High-throughput Transcriptome Research Articles

Multi-well plate lid for single-step pooling of 96 samples for high-throughput barcode-based sequencing

High-throughput transcriptomics is of increasing fundamental biological and clinical interest. The generation of molecular data from large collections of samples, such as biobanks and drug libraries, is boosting the development of new biomarkers and treatments. Focusing on gene expression, the transcriptomic market exploits the benefits of next-generation sequencing (NGS), leveraging RNA sequencing (RNA-seq) as standard for measuring genome-wide gene expression in biological samples. The cumbersome sample preparation, including RNA extraction, conversion to cDNA and amplification, prevents high-throughput translation of RNA-seq technologies. Bulk RNA barcoding and sequencing (BRB-seq) addresses this limitation by enabling sample preparation in multi-well plate format. Sample multiplexing combined with early pooling into a single tube reduces reagents consumption and manual steps. Enabling simultaneous pooling of all samples from the multi-well plate into one tube, our technology relies on smart labware: a pooling lid comprising fluidic features and small pins to transport the liquid, adapted to standard 96-well plates. Operated with standard fluidic tubes and pump, the system enables over 90% recovery of liquid in a single step in less than a minute. Large scale manufacturing of the lid is demonstrated with the transition from a milled polycarbonate/steel prototype into an injection molded polystyrene lid. The pooling lid demonstrated its value in supporting high-throughput barcode-based sequencing by pooling 96 different DNA barcodes directly from a standard 96-well plate, followed by processing within the single sample pool. This new pooling technology shows great potential to address medium throughput needs in the BRB-seq workflow, thereby addressing the challenge of large-scale and cost-efficient sample preparation for RNA-seq.Graphical abstract

Integrating high-throughput phenotypic profiling and transcriptomic analyses to predict the hepatosteatosis effects induced by per- and polyfluoroalkyl substances

Per- and polyfluoroalkyl substances (PFAS) is a large compound class (n > 12,000) that is extensively present in food, drinking water, and aquatic environments. Reduced serum triglycerides and hepatosteatosis appear to be the common phenotypes for different PFAS chemicals. However, the hepatosteatosis potential of most PFAS chemicals remains largely unknown. This study aims to investigate PFAS-induced hepatosteatosis using in vitro high-throughput phenotype profiling (HTPP) and high-throughput transcriptomic (HTTr) data. We quantified the in vitro hepatosteatosis effects and mitochondrial damage using high-content imaging, curated the transcriptomic data from the Gene Expression Omnibus (GEO) database, and then calculated the point of departure (POD) values for HTPP phenotypes or HTTr transcripts, using the Bayesian benchmark dose modeling approach. Our results indicated that PFAS compounds with fully saturated C-F bonds, sulfur- and nitrogen-containing functional groups, and a fluorinated carbon chain length greater than 8 have the potential to produce biological effects consistent with hepatosteatosis. PFAS primarily induced hepatosteatosis via disturbance in lipid transport and storage. The potency rankings of PFAS compounds are highly concordant among in vitro HTPP, HTTr, and in vivo hepatosteatosis phenotypes (ρ = 0.60–0.73). In conclusion, integrating the information from in vitro HTPP and HTTr analyses can accurately project in vivo hepatosteatosis effects induced by PFAS compounds.

Why do we age? The main signs of aging

Aging is a gradual and irreversible pathophysiological process. This is manifested in the decline of tissue and cell functions and a significant variety of pathologies associated with aging, including neurodegenerative diseases, cardiovascular diseases, metabolic disorders, increased risk of diseases of the skeleton and the immune system. Although modern medical advances have made a certain contribution to human health and greatly extended life expectancy, along with the aging of society, various chronic diseases are gradually emerging, which are the most important causes of disability and death of the elderly. Aim. The aim of the work is to analyze modern ideas in the field of causes and manifestations of aging processes for the possibility of developing a strategy to slow down its course. At the molecular and cellular level, aging is a complex biological process involving the gradual deterioration of various cellular and molecular processes in the body over time. The length of a person’s life is closely related to the decrease in the possibilities of repair and regeneration of tissues and organs. In response to stress at the molecular, cellular, and systemic levels, genetic, epigenetic, and environmental regulatory factors cause a decrease in the body’s physiological capabilities. They use complex molecular mechanisms that together contribute to aging. Molecular mechanisms (such as telomere shortening, accumulation of DNA damage, metabolic changes, and excessive free radical generation) strongly link various factors to the rate of aging. Collectively, these mechanisms inhibit cell proliferation, alter metabolism and gene expression, and induce high levels of free radicals, maintaining a senescent cell phenotype. Although the number of early senescent cells is low, they can limit the regenerative capacity of tissue stem cells and lead to the accumulation of cellular damage, thereby contributing to age-related diseases. Conclusions. Current advances in high-throughput genomics, transcriptomics, proteomics, and metabolomics enable the characterization and quantification of thousands of epigenetic markers, transcripts, proteins, metabolites, and can reveal general changes that occur with age in complex organisms at the molecular level. Thus, the integration of these molecular markers and related molecular mechanisms into a comprehensive assessment of biological age to prevent age-related functional decline and morbidity is becoming an increasingly pressing issue in medical science and should be implemented in practice as soon as possible.

