High-throughput mRNA Sequencing Research Articles

The role of AKR1B10 in osteogenic differentiation of mesenchymal stem cells and atrophic nonunion

Atrophic nonunion is a chronic disease without effective medications. Here, high-throughput mRNA sequencing was used to explore the novel targets in atrophic nonunion. AKR1B10, a member of aldo-keto reductase family 1, is upregulated in atrophic nonunion tissues. There are currently no studies to reveal the role of AKR1B10 in atrophic nonunion. We used rat bone marrow-derived mesenchymal stem cells (BMSCs) to explore the effect of AKR1B10 on the osteogenic differentiation and autophagy. In vivo, we implanted collagen sponges loaded with LV-shAKR1B10-transduced BMSCs into rat fractured femurs to explore the role of AKR1B10 in fracture healing. The results showed that AKR1B10 reduced the activity of ALP, suppressed the expression of COL1A1, RUNX2 and OCN, and inhibited calcification deposition in osteogenically differentiated BMSCs. AKR1B10 reduced the expression of LC3II, decreased the number of autophagosomes, and promoted the expression of p62. In addition, the promoting effect of AKR1B10 knockdown on osteogenic differentiation of BMSCs was attenuated by 3-MA treatment. Implantation of collagen sponges found that knockdown of AKR1B10 promoted bone fracture healing. In conclusion, AKR1B10 inhibited the osteogenic differentiation and autophagy, and delayed the bone fracture healing. These results provide a new perspective on revealing the role of AKR1B10 in nonunion and may also provide a new therapeutic target for the treatment of nonunion.

RNA sequencing reveals novel molecular mechanisms of endometriosis lesion development in a mouse model.

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

Integrated transcriptomic and metabolic analyses reveal the early response mechanism of Pinus tabulaeformis to pine wood nematodes

Pine wilt disease (PWD) is a devastating disease of pine trees caused by the pine wood nematode (Bursapherenchus xylophilus, PWN). To study how Pinus tabulaeformis responds to PWD infection, we collected 3-year-old P. tabulaeformis seedlings at 2 days, 5 days, and 8 days after being infected with B. xylophilus. We identified genes and metabolites early responding to infection using transcriptome and metabolomic data obtained by high-throughput mRNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays, respectively. The following results were obtained: (1) After inoculation with PWN, the average number of days taken for 3-year-old P. tabulaeformis seedlings to develop symptoms was 8 days. (2) Combined transcriptome and metabolome analysis revealed that phenylpropanoid biosynthesis and flavonoid biosynthesis are critically important pathways for P. tabulaeformis to respond to PWD. (3) The response of P. tabulaeformis to stress was mainly through positive regulation of gene expression, including some key genes related to plant hormones or transcription factors that have been widely studied. Genes related to pathways such as photosynthesis, plant-pathogen interactions, and DNA replication were downregulated. (4) Terpenoid biosynthesis genes involved during the development of pine wilt disease. This study demonstrated the defence and pathogenic mechanisms of P. tabulaeformis against PWD, providing a reference for the early diagnosis of PWD.

SEV-mediated lipid droplets transferred from bone marrow adipocytes promote ferroptosis and impair osteoblast function

Osteoporosis is linked to increased bone marrow adipocyte (BMAd) proliferation, which displaces bone-forming cells and alters the local environment. The impact of BMAd lipid droplets on bone health and osteoblast function remains unclear. This study investigates the interplay between BMAd-derived lipid droplets and osteoblast functionality, focusing on ferroptosis pathways. Osteoblast cultures were treated with conditioned media from adipocytes to simulate in vivo conditions. High-throughput mRNA sequencing and Western blot analysis were used to profile changes in gene expression and protein levels related to ferroptosis, oxidative phosphorylation, and osteogenic markers. Cellular assays assessed the direct impact of lipid droplets on osteoblast activity. Results showed that osteoblasts exposed to adipocyte-conditioned media had increased intracellular lipid droplet accumulation, upregulation of ferroptosis-related genes and proteins, and downregulation of oxidative phosphorylation and osteoblast differentiation markers. Treatment with ferroptosis inhibitors reversed the detrimental effects on osteoblasts, indicating the functional relevance of this pathway in osteoporosis. BMAd-derived lipid droplets contribute to osteoblast dysfunction through ferroptosis induction. Inhibiting ferroptosis could preserve osteoblast function and combat osteoporosis-related bone issues, suggesting that modulating lipid metabolism and redox balance in bone cells may be promising for future treatments.

