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Despite substantial advancements in the field of cryobiology, oocyte and embryo cryopreservation still compromise developmental competence. Furthermore, dimethyl sulfoxide (DMSO), one of the most commonly used cryoprotectants, has been found to exert potent effects on the epigenetic landscape of cultured human cells, as well as mouse oocytes and embryos. Little is known about its impact on human oocytes. Additionally, few studies investigate the effects of DMSO on transposable elements (TE), the control of which is essential for the maintenance of genomic instability. The objective of this study was to investigate the impact of vitrification with DMSO-containing cryoprotectant on the transcriptome, including on TEs, of human oocytes. Twenty-four oocytes at the GV stage were donated by four healthy women undergoing elective oocyte cryopreservation. Oocytes were paired such that half from each patient were vitrified with DMSO-containing cryoprotectant (Vitrified Cohort), while the other half were snap frozen in phosphate buffer, unexposed to DMSO (Non-Vitrified Cohort). All oocytes underwent RNA sequencing via a method with high fidelity for single cell analysis, and which allows for the analysis of TE expression through Switching Mechanism at the 5′-end of the RNA Transcript sequencing 2 (SMARTseq2), followed by functional enrichment analysis. Of the 27,837 genes identified by SMARTseq2, 7331 (26.3%) were differentially expressed (p < 0.05). There was a significant dysregulation of genes involved in chromatin and histone modification. Mitochondrial function, as well as the Wnt, insulin, mTOR, HIPPO, and MAPK signaling pathways were also altered. The expression of TEs was positively correlated with the expression of PIWIL2, DNMT3A, and DNMT3B, and negatively correlated with age. These findings suggest that the current standard process of oocyte vitrification, involving DMSO-containing cryoprotectant, induces significant transcriptome changes, including those involving TEs.

Embryos are diagnosed as mosaic if their chromosomal copy number falls between euploid and aneuploid. The purpose of this study was to investigate the impact of mosaicism on global gene expression. This study included 42 blastocysts that underwent preimplantation genetic testing for aneuploidy (PGT-A) and were donated for IRB approved research. Fourteen blastocysts were diagnosed as mosaic with Next-generation Sequencing (NGS). Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 array Comparative Genomic Hybridization, 2 Single Nucleotide Polymorphism array) were used as comparisons. RNA-sequencing was performed on all of the blastocysts. Differentially expressed genes (DEGs) were calculated using DESeq2/3.5 (R Bioconductor Package) with p 0.05 considered significantly differentially expressed. Pathway analysis was performed on mosaic embryos using EnrichR with p 0.05 considered significant. With euploid embryo gene expression used as a control, 12 of 14 mosaic embryos had fewer DEGs compared to aneuploid embryos involving the same chromosome. On principal component analysis (PCA), mosaic embryos mapped separately from aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted within mosaic embryos. Mosaic embryos have decreased disruption of global gene expression compared to aneuploid embryos. This study was limited by the small sample size, lack of replicate samples for each mosaic abnormality, and use of multiple different PGT-A platforms for the diagnosis of aneuploid embryos.

PURPOSE: To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development. METHODS: Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p. RESULTS: LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P

BackgroundCellular RNA-binding proteins (RBPs) have multiple roles in post-transcriptional control, and some are shown to bind DNA. However, the global localization and the general chromatin-binding ability of RBPs are not well-characterized and remain undefined in hematopoietic cells.ResultsWe first provide a full view of RBPs’ distribution pattern in the nucleus and screen for chromatin-enriched RBPs (Che-RBPs) in different human cells. Subsequently, by generating ChIP-seq, CLIP-seq, and RNA-seq datasets and conducting combined analysis, the transcriptional regulatory potentials of certain hematopoietic Che-RBPs are predicted. From this analysis, quaking (QKI5) emerges as a potential transcriptional activator during monocytic differentiation. QKI5 is over-represented in gene promoter regions, independent of RNA or transcription factors. Furthermore, DNA-bound QKI5 activates the transcription of several critical monocytic differentiation-associated genes, including CXCL2, IL16, and PTPN6. Finally, we show that the differentiation-promoting activity of QKI5 is largely dependent on CXCL2, irrespective of its RNA-binding capacity.ConclusionsOur study indicates that Che-RBPs are versatile factors that orchestrate gene expression in different cellular contexts, and identifies QKI5, a classic RBP regulating RNA processing, as a novel transcriptional activator during monocytic differentiation.