Color components determination and full-length comparative transcriptomic analyses reveal the potential mechanism of carotenoid synthesis during Paphiopedilum armeniacum flowering.

Paphiopedilum armeniacum (P. armeniacum), an ornamental plant native to China, is known for its distinctive yellow blossoms. However, the mechanisms underlying P. armeniacum flower coloration remain unclear. We selected P. armeniacum samples from different flowering stages and conducted rigorous physicochemical analyses. The specimens were differentiated based on their chemical properties, specifically their solubilities in polar solvents. This key step enabled us to identify the main metabolite of flower color development of P. armeniacum, and to complete the identification by High-performance liquid chromatography (HPLC) based on the results. Additionally, we employed a combined approach, integrating both third-generation full-length transcriptome sequencing and second-generation high-throughput transcriptome sequencing, to comprehensively explore the molecular components involved. We combined physical and chemical analysis with transcriptome sequencing to reveal that carotenoid is the main pigment of P. armeniacum flower color. Extraction colorimetric method and HPLC were used to explore the characteristics of carotenoid accumulation during flowering. We identified 28 differentially expressed carotenoid biosynthesis genes throughout the flowering process, validated their expression through fluorescence quantification, and discovered 19 potential positive regulators involved in carotenoid synthesis. Among these candidates, three RCP2 genes showed a strong potential for governing the PDS and ZDS gene families. In summary, our study elucidates the fundamental mechanisms governing carotenoid synthesis during P. armeniacum flowering, enhancing our understanding of this process and providing a foundation for future research on the molecular mechanisms driving P. armeniacum flowering.

Uncovering lupus nephritis-specific genes and the potential of TNFRSF17-targeted immunotherapy: a high-throughput sequencing study.

Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE). This study aimed to identify LN specific-genes and potential therapeutic targets. We performed high-throughput transcriptome sequencing on peripheral blood mononuclear cells (PBMCs) from LN patients. Healthy individuals and SLE patients without LN were used as controls. To validate the sequencing results, qRT-PCR was performed for 5 upregulated and 5 downregulated genes. Furthermore, the effect of the TNFRSF17-targeting drug IBI379 on patient plasma cells and B cells was evaluated by flow cytometry. Our analysis identified 1493 and 205 differential genes in the LN group compared to the control and SLE without LN groups respectively, with 70 genes common to both sets, marking them as LN-specific. These LN-specific genes were significantly enriched in the 'regulation of biological quality' GO term and the cell cycle pathway. Notably, several genes including TNFRSF17 were significantly overexpressed in the kidneys of both LN patients and NZB/W mice. TNFRSF17 levels correlated positively with urinary protein levels, and negatively with complement C3 and C4 levels in LN patients. The TNFRSF17-targeting drug IBI379 effectively induced apoptosis in patient plasma cells without significantly affecting B cells. Our findings suggest that TNFRSF17 could serve as a potential therapeutic target for LN. Moreover, IBI379 is presented as a promising treatment option for LN.

Controlling arbovirus infection: high-throughput transcriptome and proteome insights.

Arboviruses pose a significant threat to public health globally, demanding innovative approaches for their control. For this, a better understanding of the complex web of interactions established in arbovirus-infected mosquitoes is fundamental. High-throughput analyses allow a genome-wide view of arbovirus-induced alterations at different gene expression levels. This review provides a comprehensive perspective into the current literature in transcriptome and proteome landscapes in mosquitoes infected with arboviruses. It also proposes a coordinated research effort to define the critical nodes that determine arbovirus infection and transmission.