Genome-Wide Identification of microRNAs Associated with Starch Biosynthesis and Endosperm Development in Foxtail Millet.

Foxtail millet is one of the oldest crops, and its endosperm contains up to 70% of starch. Grain filling is an important starch accumulation process associated with foxtail millet yield and quality. However, the molecular mechanisms of grain filling in foxtail millet are relatively unclear. Here, we investigate the genes and regulated miRNAs associated with starch synthesis and metabolism in foxtail millet using high-throughput small RNA, mRNA and degradome sequencing. The regulation of starch synthesis and quality is carried out mainly at the 15 DAA to 35 DAA stage during grain filling. The DEGs between waxy and non-waxy foxtail millet were significant, especially for GBSS. Additionally, ptc-miR169i_R+2_1ss21GA, fve-miR396e_L-1R+1, mtr-miR162 and PC-5p-221_23413 regulate the expression of genes associated with the starch synthesis pathway in foxtail millet. This study provides new insights into the molecular mechanisms of starch synthesis and quality formation in foxtail millet.

Systematic bioinformatics analysis reveals the role of shikonin in blocking colon cancer progression by identifying senescence-induced genes.

Shikonin, a naturally occurring naphthoquinone compound extracted from comfrey plants, has antitumor, anti-inflammatory, and antimicrobial properties. Cell senescence plays a key role in preventing tumor progression. It is unclear whether shikonin has an effect on cell senescence in colon cancer. In the current study, we first determine the IC50 values of shikonin on colon cancer cell lines HT29 and HCT116. Then, we verified the inhibitory effects of shikonin on the proliferation and migration abilities of colon cancer cell lines HT29 and HCT116 using cell counting kit-8, colony formation, and wound healing assays. Next, we identified a series of potential targets using high-throughput mRNA sequencing and identified 210 upregulated and 296 downregulated genes. KEGG profiling revealed eight downregulated genes associated with cell senescence: CCNB3, IL-1α, CXCL8, CDKN2A, MYC, IGFBP3, SQSTM1, and GADD45G. Among them, CXCL8 and CDKN2A were associated with poor prognosis in patients with colon cancer, suggesting that their downregulation by shikonin could improve patient survival. Furthermore, SA-β-galactosidase staining revealed that the percentage of cellular senescence in colon cancer cells was significantly increased after shikonin treatment. Molecular docking revealed that shikonin suppressed colon cancer progression by blocking CXCL8 activity. Based on these findings, we deem that shikonin might induce senescence and exert antitumor activity in colon cancer cells by downregulating CDKN2A and CXCL8. This provides a new molecular mechanism and potential therapeutic target for shikonin to inhibit colon cancer progression.

Fuzheng Huayu recipe inhibits bleomycin-induced pulmonary fibrosis in rats by inhibiting M2 polarization of macrophages via the oxidative phosphorylation pathway

Fuzheng Huayu recipe (FZHYR) is a Chinese patent medicine for the treatment of fibrosis. The effects of FZHYR on pulmonary fibrosis and macrophage polarization were investigated in vitro. FZHYR inhibited pulmonary inflammation and fibrosis and M2 polarization of macrophages in bleomycin-induced pulmonary fibrosis (BPF) of rat model. Differentially expressed genes were screened by high-throughput mRNA sequencing and GSEA showed that oxidative phosphorylation (OXPHOS) was correlated with BPF. FZHYR inhibited expressions of Ndufa2 and Ndufa6 in lung tissues of BPF rats. These findings suggest that OXPHOS pathway serves as a possible target for pulmonary fibrosis therapy by FZHYR.

Alterations in tryptophan metabolism and de novo NAD+ biosynthesis within the microbiota-gut-brain axis in chronic intestinal inflammation.