Even though aberrant mechanistic target of rapamycin (mTOR) signaling is known to cause cardiomyopathy, its underlying mechanism remains poorly understood. Because augmentation of αB-crystallin and hspB2 was presented in the cortical tubers and lymphangioleiomyomatosis of tuberous sclerosis complex patients, we deciphered the role of αB-crystallin and its adjacent duplicate gene, hspB2, in hyperactive mTOR-induced cardiomyopathy. Cardiac Tsc1 deletion (T1-hKO) caused mouse mTOR activation and cardiomyopathy. Overexpression of αB-crystallin and hspB2 was presented in the hearts of these mice. Knockout of αB-crystallin/hspB2 reversed deficient Tsc1-mediated fetal gene expression, mTOR activation, mitochondrial damage, cardiomyocyte vacuolar degeneration, cardiomyocyte size, and fibrosis of T1-hKO mice. These cardiac-Tsc1; αB-crystallin; hspB2 triple knockout (tKO) mice had improved cardiac function, smaller heart weight to body weight ratio, and reduced lethality compared with T1-hKO mice. Even though activated mTOR suppressed autophagy in T1-hKO mice, ablation of αB-crystallin and hspB2 failed to restore autophagy in tKO mice. mTOR inhibitors suppressed αB-crystallin expression in T1-hKO mice and rat cardiomyocyte line H9C2. Starvation of H9C2 cells activated autophagy and suppressed αB-crystallin expression. Since inhibition of autophagy restored αB-crystallin expression in starved H9C2 cells, autophagy is a negative regulator of αB-crystallin expression. mTOR thus stimulates αB-crystallin expression through suppression of autophagy. In conclusion, αB-crystallin and hspB2 play a pivotal role in Tsc1 knockout-related cardiomyopathy and are therapeutic targets of hyperactive mTOR-associated cardiomyopathy.

Introduction: Disturbances in myocardial lipid metabolism are increasingly being recognized as drivers of the development and progression of heart disease. Therefore, there is a need for treatments that can directly target lipid metabolic defects in heart failure. The membrane-associated glycoprotein CD36 plays a pivotal role in governing myocardial lipid metabolism by mediating lipid signaling and facilitating the cellular uptake of long-chain fatty acids. Emerging evidence suggests that CD36 is a prominent target in the treatment of heart failure. Areas covered: This article provides an overview of the key role of CD36 for proper contractile functioning of a healthy heart, its implications in the development of cardiac disease (ischemia/reperfusion, cardiac hypertrophy, and diabetic cardiomyopathy), and its application as a target to normalize cardiac metabolism as part of so-called metabolic modulation therapy. Expert opinion: CD36 appears a promising and effective therapeutic target in the treatment of heart failure. Natural compounds and chemical agents known to alter the amount or subcellular distribution of CD36 or inhibit its functioning, should be evaluated for their potency to correct cardiac metabolism and cure heart disease.

Rationale: The invasive behavior of non-functioning pituitary neuroendocrine tumors (NF-PitNEts) presents obstacles for complete surgical resection and is indicative of poor prognosis. Therefore, developing reliable diagnostic tools for identifying invasive PitNEts would be helpful in guiding surgical decisions and, in particular, the follow-up treatment. Methods: We analyzed differential gene expression profiles between 39 non-invasive and 22 invasive NF-PitNEts by high-throughput sequencing, gene co-expression, and functional annotation. Twenty-one transcripts were further validated by Taqman-qPCR in another 143 NF-PitNEt samples. The histological expression and serum-exosomal mRNA of three candidate genes were examined by tissue microarray and droplet digital PCR. Results: Non-invasive and invasive NF-PitNEts were clustered into distinct groups with a few outliers because of their gonadotroph, corticotroph, or null cell lineages. The gene signature with strong invasive potential was enriched in 'Pathways in cancers' and 'MAPK pathway', with significantly higher in situ INSM1 and HSPA2 protein expression in invasive NF-PitNEts. Further integration of the 20 qPCR-validated differentially expressed genes and pituitary cell lineages provided a gene-subtype panel that performed 80.00-90.24% diagnostic accuracy for the invasiveness of NF-PitNEts. Conclusion: Our approach defined new characteristics in the core molecular network for patients at risk for invasive NF-PitNEt, representing a significant clinical advance in invasive PitNEt diagnostics.

In China, the fruits of Physalis alkekengi L. var. franchetii, which are conventionally utilized as edible berry, have attracted wide attention due to its significant biological activities. In the present study, phytochemical studies on the fruits of Physalis plants afforded six compounds, including two new withanolides (1-2) and four known agnologues (3-6). The inhibitory effects of these compounds on the formation of nitric oxide (NO) stimulated by lipopolysaccharide (LPS) in RAW264.7 macrophages were evaluated. Physapubescin M (1), with IC50 value of 1.58 μM, was selected for further study. The protein expression of COX-2 and iNOS, and LPS-induced production of cytokines (IL-6, IL-1β and TNF-α) were reduced by physapubescin M (1) in a dose-dependent way. In addition, transcriptomic analyses were conducted to profile gene expression alterations in LPS-induced RAW264.7 cells upon treatment of physapubescin M (1) and the potential antiinflammatory mechanism of withnolides was mentioned. These results provide broad view to the underlying antiinflammatory mechanism of withnolides, and give a theoretical basis for the utilization of the fruits of P. alkekengi L. var. franchetii.

