distinct subsets potentially serving diverse functions in disease progression. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages in atherosclerosis. METHODS AND RESULTS: Olfr2+ macrophages rapidly increase in Apoe-/- mice's aorta when fed a Western diet (WD). Mass cytometry showed that Olfr2+ cells are clustered within the CD64 high population and enriched for CD11c and Ccr2 markers. Olfr2+ macrophages express many pro-inflammatory cytokines, including Il1b, Il6, Il12, and Il23, and chemokines, including Ccl5, Cx3cl1, Cxcl9, and Ccl22. By extracting differentially expressed genes from bulk RNA sequencing (RNA-seq) of Olfr2+ vs. Olfr2- macrophages, we defined a signature that significantly mapped to single-cell data of plaque myeloid cells, including monocytes, subendothelial MacAir, and Trem2Gpnmb foamy macrophages. By adoptive transfer experiments, we identified that Olfr2 competent monocytes from CD45.1Apoe-/-Olfr2+/+ mice transferred into CD45.2Apoe-/-Olfr2-/- recipient mice fed WD for 12 weeks, accumulate in the atherosclerotic aorta wall already at 72 h, and differentiate in macrophages. Olfr2+ macrophages showed significantly increased BrdU incorporation compared to Olfr2- macrophages. Flow cytometry confirmed that at least 50% of aortic Olfr2+ macrophages are positive for BODIPY staining and have increased expression of both tumour necrosis factor and interleukin 6 compared to Olfr2- macrophages. Gene set enrichment analysis of the Olfr2+ macrophage signature revealed a similar enrichment pattern in human atherosclerotic plaques, particularly within foamy/TREM2hi-Mφ and monocytes. CONCLUSIONS: In summary, we conclude that Olfr2+ macrophages in the aorta originate from monocytes and can accumulate at the early stages of disease progression. These cells can undergo differentiation into MacAir and Trem2Gpnmb foamy macrophages, exhibiting proliferative and pro-inflammatory potentials. This dynamic behaviour positions them as key influencers in shaping the myeloid landscape within the atherosclerotic plaque.

Effects of Arborvitae (Thuja plicata) Essential Oil on Cervical Cancer Cells: Insights into Molecular Mechanisms.

AIMS: This study aimed to assess the effects of AEO in an in vitro model of cell lines derived from cervical cancer-namely, HeLa and SiHa-by screening for AEO's cytotoxic properties and examining its influence on the modulation of gene expression. BACKGROUND: Cervical cancer stands as a prevalent global health concern, affecting millions of women worldwide. The current treatment modalities encompass surgery, radiation, and chemotherapy, but significant limitations and adverse effects constrain their effectiveness. Therefore, exploring novel treatments that offer enhanced efficacy and reduced side effects is imperative. Arborvitae essential oil, extracted from Thuja Plicata, has garnered attention for its antimicrobial, anti-inflammatory, immunomodulatory, and tissue-remodeling properties; however, its potential in treating cervical cancer remains uncharted. OBJECTIVE: The objective of this study was to delve into the molecular mechanisms induced by arborvitae essential oil in order to learn about its anticancer effects on cervical cancer cell lines. METHODS: The methods used in this study were assessments of cell viability using WST-1 and annexin V- propidium iodide, mRNA sequencing, and subsequent bioinformatics analysis. RESULTS: The findings unveiled a dose-dependent cytotoxic effect of arborvitae essential oil on both HeLa and SiHa cell lines. Minor effects were observed only at very low doses in the HaCaT non-tumorigenic human keratinocyte cells. RNA-Seq bioinformatics analysis revealed the regulatory impact of arborvitae essential oil on genes enriched in the following pathways: proteasome, adherens junctions, nucleocytoplasmic transport, cell cycle, proteoglycans in cancer, protein processing in the endoplasmic reticulum, ribosome, spliceosome, mitophagy, cellular senescence, and viral carcinogenesis, among others, in both cell lines. It is worth noting that the ribosome and spliceosome KEGG pathways are the most significantly enriched pathways in HeLa and SiHa cells. CONCLUSION: Arborvitae essential oil shows potential as a cytotoxic and antiproliferative agent against cervical cancer cells, exerting its cytotoxic properties by regulating many KEGG pathways.