Inflammatory bowel disease is an incurable and idiopathic disease characterized by recurrent gastrointestinal tract inflammation. Tryptophan metabolism in mammalian cells and some gut microbes comprise intricate chemical networks facilitated by catalytic enzymes that affect the downstream metabolic pathways of de novo nicotinamide adenine dinucleotide (NAD+) synthesis. It is hypothesized that a correlation exists between tryptophan de novo NAD+ synthesis and chronic intestinal inflammation. Transcriptome analysis was performed using high-throughput sequencing of mRNA extracted from the distal colon and brain tissue of Winnie mice with spontaneous chronic colitis and C57BL/6 littermates. Metabolites were assessed using ultra-fast liquid chromatography to determine differences in concentrations of tryptophan metabolites. To evaluate the relative abundance of gut microbial genera involved in tryptophan and nicotinamide metabolism, we performed 16S rRNA gene amplicon sequencing of fecal samples from C57BL/6 and Winnie mice. Tryptophan and nicotinamide metabolism-associated gene expression was altered in distal colons and brains of Winnie mice with chronic intestinal inflammation. Changes in these metabolic pathways were reflected by increases in colon tryptophan metabolites and decreases in brain tryptophan metabolites in Winnie mice. Furthermore, dysbiosis of gut microbiota involved in tryptophan and nicotinamide metabolism was evident in fecal samples from Winnie mice. Our findings shed light on the physiological alterations in tryptophan metabolism, specifically, its diversion from the serotonergic pathway toward the kynurenine pathway and consequential effects on de novo NAD+ synthesis in chronic intestinal inflammation. The results of this study reveal differential expression of tryptophan and nicotinamide metabolism-associated genes in the distal colon and brain in Winnie mice with chronic intestinal inflammation. These data provide evidence supporting the role of tryptophan metabolism and de novo NAD+ synthesis in IBD pathophysiology.

Transcriptomic Analysis Reveals the Effect of Urea on Metabolism of Nannochloropsis oceanica.

The eukaryotic microalga Nannochloropsis oceanica represents a promising bioresource for the production of biofuels and pharmaceuticals. Urea, a crucial nutrient for the photosynthetic N. oceanica, stimulates the accumulation of substances such as lipids, which influence growth and physiology. However, the specific mechanisms by which N. oceanica responds and adapts to urea addition remain unknown. High-throughput mRNA sequencing and differential gene expression analysis under control and urea-added conditions revealed significant metabolic changes. This involved the differential expression of 2104 genes, with 1354 being upregulated and 750 downregulated, resulting in the reprogramming of crucial pathways such as carbon and nitrogen metabolism, photosynthesis, and lipid metabolism. The results specifically showed that genes associated with photosynthesis in N. oceanica were significantly downregulated, particularly those related to light-harvesting proteins. Interestingly, urea absorption and transport may depend not only on specialized transport channels such as urease but also on alternative transport channels such as the ABC transporter family and the CLC protein family. In addition, urea caused specific changes in carbon and lipid metabolism. Genes associated with the Calvin cycle and carbon concentration mechanisms were significantly upregulated. In lipid metabolism, the expression of genes associated with lipases and polyunsaturated fatty acid synthesis was highly activated. Furthermore, the expression of several genes involved in the tricarboxylic acid cycle and folate metabolism was enhanced, making important contributions to energy supply and the synthesis and modification of genes and macromolecules. Our observations indicate that N. oceanica actively and dynamically regulates the redistribution of carbon and nitrogen after urea addition, providing references for further research on the effects of urea on N. oceanica.

Unraveling the molecular mechanisms of fish physiological response to freshwater salinization: A comparative multi-tissue transcriptomic study in a river polluted by potash mining

Freshwater salinization is an escalating global environmental issue that threatens freshwater biodiversity, including fish populations. This study aims to uncover the molecular basis of salinity physiological responses in a non-native minnow species (Phoxinus septimaniae x P. dragarum) exposed to saline effluents from potash mines in the Llobregat River, Barcelona, Spain. Employing high-throughput mRNA sequencing and differential gene expression analyses, brain, gills, and liver tissues collected from fish at two stations (upstream and downstream of saline effluent discharge) were examined. Salinization markedly influenced global gene expression profiles, with the brain exhibiting the most differentially expressed genes, emphasizing its unique sensitivity to salinity fluctuations. Pathway analyses revealed the expected enrichment of ion transport and osmoregulation pathways across all tissues. Furthermore, tissue-specific pathways associated with stress, reproduction, growth, immune responses, methylation, and neurological development were identified in the context of salinization. Rigorous validation of RNA-seq data through quantitative PCR (qPCR) underscored the robustness and consistency of our findings across platforms. This investigation unveils intricate molecular mechanisms steering salinity physiological response in non-native minnows confronting diverse environmental stressors. This comprehensive analysis sheds light on the underlying genetic and physiological mechanisms governing fish physiological response in salinity-stressed environments, offering essential knowledge for the conservation and management of freshwater ecosystems facing salinization.