Long noncoding RNAs (lncRNAs) have emerged as diverse functional regulators involved in mammalian development; however, large-scale functional investigation of lncRNAs in mammalian spermatogenesis in vivo is lacking. Here, we delineated the global lncRNA expression landscape in mouse spermatogenesis and identified 968 germ cell signature lncRNAs. By combining bioinformatics and functional screening, we identified three functional lncRNAs (Gm4665, 1700027A15Rik, and 1700052I22Rik) that directly influence spermatogenesis in vivo. Knocking down Gm4665 hampered the development of round spermatids into elongating spermatids and disrupted key spermatogenic gene expression. Mechanistically, lncRNA Gm4665 localized in the nucleus of round spermatids and occupied the genomic regulatory region of important spermatogenic genes including Ip6k1 and Akap3. These findings provide a valuable resource and framework for future functional analysis of lncRNAs in spermatogenesis and their potential roles in other biological processes.

Obesity is a metabolic disease and its relation with male subfertility has aroused a growing concern. However, it is unclear whether gene expression and post-translational modifications (PTMs), two vital molecular mechanisms regulating cellular functions, are associated with obesity-induced male reproductive dysfunction. In this study, male obesity with compromised sperm motility was induced by a high-fat diet (HFD) using a mouse model. The expression of motility related-genes, the level of histone modifications, and the global profiles of post-translational modifications (PTMs), were examined in testes of HFD and control mice by quantitative real-time PCR and western blot, respectively. Outer dense fiber protein 2, a major component of outer dense fibers in the sperm tail, is the most obviously down-regulated gene out of 11 evaluated genes, showing a reduction of about 50% RNA level in testes of obese male mice compared with that in control mice. Semi-quantitative analysis of the western blot demonstrated that ∼56% enrichment of di-methylated histone (H)3 lysine (K)36, ∼59% enrichment of 2-hydroxyisobutyrylated H4K8, ∼32% decrease of propionylated H3K23, ∼33% decrease of crotonylated H4K8, and ∼45% decrease of acetylated H3K122 and H4K8 were detected in testes of male HFD mice compared with that in control mice. In addition, male obesity up-regulated the testicular levels of ubiquitination by ∼18%, tyrosine nitration by ∼20%, lysine succinylation by ∼25%, lysine benzoylation by ∼28%, lysine malonylation by ∼32%, lysine glutarylation by ∼36%, lysine propionylation by ∼42%, lysine 2-hydroxyisobutyrylation by ∼45%, and SUMO1 modification by ∼59%, and down-regulated the testicular levels of O-GlcNAcylation by ∼12%, lysine crotonylation by ∼22%, and lysine acetylation by 35%. These findings indicate that altered gene expression and PTMs are associated with the obesity-induced male reproductive dysfunction.

MicroRNAs (miRNAs) are important regulators of gene expression in eukaryotes. Studies have shown that plant-derived miRNAs can be absorbed through diets and regulate gene expression in mammals. Although soybean-derived miRNAs have been reported, their biological functions are still unclear. In this study, we found that soybean-derived small RNAs (sRNAs) significantly inhibited the proliferation and stimulated the apoptosis of Caco-2 cells. Bioinformatics analysis indicated that the target gene set of soybean miRNAs was extensively enriched in cancer pathways. Besides, we obtained 8 target genes, including Transcription factor 7 (TCF7), associated with colon cancer through prediction. Further studies showed that gma-miR159a inhibited the proliferation of Caco-2 cells and played an important role in the inhibitory effect of sRNAs by inhibiting TCF7 protein, which are upregulated in colon cancer cells but not normal mucosal cells in culture. These findings provide a novel molecular mechanism of soybean-derived miRNAs for potential application in tumor prevention.