Transcriptomic analysis of Asparagus officinalis cultivars with varying levels of freezing tolerance over fall acclimation and spring deacclimation periods.

Asparagus (Asparagus officinalis L.) is an important vegetable crop in southern Ontario, Canada, where winter air and soil temperatures below 0°C are common. Consequently, cultivars growing in this area must possess winterhardiness and freezing tolerance for survival. Asparagus acquires freezing tolerance in the fall through cold acclimation and loses freezing tolerance in the spring through deacclimation. To understand the molecular bases of these processes, transcriptomic analysis (RNA-Seq) was conducted on two cultivars, one adapted, 'Guelph Millennium' (GM), and one unadapted, 'UC157' (UC), to the winter conditions of southern Ontario. RNA extracted from bud and rhizome tissues, sampled on three dates during early spring and late fall, was subjected to sequencing. In the fall, the numbers of differentially expressed (DE) genes at the second and third harvests increased, relative to the first harvest, in dormant buds and rhizomes as freezing tolerance of cultivars increased, and the majority of DE genes were downregulated. In spring, freezing tolerance decreased as plants deacclimated and most genes DE at second and third harvests were upregulated in both cultivars. GM had lower LT50 (lethal temperature at which 50% of plants die) values and hence higher freezing tolerance than UC on specific sampling dates during both spring and fall, and expression patterns of specific genes were correlated with LT50 differences. Functional analysis revealed that these genes were involved in carbohydrate metabolic process, plant hormone signal transduction (auxin and gibberellin), proline metabolism, biosynthesis of secondary metabolites, circadian rhythm, and late embryogenesis abundant proteins and could be associated with cold acclimation and deacclimation processes. These findings will help researchers understand the molecular mechanisms of freezing tolerance in asparagus, leading to breeding and genetic strategies to improve the trait.

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.

Genomic changes and stabilization following homoploid hybrid speciation of the Oxford ragwort Senecio squalidus.

Oxford ragwort (Senecio squalidus) is one of only two homoploid hybrid species known to have originated very recently, so it is a unique model for determining genomic changes and stabilization following homoploid hybrid speciation. Here, we provide a chromosome-level genome assembly of S. squalidus with 95% of the assembly contained in the 10 longest scaffolds, corresponding to its haploid chromosome number. We annotated 30,249 protein-coding genes and estimated that ∼62% of the genome consists of repetitive elements. We then characterized genome-wide patterns of linkage disequilibrium, polymorphism, and divergence in S. squalidus and its two parental species, finding that (1) linkage disequilibrium is highly heterogeneous, with a region on chromosome 4 showing increased values across all three species but especially in S. squalidus; (2) regions harboring genetic incompatibilities between the two parental species tend to be large, show reduced recombination, and have lower polymorphism in S. squalidus; (3) the two parental species have an unequal contribution (70:30) to the genome of S. squalidus, with long blocks of parent-specific ancestry supporting a very rapid stabilization of the hybrid lineage after hybrid formation; and (4) genomic regions with major parent ancestry exhibit an overrepresentation of loci with evidence for divergent selection occurring between the two parental species on Mount Etna. Our results show that both genetic incompatibilities and natural selection play a role in determining genome-wide reorganization following hybrid speciation and that patterns associated with homoploid hybrid speciation-typically seen in much older systems-can evolve very quickly following hybridization.

Unveiling the Genomic Symphony: Identification Cultivar-Specific Genes and Enhanced Insights on Sweet Sorghum Genomes Through Comprehensive superTranscriptomic Analysis.