Differential gene expression and miRNA regulatory network in coronary slow flow

Coronary slow flow (CSF) is characterized by slow progression of coronary angiography without epicardial stenosis. The aim of this study was to explore the potential biomarkers and regulatory mechanism for CSF. Peripheral blood mononuclear cells from 3 cases of CSF and 3 healthy controls were collected for high-throughput sequencing of mRNA and miRNA, respectively. The differentially expressed mRNAs (DE-mRNAs) and miRNAs (DE-miRNAs) was identified. A total of 117 DE-mRNAs and 32 DE-miRNAs were obtained and they were mainly enriched in immune and inflammatory responses. Twenty-six DE-mRNAs were the predicted target genes for miRNAs by RAID, and then the regulatory network of 15 miRNAs were constructed. In addition, through the PPI network, we identified the three genes (FPR1, FPR2 and CXCR4) with larger degrees as hub genes. Among them, FPR1 was regulated by hsa-miR-342-3p, hsa-let-7c-5p and hsa-miR-197-3p and participated in the immune response. Finally, we validated the differential expression of hub genes and key miRNAs between 20 CSF and 20 control. Moreover, we found that miR-342-3p has a targeted regulatory relationship with FPR1, and their expression is negatively correlated. Then we established a hypoxia/reoxygenation (H/R) HUVEC model and detected FPR1, cell proliferation and apoptosis. Transfection with miR-342-3p mimics can significantly promote the proliferation of HUVEC under H/R conditions. FPR1 were associated with CSF as a biomarker and may be regulated by miR-342-3p potential biomarkers.

WITHDRAWN: Transdifferentiation of Human Neonatal Foreskin Fibroblasts into Keratinocyte-like Cells via Regulating the AKT/P53/WNT/LEF1 Axis

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High-throughput mRNA sequencing of human placenta shows sex differences across gestation

IntroductionFetal sex affects fetal and maternal health outcomes in pregnancy, but this connection remains poorly understood. As the placenta is the route of fetomaternal communication and derives from the fetal genome, placental gene expression sex differences may explain these outcomes. ObjectivesWe utilized next generation sequencing to study the normal human placenta in both sexes in first and third trimester to generate a normative transcriptome based on sex and gestation. Study designWe analyzed 124 first trimester (T1, 59 female and 65 male) and 43 third trimester (T3, 18 female and 25 male) samples for sex differences within each trimester and sex-specific gestational differences. ResultsPlacenta shows more significant sexual dimorphism in T1, with 94 T1 and 26 T3 differentially expressed genes (DEGs). The sex chromosomes contributed 60.6% of DEGs in T1 and 80.8% of DEGs in T3, excluding X/Y pseudoautosomal regions. There were 6 DEGs from the pseudoautosomal regions, only significant in T1 and all upregulated in males. The distribution of DEGs on the X chromosome suggests genes on Xp (the short arm) may be particularly important in placental sex differences. Dosage compensation analysis of X/Y homolog genes shows expression is primarily contributed by the X chromosome. In sex-specific analyses of first versus third trimester, there were 2815 DEGs common to both sexes upregulated in T1, and 3263 common DEGs upregulated in T3. There were 7 female-exclusive DEGs upregulated in T1, 15 female-exclusive DEGs upregulated in T3, 10 male-exclusive DEGs upregulated in T1, and 20 male-exclusive DEGs upregulated in T3. DiscussionThis is the largest cohort of placentas across gestation from healthy pregnancies defining the normative sex dimorphic gene expression and sex common, sex specific and sex exclusive gene expression across gestation. The first trimester has the most sexually dimorphic transcripts, and the majority were upregulated in females compared to males in both trimesters. The short arm of the X chromosome and the pseudoautosomal region is particularly critical in defining sex differences in the first trimester placenta. As pregnancy is a dynamic state, sex specific DEGs across gestation may contribute to sex dimorphic changes in overall outcomes.