Pre-eclampsia (PE) is a disorder of pregnancy characterized by proteinuria and high blood pressure, affecting 2-8% of pregnancies worldwide. Previous studies have shown that PE is closely associated with trophoblast cell dysfunction. Here, we investigated the role of tissue factor pathway inhibitor-2 (TFPI-2) in regulating the biological processes of trophoblast cells. The TFPI-2 levels in plasma samples and placental tissues were tested by ELISA, immunohistochemistry, qRT-PCR, and western blot. HTR8/Svneo cell line was used to simulate the primary trophoblast cells and H/R culture was applied to mimic the oxidative stress state of PE. MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and transwell assay were used to determine the cell proliferation, apoptosis, and invasion. The expression levels of matrix metalloproteinases (MMPs) were evaluated by western blot. The expression of TFPI-2 was remarkably up-regulated in both the serum and placenta of PE patients. Hypoxia/reoxygenation increased the expression of TFPI-2 in HTR-8/SVneo cell line. TFPI-2 promoted that cell proliferation and inhibited the cell apoptosis of HTR8/SVneo cells in H/R condition. In addition, downregulation of TFPI-2 increased the cell invasion and the expression of MMP2 and MMP9. This study reveals that TFPI-2 plays a crucial role in monitoring the biological function of trophoblast cells, which might provide theoretical basis and therapeutic targets for the treatment of PE.

In this report, we investigated the hepatocytic uptake of rosuvastatin when administered with scutellarin (a Chinese herbal medicine) in rats and the role of organic anion transporting polypeptide 1B1 (OATP1B1) plays in the uptake. Forty-eight rats were randomly divided into two groups according to the medicine administered: rosuvastatin alone and rosuvastatin in combination with a series concentration of scutellarin. Rosuvastatin concentrations in blood and liver were measured using the liquid chromatography–tandem mass spectrometry (LC-MS) method. The uptake was also measured in rat primary hepatocytes and OATP1B1 transfected human embryonic kidney 293 T (HEK293T) cells. The uptake was investigated under the optimal intake conditions. The rosuvastatin Cmax and AUC0−∞ in rat plasma increased 55% and 61%, respectively in the combination treatment group; and the liver scutellarin concentrations decreased 32%, 34%, and 33% at 1 h, 2 h, and 6 h, respectively. All scutellarin dosages (20, 50, and 100 μM) inhibited the uptake of rosuvastatin in rat primary hepatocytes (4.71%, 22.73%, and 45.89%). Scutellarin of 10 μM significantly inhibited the in vitro uptake of rosuvastatin in OATP1B1-HEK293T cells (P

Background: Alzheimer’s disease (AD) is one of many common neurodegenerative diseases without ideal treatment, but early detection and intervention can prevent the disease progression. Objective: This study aimed to identify AD-related glycolysis gene for AD diagnosis and further investigation by integrated bioinformatics analysis. Methods: 122 subjects were recruited from the affiliated hospitals of Ningbo University between 1 October 2015 and 31 December 2016. Their clinical information and methylation levels of 8 glycolysis genes were assessed. Machine learning algorithms were used to establish an AD prediction model. Receiver operating characteristic curve (AUC) and decision curve analysis (DCA) were used to assess the model. An AD risk factor model was developed by SHapley Additive exPlanations (SHAP) to extract features that had important impacts on AD. Finally, gene expression of AD-related glycolysis genes were validated by AlzData. Results: An AD prediction model was developed using random forest algorithm with the best average ROC_AUC (0.969544). The threshold probability of the model was positive in the range of 0∼0.9875 by DCA. Eight glycolysis genes (GAPDHS, PKLR, PFKFB3, LDHC, DLD, ALDOC, LDHB, HK3) were identified by SHAP. Five of these genes (PFKFB3, DLD, ALDOC, LDHB, LDHC) have significant differences in gene expression between AD and control groups by Alzdata, while three of the genes (HK3, ALDOC, PKLR) are related to the pathogenesis of AD. GAPDHS is involved in the regulatory network of AD risk genes. Conclusion: We identified 8 AD-related glycolysis genes (GAPDHS, PFKFB3, LDHC, HK3, ALDOC, LDHB, PKLR, DLD) as promising candidate biomarkers for early diagnosis of AD by integrated bioinformatics analysis. Machine learning has the advantage in identifying genes.

Ganoderic acids (GAs), a group of highly oxygenated lanostane-type triterpenoids from the traditional Chinese medicinal mushroom Ganoderma lucidum, possessed significant pharmacological activities. Due to the difficulty in its genetic manipulation, low yield, and slow growth of G. lucidum, biosynthesis of GAs in a heterologous host is a promising alternative for their efficient production. Heterologous production of a GA, 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA), was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA to biosynthesize other GAs remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new GA 3,28-dihydroxy-lanosta-8,24-dien-26-oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by UPLC-APCI-HRMS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27 mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmids pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR, and hygromycin-resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418-resistant gene KanMx) was adopted. Finally, 2.2 mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work enriches the GA biosynthetic enzyme library, and is helpful to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum. KEY POINTS: • Another P450 gene responsible for GA's post-modification was discovered and identified. • One new GA, DHLDOA, was identified and produced via engineered yeast. • With the balance of the two CYP genes expression, DHLDOA production was significantly improved.