Sorghum (Sorghum bicolor (L.) Moench) is a multipurpose crop grown for food, fodder, and bioenergy production. Its cultivated varieties, along with their wild counterparts, contribute to the core genetic pool. Despite the availability of several re-sequenced sorghum genomes, a variable portion of sorghum genomes is not reported during reference genome assembly and annotation. The present analysis used 223 publicly available RNA-seq datasets from seven sweet sorghum cultivars to construct superTranscriptome. This approach yielded 45,864 Representative Transcript Assemblies (RTAs) that showcased intriguing Presence/Absence Variation (PAV) across 15 published sorghum genomes. We found 301 superTranscripts were exclusive to sweet sorghum, including 58 de novo genes encoded core and linker histones, zinc finger domains, glucosyl transferases, cellulose synthase, etc. The superTranscriptome added 2,802 new protein-coding genes to the Sweet Sorghum Reference Genome (SSRG), of which 559 code for different transcription factors (TFs). Our analysis revealed that MULE-like transposases were abundant in the sweet sorghum genome and could play a hidden role in the evolution of sweet sorghum. We observed large deletions in the D locus and terminal deletions in four other NAC encoding loci in the SSRG compared to its wild progenitor (353) suggesting non-functional NAC genes contributed to trait development in sweet sorghum. Moreover, superTranscript-based methods for Differential Exon Usage (DEU) and Differential Gene Expression (DGE) analyses were more accurate than those based on the SSRG. This study demonstrates that the superTranscriptome can enhance our understanding of fundamental sorghum mechanisms, improve genome annotations, and potentially even replace the reference genome.

The opposite aging effect to single cell transcriptome profile among cell subsets.

Comparing transcriptome profiling between younger and older samples reveals genes related to aging and provides insight into the biological functions affected by aging. Recent research has identified sex, tissue, and cell type-specific age-related changes in gene expression. This study reports the overall picture of the opposite aging effect, in which aging increases gene expression in one cell subset and decreases it in another cell subset. Using the Tabula Muris Senis dataset, a large public single-cell RNA sequencing dataset from mice, we compared the effects of aging in different cell subsets. As a result, the opposite aging effect was observed widely in the genes, particularly enriched in genes related to ribosomal function and translation. The opposite aging effect was observed in the known aging-related genes. Furthermore, the opposite aging effect was observed in the transcriptome diversity quantified by the number of expressed genes and the Shannon entropy. This study highlights the importance of considering the cell subset when intervening with aging-related genes.

Genomic and transcriptomic landscape to decipher the genetic basis of hyperpigmentation in Lanping black-boned sheep (Ovis aries).

BACKGROUND: Lanping black-boned sheep (LPB) represent a distinctive mammalian species characterized by hyperpigmentation, resulting in black bone and muscle features, in contrast to their conventional counterparts exhibiting red muscle and white bone. The genetic basis underlying LPB hyperpigmentation has remained enigmatic. METHODS: In this study, we conducted whole-genome sequencing of 100 LPB and 50 Lanping normal sheep (LPN), and integrated this data with 421 sequenced datasets from wild and domestic sheep, shedding light on the genetic backdrop and genomic variations associated with LPB. Furthermore, we performed comparative RNA-Seq analysis using liver sample to pinpoint genes implicated in the pigmentation process. We generated a comprehensive dataset comprising 97,944,357 SNPs from 571 sheep, facilitating an in-depth exploration of genetic factors. RESULTS: Population genetic structure analysis revealed that the LPB breed traces its origin back to LPN, having evolved into a distinct breed. The integration of positively selected genes with differentially expressed genes identified two candidates, ERBB4 and ROR1, potentially linked to LPB hyperpigmentation. Comparative analysis of ERBB4 and ROR1 mRNA relative expression levels in liver, spleen, and kidney tissues of LPB, in comparison to Diqing sheep, revealed significant upregulation, except for ERBB4 in the liver. Gene expression heatmaps further underscored marked allelic frequency disparities in different populations. CONCLUSION: Our findings establish the evolutionary lineage of the LPB breed from LPN and underscore the involvement of ERBB4 and ROR1 genes in melanin synthesis. These results enhance our comprehension of the molecular basis of hyperpigmentation and contribute to a more comprehensive depiction of sheep diversity.