Cai’s prescription inhibits granulosa cell apoptosis through ARHGAP4 on poor ovarian responders

PurposePoor ovarian response (POR) is a big challenge for in vitro fertilization. The traditional Chinese medicine, Cai’s Prescription of Tonifying Kidney and Strengthening Vitals (Cai’s Prescription) has yielded satisfactory results for POR treatment clinically, but systematic scientific research of Cai’s Prescription is not well reported. This study aimed to investigate the clinical effect of Cai’s Prescription on poor ovarian responders and its biological mechanism.MethodsSerum was collected from poor ovarian responders, and IL-1β, INFγ, FSH, E2 and AMH levels were analyzed by ELISA. Ovarian antral follicles were identified and counted using transvaginal ultrasound. The embryo quality grading were done on day 3 after retrieval. We used high-throughput sequencing of granulosa cells to investigate the gene transcription patterns of ovarian granulosa cells in poor ovarian responders after Cai’s Prescription pretreatment. The expression level of ARHGAP4 was analyzed by quantitative real-time PCR and western blot. The effects of ARHGAP4 for granulosa cells were analyzed by CCK-8 assay, annexin-V and PI staining, ELISA and western blot. The effects of Cai’s Prescription on the expression of PI3K-Akt pathway and apoptosis were analyzed by western blot.ResultsIn this study, we found that Cai’s Prescription pretreatment had the tendency to improve the ovarian reserve function and could increase the number of high quality embryos for poor ovarian responders. Through high-throughput sequencing of mRNA in granulosa cells, we discovered ARHGAP4, which is a member of GTPase-activating proteins (GAPs) may be a candidate target for POR treatment. ARHGAP4 was significantly increased in poor ovarian responders and can be recovered after Cai’s Prescription pretreatment. Mechanically, combining the cell line model and clinical tissue samples, we found that ARHGAP4 can accelerate cell apoptosis and inflammation response in granulosa cells via PI3K-Akt signaling pathway. In addition, Cai’s Prescription pretreatment for three months significantly reduced the high level of ARHGAP4 in poor ovarian responders.ConclusionThis study shows that the traditional Chinese medicine, Cai’s Prescription yielded satisfactory results for poor ovarian responders clinically and ARHGAP4 may be a candidate target for POR treatment.

High throughput mRNA sequencing reveals potential therapeutic targets of Si-Ni-San in the pons for a stress-induced depression model.

An accumulating body of research indicates that the pons is related to the occurrence of depression. Si-Ni-San (SNS) is a well-known Chinese herbal formula that is used to treat depression. Chinese herbal formulae have multiple therapeutic characteristics. Although it has been proven that SNS can exert antidepressant effects by improving changes in the limbic system, it is currently unclear whether SNS has therapeutic targets in the pons. This study aimed to explore the therapeutic targets of SNS in the pons for depression treatment. Two experiments were conducted. In Experiment 1, 32 rats were divided into four groups: (1) a Control (C) group that received distilled water as a vehicle; (2) a Model (M) group that received the chronic unpredictable mild stress (CUMS) procedure and was administered distilled water; (3) a Stress + SNS (MS) group that received the CUMS procedure and was administered SNS dissolved in distilled water; and (4) a Stress + Fluoxetine (MF) group that received the CUMS procedure and was administered fluoxetine dissolved in distilled water. The open field test (OFT), the sucrose preference test (SPT), and the novel object recognition test (NOR) were performed to test the antidepressant effects of SNS. High-throughput mRNA sequencing (RNA-seq) was used to explore possible gene targets of SNS in the pons, and quantitative real-time PCR was performed to verify the results. High-performance liquid chromatography was used to detect neurotransmitters. Finally, correlation analyses were conducted between behaviors, genes expression, and neurotransmitters. In Experiment 2, 18 rats were divided into the same three groups as in Experiment 1: (1) C, (2) M, and (3) MS. fMRI was used to confirm whether SNS altered the pons in a rat model of depression. SNS significantly improved sucrose preference in the SPT and TN-TO in the NOR compared to the M group (P < 0.05). RNA-seq filtered 49 differentially expressed genes(DEGs) that SNS could reverse in the pons of the CUMS depression model. Real-time PCR detected six genes, including Complexin2 (Cplx2), Serpinf1, Neuregulin1 (Nrg1), Annexin A1 (Anxa1), β-arrestin 1 (Arrb1) and presenilin 1 (Psen1). SNS significantly reversed changes in the expression of Anxa1, Nrg1, and Psen1 caused by CUMS (P < 0.05), which is consistent with the DEGs results. Additionally, SNS significantly reversed norepinephrine (NE) changes in the pons. There were 18 noteworthy correlations between behavior, genes, and neurotransmitters (P < 0.05). fMRI showed that SNS can decrease the amplitude of low-frequency fluctuations (ALFF) in the pons of living depressed rats. The pons is an important target brain region for SNS to exert its antidepressant effects. SNS may improve pontine NE levels by regulating the Anxa1, Nrg1, and Psen1 genes, thereby exerting antidepressant effects and improving cognitive function.