Co-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3−/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22−/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3−/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3−/− mESCs, or Atxn7l3−/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3−/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

The N6-methyladenosine (m6A) modification is the most common internal post-transcriptional modification, with important regulatory effects on RNA export, splicing, stability, and translation. Studies on the m6A modifications in plants have focused on Arabidopsis thaliana growth and development. However, A. thaliana is a salt-sensitive and model plant species. Thus, studies aimed at characterizing the role of the m6A modification in the salt stress responses of highly salt-tolerant crop species are needed. Sweet sorghum is cultivated as an energy and forage crop, which is highly suitable for growth on saline-alkaline land. Exploring the m6A modification in sweet sorghum may be important for elucidating the salt-resistance mechanism of crops. In this study, we mapped the m6A modifications in two sorghum genotypes (salt-tolerant M-81E and salt-sensitive Roma) that differ regarding salt tolerance. The m6A modification in sweet sorghum under salt stress was drastically altered, especially in Roma, where the m6A modification on mRNAs of some salt-resistant related transcripts increased, resulting in enhanced mRNA stability, which in turn was involved in the regulation of salt tolerance in sweet sorghum. Although m6A modifications are important for regulating sweet sorghum salt tolerance, the regulatory activity is limited by the initial m6A modification level. Additionally, in M-81E and Roma, the differences in the m6A modifications were much greater than the differences in gene expression levels and are more sensitive. Our study suggests that the number and extent of m6A modifications on the transcripts of salt-resistance genes may be important factors for determining and assessing the salt tolerance of crops.

Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.

Background Reversible N6-methyladenosine (m6A) modifications in messenger RNAs can be categorized under the field of “RNA epigenetics.” However, the potential role of m6A-related genes in gastric cancer (GC) prognosis has not been systematically researched. Aims This study was aimed at providing insights into the prognostic role of m6A-related gene expression, at both mRNA and protein levels. Methods Kaplan–Meier (KM) plotter database and The Cancer Genome Atlas (TCGA) database were used to explore the prognostic significance of individual m6A-related genes in overall survival (OS) and progression-free survival at the mRNA level. For independent validation, the protein level of genes significantly associated with prognosis in both databases was further detected in 450 paired GC and corresponding adjacent non-tumor tissues using tissue microarray (TMA)-based immunohistochemistry (IHC). The relationship between the FTO and ALKBH1 expression and the clinicopathological characteristics was explored. Results Among nine m6A-related genes, aberrantly high mRNA expression of FTO and ALKBH1 was associated with poor OS in the KM and TCGA cohorts. However, the TMA-IHC indicated that protein expression of FTO and ALKBH1 was markedly downregulated in GC tissues. A lower protein level of ALKBH1 was closely correlated with larger tumor sizes (≥ 5 cm) and more advanced TNM stages, while lower FTO protein expression was associated with shorter OS in GC patients. Conclusions Aberrant expression of demethylase genes, FTO and ALKBH1, has a distinct prognostic value in GC patients, indicating that FTO and ALKBH1 may play vital roles in GC progression and metastasis. Electronic supplementary material The online version of this article (10.1007/s10620-018-5452-2) contains supplementary material, which is available to authorized users.

Acute influenza infection has been reported to be associated with neurological symptoms such as influenza-associated encephalopathy (IAE). Although the pathophysiology of this condition remain unclear, neuroinflammation and associated alterations in the central nervous system (CNS) are usually induced. Microglia (MGs), CNS-resident macrophages, are generally the first cells to be activated in response to brain infection or damage. We performed reverse transcriptase droplet digital PCR (RT-ddPCR) and luminex assays to investigate virus proliferation and immune reactions in BV2 MGs infected with influenza A(H1N1)pdm09 virus. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to investigate the dynamic change in the protein expression profile in BV2 MGs to gain insight into the CNS response to influenza A (H1N1) pdm09 infection. Our results showed that the influenza A(H1N1)pdm09 virus was replicative and productive in BV2 MG cells, which produced cytokines such as interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. The expression of osteopontin (OPN) in the influenza A (H1N1) pdm09-infected BV2 MGs was upregulated at 16 and 32 h post-infection (hpi) compared to that in the control group, resulting in aggravated brain damage and inflammation. Our study indicates that OPN signalling might provide new insights into the treatment of CNS injury and neurodegenerative diseases in IAE.

As shown in our previous study, two alternatively spliced androgen receptor (AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90 (HSP90) and importin α (the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone (DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR (Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR (Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.