Extracting high-quality RNA from formaldehyde-fixed naturally aged neuromusculoskeletal tissues.

Modern approaches to discovering molecular mechanisms and validating treatments for age-related neuromusculoskeletal dysfunction typically rely on high-throughput transcriptome analysis. Previously harvested and fixed tissues offer an incredible reservoir of untapped molecular information. However, obtaining RNA from such formaldehyde-fixed neuromusculoskeletal tissues, especially fibrotic aged tissues, is technically challenging and often results in RNA degradation, chemical modification and yield reduction, prohibiting further analysis. Therefore, we developed a protocol to extract high-quality RNA from formaldehyde-fixed brain, cartilage, muscle and peripheral nerve isolated from naturally aged mice. Isolated RNA produced reliable gene expression data comparable to fresh and flash-frozen tissues and was sensitive enough to detect age-related changes, making our protocol valuable to researchers in the field of aging.

Molecular mechanism of Serratia marcescens Bizio infection in Reticulitermes chinensis Snyder based on full-length SMRT transcriptome sequencing.

Reticulitermes chinensis Snyder is an important pest in forestry and construction and is widely distributed in China. We found that Serratia marcescens Bizio strain SM1 has insecticidal activity to R. chinensis, but the pathogenic mechanism of SM1 to R. chinensis is not clear. Therefore, full-length transcriptome sequencing was performed on R. chinensis infected with SM1 and the control group. A total of 230 differentially expressed genes were identified by comparing SM1 infection group and the control group, among which 103 were downregulated and 127 were upregulated. We found downregulated genes in nine metabolic pathway categories, among which carbohydrate metabolism had the most downregulated genes, followed by energy metabolism and amino acid metabolism. We also found that some downregulated genes were related to pattern recognition receptors, cellular immunity, and humoral immunity, indicating that R. chinensis immunity was negatively affected by SM1 infection. In addition, some genes in signal transduction and genetic information processing pathways were downregulated. In this study, high-throughput full-length transcriptome analysis was used to analyse the pathogenic mechanism of SM1 to R. chinensis. The results of this study provide useful information for exploring the relationship between SM1 and R. chinensis, and provide theoretical support for the future application of SM1 and the prevention and treatment of R. chinensis.

Uncovering the potential molecular mechanism of liraglutide to alleviate the effects of high glucose on myoblasts based on high-throughput transcriptome sequencing technique

BackgroundMyoblasts play an important role in muscle growth and repair, but the high glucose environment severely affects their function. The purpose of this study is to explore the potential molecular mechanism of liraglutide in alleviating the effects of high glucose environments on myoblasts.MethodsMTT, western blot, and ELISA methods were used to investigate the role of liraglutide on C2C12 myoblasts induced by high glucose. The high-throughput transcriptome sequencing technique was used to sequence C2C12 myoblasts from different treated groups. The DESeq2 package was used to identify differentially expressed-mRNAs (DE-mRNAs). Then, functional annotations and alternative splicing (AS) were performed. The Cytoscape-CytoHubba plug-in was used to identify multicentric DE-mRNAs.ResultsThe MTT assay results showed that liraglutide can alleviate the decrease of myoblasts viability caused by high glucose. Western blot and ELISA tests showed that liraglutide can promote the expression of AMPKα and inhibit the expression of MAFbx, MuRF1 and 3-MH in myoblasts. A total of 15 multicentric DE-mRNAs were identified based on the Cytoscape-CytoHubba plug-in. Among them, Top2a had A3SS type AS. Functional annotation identifies multiple signaling pathways such as metabolic pathways, cytokine-cytokine receptor interaction, cAMP signaling pathway and cell cycle.ConclusionLiraglutide can alleviate the decrease of cell viability and degradation of muscle protein caused by high glucose, and improves cell metabolism and mitochondrial activity. The molecular mechanism of liraglutide to alleviate the effect of high glucose on myoblasts is complex. This study provides a theoretical basis for the clinical effectiveness of liraglutide in the treatment of skeletal muscle lesions in diabetes.