Modification of Gene Expression of Tomato Plants through Foliar Flavonoid Application in Relation to Enhanced Growth.

The exogenous application of phenolic compounds is increasingly recognized as a valuable strategy for promoting growth and mitigating the adverse effects of abiotic stress. However, the biostimulant effect under optimal conditions has not been thoroughly explored. In this study, we investigated the impact of foliar application of flavonoids, specifically CropBioLife (CBL), on tomato plants grown under controlled conditions. Our study focused on determining growth parameters, such as cell size, and assessing the concentration of hormones. Principal component analysis (PCA) from all physiological variables was determined. Additionally, we utilized high-throughput mRNA-sequencing technology and bioinformatic methodologies to robustly analyze the transcriptomes of tomato leaves regulated by flavonoids. The findings revealed that CBL primarily influenced cell enlargement by 60%, leading to increased growth. Furthermore, CBL-treated plants exhibited higher concentrations of the hormone zeatin, but lower concentrations of IAA (changes of 50%). Moreover, RNA-seq analysis indicated that CBL-treated plants required increased mineral transport and water uptake, as evidenced by gene expression patterns. Genes related to pathways such as fatty acid degradation, phenylpropanoid biosynthesis, and ABC transporters showed regulatory mechanisms governing internal flavonoid biosynthesis, transport, and tissue concentration, ultimately resulting in higher flavonoid concentrations in tomato leaves.

Elucidating the pharmacodynamic mechanisms of Yuquan pill in T2DM rats through comprehensive multi-omics analyses.

Background: Yuquan Pill (YQW) is a modern concentrated pill preparation of six herbs, namely, Ge Gen (Pueraria lobata Ohwi), Di huang (Rehmannia glutinosa Libosch.), Tian Huafen (Trichosanthes kirilowii Maxim.), Mai Dong (Ophiopogon japonicus (L. f.) Ker Gawl.), Wu Weizi (Schisandra chinensis (Turcz.) Baill.) and Gan Cao (Glycyrrhiza uralensis Fisch.). It is extensively used to treat type 2 diabetes-related glucose and lipid metabolism disorders. But what's the pharmacodynamic substance and how it works in the treatment of Type 2 diabetes mellitus (T2DM) are still unclear. Purpose: The purpose of this study is to determine the likely pharmacological components and molecular mechanism of YQW's intervention on T2DM by combining serum pharmacochemistry, network analysis and transcriptomics. Methods: The efficacy and prototypical components of blood entry were determined after oral administration of YQW aqueous solution to T2DM rats induced by high-fat feed and low-dose streptozotocin (STZ), and the key targets and pathways for these compounds to intervene in T2DM rats were predicted and integrated using network analysis and transcriptomics techniques. Results: In diabetic rats, YQW can lower TG, CHO, NO, and MDA levels (p < 0.05) while increasing HDL-C levels (p < 0.01), and protecting the liver and kidney. 22 prototype components (including puerarin, daidzein, 3'-methoxypuerarin, and liquiritigenin, among others) were found in the serum of rats after oral administration of YQW for 90min, which might be used as a possible important ingredient for YQW to intervene in T2DM rats. 538 YQW pharmacodynamic components-related targets and 1,667 disease-related targets were projected through the PharmMapper database, with 217 common targets between the two, all of which were engaged in regulating PI3K-Akt, MAPK, Ras and FoxO signal pathway. Finally, the mRNA expression profiles of liver tissues from rats in the control, model, and YQW groups were investigated using high-throughput mRNA sequencing technology. YQW can regulate the abnormal expression of 89 differential genes in a disease state, including 28 genes with abnormally high expression and 61 genes with abnormally low expression. Five common genes (Kit, Ppard, Ppara, Fabp4, and Tymp) and two extensively used regulatory pathways (PI3K-Akt and MAPK signaling pathways) were revealed by the integrated transcriptomics and network analysis study. Conclusion: The mechanism of YQW's intervention in T2DM rats could be linked to 22 important components like puerarin, daidzein, and glycyrrhetinic acid further activating PI3K-Akt and MAPK signaling pathways by regulating key targets Kit, Ppard, Ppara, Fabp4, and Tymp, and thus improving lipid metabolism disorder, oxidative stress, and inflammation levels in T2DM rats. On the topic, more research into the pharmacological ingredient foundation and mechanism of YQW intervention in T2DM rats can be done.