Cohesin and CTCF are major drivers of 3D genome organization. Even though human mutations underscore the importance of cohesin and CTCF for neurodevelopment, their role in neurons is only just beginning to be addressed. Here we conditionally ablate Rad21 in cortical neurons, revealing a prominent role for cohesin in the expression of genes that facilitate neuronal maturation, homeostasis, and activation. In agreement with recent reports, activity-dependent genes were downregulated at baseline. However, in contrast to current models that attribute impaired activity-dependent gene expression to a role for cohesin and CTCF in anchoring enhancer-promoter contacts, we show that nearly all activity-dependent genes remain inducible in the absence of cohesin. While CTCF-based chromatin loops were substantially weakened, long-range contacts still formed robustly between activity-dependent enhancers and their target immediate early gene promoters. We suggest a model where neuronal cohesin facilitates the precise level of activity-dependent gene expression, rather than inducibility per se, and where inducibility of activity-dependent gene expression is linked to cohesin-independent enhancer-promoter contacts. These data expand our understanding of the importance of cohesin-independent enhancer-promoter contacts in regulating gene expression.

Human umbilical vein endothelial cells (HUVECs) serve a critical role in maintaining normal vascular function. Lipopolysaccharide (LPS), which is released from pathogenic bacteria in the blood, induces HUVEC apoptosis and injury to cause vascular dysfunction and infectious vascular diseases. Procyanidin B2 (PB2) possesses numerous functions, including antioxidant, antitumor, anti‑inflammatory and antiapoptosis effects, but the molecular mechanism is not completely understood. The present study investigated the effects of PB2 on LPS‑induced cytotoxicity and apoptosis in HUVECs, as well as the underlying mechanisms. The effects of PB2 on LPS‑mediated alterations to cytotoxicity, mitochondrial membrane potential, apoptosis were assessed by performing Cell Counting Kit‑8, JC‑1 fluorescence, Hoechst 33258 staining assays, respectively. IL‑1β, IL‑6 and TNF‑α mRNA expression and protein levels were measured by performing reverse transcription‑quantitative PCR and ELISAs, respectively. Bcl‑2, Bax, cleaved caspase‑3, cleaved caspase‑7, cleaved caspase‑9, phosphorylated (p)‑IκB‑α, p‑IκB‑β, p‑NF‑κB‑p65 and total NF‑κB p65 protein expression levels were determined via western blotting. NF‑κB p65 nuclear translocation was assessed via immunofluorescence. PB2 pretreatment markedly attenuated LPS‑induced cytotoxicity and apoptosis in HUVECs. PB2 also significantly downregulated the expression levels of IL‑1β, IL‑6, TNF‑α, Bax, cleaved caspase‑3, cleaved caspase‑7, cleaved caspase‑9 and p‑NF‑κB‑p65, but upregulated the expression levels of Bcl‑2, p‑IκB‑α and p‑IκB‑β in LPS‑induced HUVECs. Moreover, PB2 markedly inhibited LPS‑induced NF‑κB p65 nuclear translocation in HUVECs. The results suggested that the potential molecular mechanism underlying PB2 was associated with the Bax/Bcl‑2 and NF‑κB signalling pathways. Therefore, PB2 may serve as a useful therapeutic for infectious vascular diseases.

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS., A feature of cohesin mutations in patients with Cornelia de Lange Syndrome (CdLS) is intellectual disability, but the underlying mechanisms have remained obscure. Here the authors show gene expression is deregulated in CdLS neurons and is recapitulated in a mouse model with cohesin depletion, which can be restored by re-expression of cohesin.

Hepatoblastoma is the most common tumor of liver in childhood. The onset of the disease is insidious, and the early stage is mostly asymptomatic. About 20 % of the children had distant metastasis at diagnosis. Only 50 %-60 % of the tumors can be completely resected by surgery, and the relapse percentage of children who undergo surgical treatment alone is still high. Therefore, the aim was to conduct a multimessenger RNA signature to improve hepatoblastoma progression prediction. Messenger RNA expression profiling of Gene Expression Ominus dataset (GSE131329) was analyzed in a large hepatoblastoma cohort and screened the significantly differential expressed Messenger RNAs between normal tissues and tumors. Weighted correlation network analysis was used to select genes that correlated to disease progression. Data fitting analysis was performed and three samples were removed from subsequent analyses in GSE131329. A total of 1263 differentially expressed genes were screened, consisting 569 up-regulated genes and 694 down-regulated genes. Gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analyses were performed and by using weighted correlation network analysis, 8 hub genes were selected and an 8-messenger RNA-based signature was established that was significantly related to the disease progression of hepatoblastoma. Our results might provide an efficient clinical multiple-messenger RNA signature for predicting disease progression and be potentially efficient targets for hepatoblastoma patients.