LncRNA LBX2-AS1 promotes proliferation and migratory capacity of clear cell renal cell carcinoma through mitophagy

BackgroundLong non-coding RNAs (lncRNAs) have been extensively investigated in the field of cancer, among which, lncRNA ladybird homeobox 2-antisense RNA 1 (LBX2-AS1) has been demonstrated to exert carcinogenic effects on a variety of malignancies. However, the biological functions of LBX2-AS1 in clear cell renal cell carcinoma (ccRCC) have not been explicitly elucidated.MethodsArraystar lncRNA chip and qRT-PCR verify the expression of LncRNA LBX2-AS1 in ccRCC. CCK-8 assay and cell cloning assay were used to assess the proliferative capacity of ccRCC cells. Migration abilities were quantified by scratch assay and transwell assay. Potential molecular signaling pathways were determined by high-throughput whole transcriptomics analysis. WB analysis was performed to validate the relationship between LBX2-AS1 and key molecules of mitophagy pathway. The effect of LBX2-AS1 on mitophagy was observed by laser confocal microscopy. Rescue experiments further validated the role of downstream gene FOXO3A in the LBX2-AS1 signaling pathway. Finally, the authentic effect of LBX2-AS1 was verified in vivo.ResultsLncRNA LBX2-AS1 was over expressed in ccRCC tissues and could enhance the proliferation and migration of ccRCC cells. Autophagic pathway was identified as a possible mechanism involved in the oncogenic effect of LBX2-AS1. Mitophagy levels were observed in LBX2-AS1 low-expressing cells through laser confocal microscopy. Knockdown of LBX2-AS1 significantly elevated mitophagy levels as observed using laser confocal microscopy and led to FOXOA3 decreasing in and BNIP3L and LC3 enrichment. Meanwhile, LBX2-AS1 knocking down dampened the proliferation of ccRCC cells in vivo.

SVM-Root: Identification of Root-Associated Proteins in Plants by Employing the Support Vector Machine with Sequence-Derived Features

Background: Root is a desirable trait for modern plant breeding programs, as the roots play a pivotal role in the growth and development of plants. Therefore, identification of the genes governing the root traits is an essential research component. With regard to the identification of root-associated genes/proteins, the existing wet-lab experiments are resource intensive and the gene expression studies are species-specific. Thus, we proposed a supervised learning-based computational method for the identification of root-associated proteins. Method: The problem was formulated as a binary classification, where the root-associated proteins and non-root-associated proteins constituted the two classes. Four different machine learning algorithms such as support vector machine (SVM), extreme gradient boosting, random forest, and adaptive boosting were employed for the classification of proteins of the two classes. Sequence-derived features such as AAC, DPC, CTD, PAAC, and ACF were used as input for the learning algorithms. Results: The SVM achieved higher accuracy with the 250 selected features of AAC+DPC+CTD than that of other possible combinations of feature sets and learning algorithms. Specifically, SVM with the selected features achieved overall accuracies of 0.74, 0.73, and 0.73 when evaluated with single 5-fold cross-validation (5F-CV), repeated 5F-CV, and independent test set, respectively. Conclusions: A web-enabled prediction tool SVM-Root (https://iasri-sg.icar.gov.in/svmroot/) has been developed for the computational prediction of the root-associated proteins. Being the first of its kind, the proposed model is believed to supplement the existing experimental methods and high throughput GWAS and transcriptome studies.

Current Advances in Single-Cell RNA Sequencing in Diabetic Retinopathy

With the development of high-throughput sequencing technology, humans have been able to conduct large-scale analysis of DNA sequence, chromatin structure, RNA transcripts, proteins, metabolites and other genomes and their products. Traditional high-throughput transcriptome sequencing techniques based on tissue samples (RNA Seq) are used to centrally sequence thousands of cells, each of which varies in size, protein levels, and mRNA expression transcription. Measuring the average of multiple cells grouped together can mask significant differences in gene expression between cells. Single-cell RNA sequencing is a technique for high-throughput sequencing of the genome, transcriptome, and epigenome at the single-cell level. Based on the single cell RNA transcription map, the intraocular cells can be distinguished from other subtypes, and the different subtypes are found to have significant differences in morphology, physiology and specific expression genes. In recent years, the application of single-cell RNA sequencing technology in the field of ophthalmology has increased, mainly including cell type and cell subtype identification, retinal development process, and eye disease research. This paper systematically summarized the latest application of single-cell sequencing technology in the field of diabetic retinopathy, and summarized marker genes and potential therapeutic targets. It has guiding significance for the clinical treatment of diabetic retinopathy.

A novel lncRNA TCONS_00071187 upregulated by activated GSK3β promotes high glucose-induced mesangial cell proliferation in the diabetic nephropathy.