Clonal Evolution Dissection Reveals High MSI2 Level Promotes Chemo-Resistance in T-Cell Acute Lymphoblastic Leukemia

Occurrences of T-cell acute lymphoblastic leukemia (T-ALL) arise in approximately 10-15% of pediatric ALL cases and the outcomes for high-risk T-ALL are inferior. Currently, the precise dissection of clonal evolution with intra-tumoral heterogeneity and to delineate targetable molecular events driving relapse in T-ALL subtypes have become area of interest. We carried out high-throughput droplet-based 5′-single-cell RNA-sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) of isolated CD7 + BMMCs from five patients' Dx_Rel paired samples. To investigate the gene signature variation underlying leukemic progression, we observed two distinct patterns of evolutionary trajectories in the four Dx_Rel nonETP-ALL pairs. From T956 and T723, a significant outgrowth in TCR initially negligible diagnostic subclusters at relapse, to which surrogate TCR repertoires were correspondingly linked and enumerated, suggestive robust dominant “clonal shifts” under continuous chemotherapeutic pressure. Whereas in Dx_Rel pairs T593 and T856, which contained subclusters with distinct gene signatures at diagnosis or relapse respectively, shared an identical TCR genotype (TCR Dx-Rel major clone). The clonal architecture clues gained from simultaneous scRNA-seq and scTCR-seq are strongly indicative of the leukemic “clonal drift” that commonly occurs under chemotherapeutic stress, during which somehow, hypothetically, molecular signatures within the same TCR subclones intrinsically progressed over time ( Fig. A-H). Next, we designed a targeted scDNA-seq library within a custom panel covering 127 amplicons and 102 genes via Tapastri platform to validate these two patterns of clonal evolution during leukemia progression unambiguously. Likewise, we captured that in persisted subclones in T593 and T856 leukemic cells attained reproductive fitness from maintaining a mutational static status with remarkable transcriptomic variation from diagnosis to relapse. To further interpret the transcriptomic signature drift in the persisted TCR-defined major clones from T593 and T856 pairs, differential expression analysis was employed with RNA-binding protein Musashi-2 (MSI2) showing the most prominent differential expression pattern across drifted subclones catching our attention as a potential indicator of intrinsic clonal progression related to chemo-resistance. Comprehensive analysis of RNA-seq data from the CCCG-ALL-2015 cohort (n=72) and TARGET T-ALL cohort (n = 264) revealed that higher levels of MSI2 mRNA expression in pediatric T-ALL were correlated with higher measurable residual disease (MRD) after induction, and significant correlation between elevated MIS2 mRNA and resistance to prednisone, daunorubicin, and cytarabine were observed from ex vivo drug response assay. RNA immunoprecipitation and high-throughput mRNA sequencing from T-ALL cell lines detected a consistent significant enrichment of MYC transcript peaks in three T-ALL cell lines, indicating the molecular mechanism of MSI2 upregulation on MYC in the progression of T-ALL. Finally, In vitro and in vivo pharmacotyping analysis further validated the MSI2 inhibition significantly sensitized T-ALL to the chemotherapeutic drugs. Collectively, our study leveraging single-cell multi-omics profiling has provided a snapshot of the clonal architecture evolution that occurs from diagnosis to relapse in T-ALL, proving the coexistence of “clonal shift” and “clonal drift” in leukemic progression. Systemic analysis of drifted T-ALL clones has provided compelling evidence of the posttranscriptional regulatory effect of MSI2 on MYC and its potential correlation with leukemic reoccurrence, pinpointing MSI2 as a promising drug target of T-ALL in the future practice.