Early transcriptomic response of innate immune cells to subcutaneous BCG vaccination of mice.

OBJECTIVES: Current data suggests that Bacille Calmette-Guerin (BCG) vaccination contributes to nonspecific enhancement of resistance to various infections. Thus, BCG vaccination induces both specific immunity against mycobacteria and non-specific "trained immunity" against various pathogens. To understand the fundamental mechanisms of "trained" immunity, studies of transcriptome changes occurring during BCG vaccination in innate immunity cells, as well as in their precursors, are necessary. Furthermore, this data possesses important significance for practical applications associated with the development of recombinant BCG strains aimed to enhance innate immunity against diverse infectious agents. DATA DESCRIPTION: We performed RNA sequencing of innate immune cells derived from murine bone marrow and spleen three days after subcutaneous BCG vaccination. Using fluorescence-activated cell sorting we obtained three cell populations for each mouse from both control and BCG vaccinated groups: bone marrow monocytes and neutrophils and splenic NK-cells. Then double-indexed cDNA libraries for Illumina sequencing from the collected samples were prepared, the resulting cDNA library mix was subjected to NovaSeq 6000 sequencing. This paper describes the collection of 24 RNA sequencing samples comprising 4 sets of immune cell populations obtained from subcutaneously BCG-vaccinated and control mice.

The microenvironmental factors induced invasive tumor cells in glioblastoma.

Glioblastoma (GBM) cells have the potential to switch from being "proliferative cells" to peritumoral "invasive cells". Peritumoral GBM cells have highly invasive properties that allow them to survive surgery, leading to recurrence. The mechanisms underlying the manner in which the tumor microenvironment (TME) regulates the invasiveness of GBM remain unclear. Single-cell RNA sequencing analysis revealed heterogeneity in GBM cells, microglia and macrophages. In this study, the Oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) expression indicated higher invasiveness in core GBM cells. Under environmental stress, the expression of OSMR and LIFR were up-regulated with the effect of hypoxic, acidic, and low-glucose conditions in vitro. Functional experiments revealed that TME stress significantly influences the proliferation, migration and invasion of GBM cells. The differences in core/peripheral TMEs in GBM affected the invasive properties, indicating the significant role of OSMR expression within the TME in tumor progression and postoperative therapy.

Analyzing the expression of the transcriptome in adipose tissue of fat- and thin-tailed sheep.

Significant efforts have been made to understand how fat deposition in sheep tail is regulated in genetic, transcriptomic, physiologic, biochemical, and metabolic levels in order to elucidate the complex mechanisms underlying the energy storage, lipid metabolism in adipose tissue, adaptability to harsh environments, and evolutionary domestication. Through RNA-seq data analysis, we are able to compare the gene expression of fat-tailed sheep versus thin-tailed sheep breeds in an acceptable resolution at transcriptome level. The purpose of this study was to compare the transcriptomes of Ghezel (fat-tailed) and Zel (thin-tailed) sheep. Total RNA from subcutaneous and tail tissue samples from healthy lambs was sequenced (150b PE) to identify differentially expressed genes (DEGs) between the two mentioned tissues and between the Ghezel and Zel sheep breeds. Further downstream pathway and network analyses were conducted afterwards. The results uncovered the association of the most important DEGs such as CAV1, ALB, and SOCS3 with cellular signaling pathways of lipids metabolism. It seems that the SOCS3 gene plays an important role in the differential deposition of lipid in the tails of two phenotypically different sheep breeds. Although the detail of gene expression in the tail and subcutaneous tissues of two morphologically different breeds was decoded here, to fully understand how differential expression of the SOCS3 gene affects the fat synthesis, further studies are needed.

Single-cell RNA-seq in diabetic foot ulcer wound healing.