Inhibiting mesangial cell proliferation is one of the strategies to control the early progression of diabetic nephropathy (DN). GSK3β is closely related to cell apoptosis as well as the development of DN, but whether it acts on the proliferation of mesangial cells is unclear. This study aimed to elucidate the role and mechanism of GSK3β-mediated lncRNA in high glucose-induced mesangial cell proliferation. HBZY-1 cells were used to establish the cell model of DN. The automatic cell counter was applied to assess cell proliferation. Flow cytometry was used to detect cell apoptosis and intracellular ROS levels. High-throughput transcriptomics sequencing was performed to detect the different expressions of long noncoding RNAs (lncRNAs) in the cell model of DN after knocking down the expression of GSK3β by the transfection of siRNA. The expression of RNA was detected by real-time PCR. In the cell model of DN using HBZY-1 cells, cell proliferation was enhanced accompanied by GSK3β activation and elevated apoptosis rate and reactive oxygen species (ROS) levels. A panel of novel lncRNAs, which were differentially expressed after GSK3β knockdown in the cell model of DN, were identified by high-throughput transcriptomics sequencing. Among them, the expression of TCONS_00071187 was upregulated under high glucose conditions while the knockdown of the GSK3β expression led to the downregulation of TCONS_00071187. The knockdown of TCONS_00071187 resulted in reduced mesangial cell proliferation, and decreased apoptosis rates and ROS levels. In conclusion, GSK3β promoted mesangial cell proliferation by upregulating TCONS_00071187, which led to enhanced ROS production under high glucose conditions in the cell model of DN. This study revealed the role of GSK3β medicated lncRNAs in the development of DN.

Genome-wide identification of the B3 transcription factor family in pepper (Capsicum annuum) and expression patterns during fruit ripening

In plants, B3 transcription factors play important roles in a variety of aspects of their growth and development. While the B3 transcription factor has been extensively identified and studied in numerous species, there is limited knowledge regarding its B3 superfamily in pepper. Through the utilization of genome-wide sequence analysis, we identified a total of 106 B3 genes from pepper (Capsicum annuum), they are categorized into four subfamilies: RAV, ARF, LAV, and REM. Chromosome distribution, genetic structure, motif, and cis-acting element of the pepper B3 protein were analyzed. Conserved gene structure and motifs outside the B3 domain provided strong evidence for phylogenetic relationships, allowing potential functions to be deduced by comparison with homologous genes from Arabidopsis. According to the high-throughput transcriptome sequencing analysis, expression patterns differ during different phases of fruit development in the majority of the 106 B3 pepper genes. By using qRT-PCR analysis, similar expression patterns in fruits from various time periods were discovered. In addition, further analysis of the CaRAV4 gene showed that its expression level decreased with fruit ripening and located in the nucleus. B3 transcription factors have been genome-wide characterized in a variety of crops, but the present study is the first genome-wide analysis of the B3 superfamily in pepper. More importantly, although B3 transcription factors play key regulatory roles in fruit development, it is uncertain whether B3 transcription factors are involved in the regulation of the fruit development and ripening process in pepper and their specific regulatory mechanisms because the molecular mechanisms of the process have not been fully explained. The results of the study provide a foundation and new insights into the potential regulatory functions and molecular mechanisms of B3 genes in the development and ripening process of pepper fruits, and provide a solid theoretical foundation for the enhancement of the quality of peppers and their selection and breeding of high-yield varieties.

Differential analysis of transcriptome of psychrophilic bacteria under different culture temperatures.

Psychrophilic bacteria can survive in a unique living environment. To explore the mechanism of low temperature adaptation and the physiological function of thermophilic metabolic genes. Serratia marcescens strain F13 stored in microbial laboratory was cultured at 5∘C, 10∘C and 25∘C respectively, and the obtained strains were sequenced by high-throughput transcriptome. Serratia marcescens strain CAV1761 was used as the reference strain. The data produced by transcriptome sequencing were statistically analyzed by biostatistics software such as soapnuke, soap and edger. The differentially expressed genes were found based on the gene expression, and analyzed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The results showed that there were 718 differential genes in F13-10 vs F13-5 comparison group, 1614 differential genes in F13-25 vs F13-5 comparison group and 1636 differential genes in F13-25 vs F13-10 comparison group. GO function enrichment analysis showed that the GO term mainly enriched by different genes in the three comparison groups was mostly related to the migration and transport of cellular or subcellular components, cell localization and transmembrane transporter activity, as well as cilia or flagella dependent cell movement. In the enrichment analysis of KEGG pathway, the three comparison groups all enriched the largest number of differential genes in the branch pathway of KEGG metabolism, followed by the branch pathway of environmental information processing. In F13-10 vs F13-5, the differential genes were mainly concentrated in 20 pathways such as ATP-binding cassette transport (ABC) transporters, thiamine metabolism and flagella assembly; In F13-25 vs F13-5, the differential genes are mainly concentrated in 20 pathways, such as (ABC) transporters, arginine and proline metabolism, two-component system and so on; In F13-25 vs F13-10, the differential genes are mainly concentrated in 20 pathways such as various types of glycan synthesis, two-component system and arginine metabolism.