Mouse homozygous mutants in Wnt/planar cell polarity (PCP) pathway genes have been shown to cause neural tube defects (NTDs) through the disruption of normal morphogenetic processes critical to neural tube closure (NTC). Knockout mice that are heterozygotes of single PCP genes likely fail to produce NTD phenotypes, yet damaging variants detected in human NTDs are almost always heterozygous, suggesting that other deleterious interacting variants are likely to be present. Nonetheless, the Wnt/PCP pathway remains a genetic hotspot. Addressing these issues is essential for understanding the genetic etiology of human NTDs.We performed targeted next-generation sequencing (NGS) on 30 NTD-predisposing Wnt/PCP pathway genes in 184 Chinese NTD cases. We subsequently replicated our findings for the CELSR1 gene in an independent cohort of 292 Caucasian NTD samples from the USA. Functional validations were confirmed using in vitro assays.CELSR1, CELSR2 and CELSR3 genes were significantly clustered with rare driver coding mutations (q-value 0.05) demonstrated by OncodriveCLUST. During the validation stage, the number of rare loss of function (LoF) variants in CELSR1 was significantly enriched in NTDs compared with the LoF counts in the ExAC database (p 0.001). Functional studies indicated compound heterozygote variants of CELSR2 p.Thr2026Met and DVL3 p.Asp403Asn result in down regulation of PCP signals.These data indicate rare damaging variants of the CELSR genes, identified in ~ 14% of NTD cases, are expected to be driver genes in the Wnt/PCP pathway. Compound damaging variants of CELSR genes and other Wnt/PCP genes, which were observed in 3.3% of the studied NTD cohort, are also expected to amplify these effects at the pathway level.

BackgroundCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a preliminary understanding of the replication and transcription of SARS-CoV-2 has recently emerged, their regulation remains unknown.ResultsBy comprehensive analysis of genome sequence and protein structure data, we propose a negative feedback model to explain the regulation of CoV replication and transcription, providing a molecular basis of the “leader-to-body fusion” model. The key step leading to the proposal of our model was that the transcription regulatory sequence (TRS) motifs were identified as the cleavage sites of nsp15, a nidoviral RNA uridylate-specific endoribonuclease (NendoU). According to this model, nsp15 regulates the synthesis of subgenomic RNAs (sgRNAs), and genomic RNAs (gRNAs) by cleaving TRSs. The expression level of nsp15 controls the relative proportions of sgRNAs and gRNAs, which in turn change the expression level of nsp15 to reach equilibrium between the CoV replication and transcription.ConclusionThe replication and transcription of CoVs are regulated by a negative feedback mechanism that influences the persistence of CoVs in hosts. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, and provide new clues for future studies. One important clue is that nsp15 may be an important and ideal target for the development of drugs (e.g., uridine derivatives) against CoVs.

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice., Developing T cells commit to either CD4/helper or CD8/cytotoxic lineage in the thymus, but how CD4 and CD8 coreceptors and TCR signaling dictate this selection process is still unclear. Here the authors use single cell RNA sequencing of mouse thymocytes to show that, in selection intermediates, TCR signaling strength informs coreceptor expression timing.

Long noncoding RNAs (lncRNAs) have gained much attention in the past few years. Long intergenic non-protein coding RNA 520 (LINC00520) was one of the newly discovered lncRNA which has been demonstrated to be dysregulated in several cancers. So far, the function and mechanism of LINC00520 in non-small cell lung cancer (NSCLC) are unclear. In this paper, our group first showed that LINC00520 level was elevated in non-small cell lung cancer (NSCLC) tissue and cells. In addition, SP1 could bind directly to the promoter region of LINC00520 and thus promote its transcription. Increased LINC00520 was distinctly correlated with advanced tumor stage and shorter survival time in NSCLC patients. Further functional investigations provided evidences that forced down regulation of LINC00520 inhibited NSCLC cell proliferation, invasion, metastasis and EMT, while contributing to cells apoptosis. Mechanistically, we found that LINC00520 serving as a competing endogenous RNA to be involved in the modulation of miR-577 expressions, and thus affected the expression of CCNE2 which was a target gene of miR-577. Moreover, in NSCLC cells with si-LINC00520, up regulation of CCNE2 led to an increase of cell growth and invasion. Taken together, LINC00520 displayed its tumor-promotive roles through modulating the miR-577/CCNE2 axis, highlighting a potential therapeutic strategy for NSCLC patients.