Diabetic foot ulcer (DFU) is a chronic and serious complication of diabetes mellitus. It is mainly caused by hyperglycaemia, diabetic peripheral vasculopathy and diabetic peripheral neuropathy. These conditions result in ulceration of foot tissues and chronic wounds. If left untreated, DFU can lead to amputation or even endanger the patient's life. Single-cell RNA sequencing (scRNA-seq) is a technique used to identify and characterise transcriptional subpopulations at the single-cell level. It provides insight into cellular function and the molecular drivers of disease. The objective of this paper is to examine the subpopulations, genes and molecules of cells associated with chronic wounds of diabetic foot by using scRNA-seq. The paper aims to explore the wound-healing mechanism of DFU from three aspects: inflammation, angiogenesis and extracellular matrix remodelling. The goal is to gain a better understanding of the mechanism of DFU wound healing and identify possible DFU therapeutic targets, providing new insights for the application of DFU personalised therapy.

Integrated physio-biochemistry and RNA-seq revealed the mechanism underlying biochar-mediated alleviation of compound heavy metals (Cd, Pb, As) toxicity in cotton.

Biochar has been recognised as an efficacious amendment for the remediation of compound heavy metal contamination in soil. However, the molecular mechanism of biochar-mediated tolerance to compound heavy metal toxicity in cotton is unknown. The objective of this research was to investigate the positive impact of biochar (10 g·kg-1) on reducing damage caused by compound heavy metals (Cd, Pb, and As) in cotton (Gossypium hirsutum L.). The results revealed that biochar reduced Cd concentrations by 24.9 % (roots), and decreased Pb concentrations by 37.1 % (roots) and 59.53 % (stems). Biochar maintained ionic homoeostasis by regulating the expression of metal transporter proteins such as ABC, HIPP, NRAMP3, PCR, and ZIP, and genes related to the carbon skeleton and plasma membrane. Biochar also downregulated genes related to photosynthesis, thereby increasing photosynthesis. Biochar re-established redox homoeostasis in cotton by activating signal transduction, which regulated the activity of the enzymes POD, SOD, and CAT activity; and the expression of related genes. This research revealed the molecular mechanism by which biochar confers resistance to the harmful effects of compound heavy metal toxicity in cotton. The application of biochar as a soil amendment to neutralise the toxicity of compound heavy metals is recommended for cash crop production.

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.

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.

Protocol for genome-scale differential flux analysis to interrogate metabolic differences from gene expression data.

Deciphering the functional differences between diseased and healthy cells requires understanding the alterations in biochemical flux patterns. We present a genome-scale differential flux analysis (GS-DFA) protocol to elucidate these metabolic disparities by integrating condition-specific gene expression data into the human genome-scale metabolic model (humanGEM). In this protocol, we describe the steps to normalize and integrate data into the humanGEM and analyze differential flux across the biochemical network between diseased and healthy cells. For complete details on the use and execution of this protocol, please refer to Nanda et al.1.

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.

GLABRA2 transcription factor integrates arsenic tolerance with epidermal cell fate determination.

Arsenic poses a global threat to living organisms, compromising crop security and yield. Limited understanding of the transcriptional network integrating arsenic-tolerance mechanisms with plant developmental responses hinders the development of strategies against this toxic metalloid. Here, we conducted a high-throughput yeast one-hybrid assay using as baits the promoter region from the arsenic-inducible genes ARQ1 and ASK18 from Arabidopsis thaliana, coupled with a transcriptomic analysis, to uncover novel transcriptional regulators of the arsenic response. We identified the GLABRA2 (GL2) transcription factor as a novel regulator of arsenic tolerance, revealing a wider regulatory role beyond its established function as a repressor of root hair formation. Furthermore, we found that ANTHOCYANINLESS2 (ANL2), a GL2 subfamily member, acts redundantly with this transcription factor in the regulation of arsenic signaling. Both transcription factors act as repressors of arsenic response. gl2 and anl2 mutants exhibit enhanced tolerance and reduced arsenic accumulation. Transcriptional analysis in the gl2 mutant unveils potential regulators of arsenic tolerance. These findings highlight GL2 and ANL2 as novel integrators of the arsenic response with developmental outcomes, offering insights for developing safer crops with reduced arsenic content and increased tolerance to this hazardous metalloid.