Transcriptome Analysis and Identification of Cadmium-Induced Oxidative Stress Response Genes in Different Meretrix meretrix Developmental Stages.

Cadmium (Cd) is one of the major pollutants in the aquatic environment, and it can easily accumulate in aquatic animals and result in toxic effects by changing the metabolism of the body, causing a serious impact on the immune system, reproductive system, and the development of offspring. The clam Meretrix meretrix is one of the commercially important species that is cultivated in large-scale aquaculture in China. To elucidate the underlying molecular mechanisms of Cd2+ in the developmental processes, fertilized eggs and larvae of M. meretrix at different developmental stages were exposed to Cd2+ (27.2 mg L-1 in natural seawater) or just natural seawater without Cd2+ (control), and high-throughput transcriptome sequencing and immunohistochemistry techniques were used to analyze the toxic effects of Cd on larvae at different early developmental stages. The results revealed 31,914 genes were differentially expressed in the different stages of M. meretrix development upon treatment with Cd2+. Ten of these genes were differentially expressed in all stages of development examined, but they comprised only six unigenes (CCO, Ndh, HPX, A2M, STF, and pro-C3), all of which were related to the oxidative stress response. Under Cd exposure, the expression levels of CCO and Ndh were significantly upregulated in D-shaped and pediveliger larvae, while pro-C3 expression was significantly upregulated in the fertilized egg, D-shaped larva, and pediveliger. Moreover, HPX, A2M, and STF expression levels in the fertilized egg and pediveliger larvae were also significantly upregulated. In contrast, CCO, Ndh, HPX, A2M, STF, and pro-C3 expression levels in the postlarva were all downregulated under Cd exposure. Besides the genes with changes in expression identified by the transcriptome, the expression of two other oxidative stress-related genes (MT and Nfr2) was also found to change significantly in the different developmental stages of M. meretrix upon Cd exposure, confirming their roles in combating oxidative stress. Overall, the findings of this study indicated that Cd would interfere with cellular respiration, ion transport, and immune response through inducing oxidative stress, and changes in the expression of oxidative stress-related genes might be an important step for M. meretrix to deal with the adverse effects of Cd at different stages of its development.

Mechanism of IL-17 Signaling Pathway in Spleen Inflammatory Response Induced by Altitude Hypoxia in Mice

To explore the mechanism of spleen tissue inflammatory response induced by altitude hypoxia in mice. C57BL/6 mice were randomly assigned to a plain, i.e., low-altitude, normoxia group and an altitude hypoxia group, with 5 mice in each group. In the plain normoxia group, the mice were kept in a normoxic environment at the altitude of 400 m above sea level (with an oxygen concentration of 19.88%). The mice in the altitude hypoxia group were kept in an environment at the altitude of 4200 m above sea level (with an oxygen concentration of 14.23%) to establish the animal model of altitude hypoxia. On day 30, spleen tissues were collected to determine the splenic index. HE staining was performed to observe the histopathological changes in the spleen tissues of the mice. Real time fluorogenic quantitative PCR (RT-qPCR) and Western blot were conducted to determine the mRNA and protein expressions of interleukin (IL)-6, IL-12, and IL-1β in the spleen tissue of the mice. High-throughput transcriptome sequencing was performed with RNA sequencing (RNA-seq). KEGG enrichment analysis was performed for the differentially expressed genes (DEGs). The DEGs in the key pathways were verified by RT-qPCR. Compared with the plain normoxia group, the mice exposed to high-altitude hypoxic environment had decreased spleen index (P<0.05) and exhibited such pathological changes as decreased white pulp, enlarged germinal center, blurred edge, and venous congestion. The mRNA and protein expression levels of IL-6, IL-12, and IL-1β in the spleen tissue of mice in the altitude hypoxia group were up-regulated (P<0.05). According to the results of transcriptome sequencing and KEGG pathway enrichment analysis, 4218 DEGs were enriched in 178 enrichment pathways (P<0.05). DEGs were significantly enriched in multiple pathways associated with immunity and inflammation, such as T cell receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway (P<0.05) in the spleen of mice exposed to high-altitude hypoxic environment. Among them, IL-17 signaling pathway and the downstream inflammatory factors were highly up-regulated (P<0.05). Compared with the plain normoxia group, the mRNA expression levels of key genes in the IL-17 signaling pathway, including IL-17, IL-17R, and mitogen-activated protein kinase genes (MAPKs), and the downstream inflammatory factors, including matrix metallopeptidase 9 (MMP9), S100 calcium binding protein A8 gene (S100A8), S100 calcium binding protein A9 gene (S100A9), and tumor necrosis factor α (TNF-α), were up-regulated or down-regulated (P<0.05) in the altitude hypoxia group. According to the validation of RT-qPCR results, the mRNA expression levels of DEGs were consistent with the RNA-seq results. Altitude hypoxia can induce inflammatory response in the mouse spleen tissue by activating IL-17 signaling pathway and promoting the release of downstream inflammatory factors.