OsSTS, a Novel Allele of Mitogen-Activated Protein Kinase Kinase 4 (OsMKK4), Controls Grain Size and Salt Tolerance in Rice

Soil salinization is one of the most common abiotic stresses of rice, which seriously affects the normal growth of rice. Breeding salt-tolerant varieties have become one of the important ways to ensure food security and sustainable agricultural development. However, the mechanisms underlying salt tolerance control still need to be clarified. In this study, we identified a mutant, termed salt-tolerant and small grains(sts), with salt tolerance and small grains. Gene cloning and physiological and biochemical experiments reveal that sts is a novel mutant allele of Mitogen-activated protein Kinase Kinase 4 (OsMKK4), which controls the grain size, and has recently been found to be related to salt tolerance in rice. Functional analysis showed that OsSTS is constitutively expressed throughout the tissue, and its proteins are localized to the nucleus, cell membrane, and cytoplasm. It was found that the loss of OsSTS function enhanced the salt tolerance of rice seedlings, and further studies showed that the loss of OsSTS function increased the ROS clearance rate of rice seedlings, independent of ionic toxicity. In order to explore the salt tolerance mechanism of sts, we found that the salt tolerance of sts is also regulated by ABA through high-throughput mRNA sequencing. Salt and ABA treatment showed that ABA might alleviate the inhibitory effect of salt stress on root length in sts. These results revealed new functions of grain size gene OsMKK4, expanded new research ideas related to salt tolerance mechanism and hormone regulation network, and provided a theoretical basis for salt-tolerant rice breeding.

TRF-003634 alleviates adriamycin-induced podocyte injury by reducing the stability of TLR4 mRNA.

Podocyte injury plays a key role in the production of proteinuria and is closely related to the progression of chronic kidney disease (CKD). Alleviating podocyte injury is beneficial to prevent the occurrence and development of CKD. tRNA-derived RNA fragments (tRFs) are associated with podocytes injury processes such as protein binding, cell adhesion, synapses, the actin cytoskeleton. Our previous data showed that tRF-003634 tightly correlated with podocyte injury, while its effect remains unclear. This study aimed to investigate the role of tRF-003634 in podocyte injury and the potential mechanisms. The expression level of tRF-003634, nephrin, podocin and tRF-003634 targeted toll-like receptor 4 (TLR4) in podocytes and kidney tissues were examined by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The biochemical indices were monitored and renal pathological changes were assessed by hematoxylin and eosin PAS staining. Furthermore, potential target genes of tRF-003634 were screened using high-throughput mRNA sequencing, and then confirmed by RNA pulse-chase analysis. The results showed that tRF-003634 was downregulated in adriamycin (Adr)-induced podocyte injury. Overexpression of tRF-003634 increased the expression of nephrin and podocin in vivo and in vitro and alleviated podocyte injury. Meanwhile, overexpression of tRF-003634 alleviated proteinuria and renal pathological damage. In addition, high-throughput sequencing after overexpression of tRF-003634 showed that TLR4 might be a downstream target gene. tRF-003634 can alleviate podocyte injury by reducing the stability of TLR4 mRNA, possibly by competing with TLR4 mRNA to bind to YTH domain-containing protein 1 (YTHDC1). In conclusion, tRF-003634 was underexpressed in Adr-induced podocyte injury, and its overexpression alleviated podocyte injury in vitro and in vivo by reducing the stability of TLR4 mRNA.