Towards high-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton.

Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we applied a microarray single-cell sequencing (MASC-seq) technology, that generates microscope images of cells in parallel with capturing their transcriptomes, on three species representing important plankton groups with different cell structures; the ciliate Tetrahymena thermophila, the diatom Phaeodactylum tricornutum, and the dinoflagellate Heterocapsa sp. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating "microbial dark matter", although further optimizations are necessary to increase the signal-to-noise ratio.

Identification and characterization of multipotential stem cells in immortalized normal ovarian surface epithelial cells.

The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal epithelial cells that experience repetitive ovulatory rupture and subsequent repair. However, the characteristics of human immortalized ovarian surface epithelial cells (IOSE80) remain elusive. This study aims to determine whether IOSE80 cells have the characteristics of stem cell proliferation and multilineage differentiation and their application in regenerative medicine. IOSE80 cells are sequenced by high-throughput transcriptome analysis, and 5 sets of public data are used to compare the differences between IOSE80 cells and bone marrow mesenchymal stem cells, pluripotent stem cells, and oocytes in transcriptome profiling. The IOSE80 cells present a cobblestone-like monolayer and express the epithelial cell marker KRT18; the stem cell markers IFITM3, ALDH1A1, and VIM; lowly express stem cell marker LGR5 and germ cell markers DDX4 and DAZL. In addition, the GO terms "regulation of stem cell proliferation", "epithelial cell proliferation", etc., are significantly enriched ( P<0.05). IOSE80 cells have the potential to act as mesenchymal stem cells to differentiate into adipocytes with lipid droplets, osteoblasts, and chondroblasts in vitro. IOSE80 cells express pluripotent stem cell markers, including OCT4, SSEA4, TRA-1-60, and TRA-1-81, and they can be induced into three germ layers in vitro. IOSE80 cells also form oocyte-like cells in vitro and in vivo. In addition, IOSE80 cells exhibit robust proliferation, migration, and ovarian repair functions after in vivo transplantation. This study demonstrates that IOSE80 cells have the characteristics of pluripotent/multipotent stem cells, indicating their important role in tissue engineering and regenerative medicine.

High-Throughput Transcriptomics of Water Extracts Detects Reductions in Biological Activity with Water Treatment Processes.

The presence of numerous chemical contaminants from industrial, agricultural, and pharmaceutical sources in water supplies poses a potential risk to human and ecological health. Current chemical analyses suffer from limitations, including chemical coverage and high cost, and broad-coverage in vitro assays such as transcriptomics may further improve water quality monitoring by assessing a large range of possible effects. Here, we used high-throughput transcriptomics to assess the activity induced by field-derived water extracts in MCF7 breast carcinoma cells. Wastewater and surface water extracts induced the largest changes in expression among cell proliferation-related genes and neurological, estrogenic, and antibiotic pathways, whereas drinking and reclaimed water extracts that underwent advanced treatment showed substantially reduced bioactivity on both gene and pathway levels. Importantly, reclaimed water extracts induced fewer changes in gene expression than laboratory blanks, which reinforces previous conclusions based on targeted assays and improves confidence in bioassay-based monitoring of water quality.