Summary Uncoupling of mRNA expression from copy number (UECN) might be a strategy for cancer cells to a tolerate high degree of aneuploidy. To test the extent and role of UECN across cancers, we perform integrative multiomic analysis of The Cancer Genome Atlas (TCGA) dataset, encompassing ∼5,000 individual tumors. We find UECN is common in cancers and is associated with increased oncogenic signaling, proliferation, and immune suppression. UECN appears to be orchestrated by complex regulatory changes, with transcription factors (TFs) playing a prominent role. To further dissect the regulatory mechanisms, we develop a systems-biology approach to identify candidate TFs, which could serve as targets to disrupt UECN and reduce tumor fitness. Applying our approach to TCGA data, we identify 21 putative targets, 42.8% of which are validated by independent sources. Together, our study indicates that UECN is likely an important mechanism in development of aneuploid tumors and might be therapeutically targetable., Graphical abstract, Highlights Gene expression is frequently uncoupled from copy number in cancers Uncoupling orchestrated by complex regulatory changes improves tumor fitness Targeting transcription factors can reestablish coupling and reduce tumor fitness, Mohanty et al. show that uncoupling of mRNA expression from copy number may be an important mechanism to maintain tumor fitness in aneuploid cancers, orchestrated by complex regulatory changes. These events present a potential therapeutic avenue and can be targeted by perturbing appropriate regulatory molecules, such as transcription factors.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains., Author summary The fungal pathogen A. fumigatus has a cosmopolitan distribution, which make its isolates genotypically and phenotypically diverse. The two commonly used reference clinical isolates, Af293 and CEA17, differ in growth, virulence and susceptibility to the immune system and drug treatment; however, their genomes are 99.8% identical with differences mainly at non-coding regions. The high genetic similarity between the two isolates implicates epigenetic controls for their physiological differences. Here, we analysed two chromatin modifications in the A. fumigatus genome and found that H3K4me3 profiles of the two isolates are similar, while H3K9me3 modification at the genome-wide level is highly variable. The H3K9me3 differences between Af293 and CEA17 are correlated with differences in transposon silencing and inactivation. In support of this, we observed independent gross chromosomal alterations in the Af293 strains of different laboratories, but not among CEA17 strains. Characterization of one of the Af293 variant strains, which has undergone chromosomal loss and amplification of two separate regions in chromosome VIII (Chr VIII), showed significant improvements in secondary metabolites production, growth and virulence compared to the original Af293 isolate. These results suggest that stochastic gross chromosomal changes occur more frequently in certain isolates. Overall, this study provides a link between epigenetic and genetic mechanisms for the adaptation of A. fumigatus to diverse environments.

Persistent supraphysiological serum estradiol (E2) levels during controlled ovarian hyper-stimulation (COH) have a detrimental effect on endometrial receptivity. In this study, we explored RNA expression and DNA methylation profiles from patients’ endometrium. The patients were divided into two groups: the COH cycle (n=3, hCG+7) group and normal cycle group (n=3, LH+5). Quantitative RT-PCR was used to validate the expression of selected differentially expressed genes (DEGs). Comparing natural and stimulated endometrium transcriptome profiles revealed 640 DEGs, with a > 2-fold change (FC) and p

Chunhe Zhang,1,* Shaowei Fu,1,* Luyue Wang,1,* Fang Wang,1 Dan Wu,1 Xiangyi Zhe,1 Huizhen Xin,1 Hongtao Li,1 Dongmei Li,1 Fuyuan Jin,1 Renfu Shao,2 Zemin Pan1 1Department of Biochemistry and Molecular Biology, School of Medicine, Shihezi University, Xinjiang Endemic and Ethnic Disease and Education Ministry Key Laboratory, Shihezi, Xinjiang 832002, People’s Republic of China; 2School of Science, Technology and Engineering, Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Queensland 4556, Australia*These authors contributed equally to this workCorrespondence: Zemin Pan; Xiangyi ZheDepartment of Biochemistry and Molecular Biology, School of Medicine, Shihezi University, Xinjiang Endemic and Ethnic Disease and Education Ministry Key Laboratory, Shihezi, People’s Republic of ChinaEmail panteacher89@sina.com; 342480821@qq.comIntroduction: The aim of this study was to determine whether ZNF582 gene methylation and tissue protein expression can be used as a tool with high sensitivity and specificity for cervical cancer screening. We analyzed the correlation between promoter methylation of ZNF582 gene and cervical cancer and high risk HPV16/18 infection.Methods: Tissue samples of normal cervical or chronic cervicitis (n=51), CIN (cervical intraepithelial neoplasia) (n=35), and cervical carcinoma (n=68) were tested for HPV16/18 infection by polymerase chain reaction (PCR). We also detected the methylation status of the ZNF582 gene promoter in the same tissues by methylation-specific PCR (MSP), then analyzed the correlation between ZNF582 promoter methylation and HPV16/18 infection. Immunohistochemistry was used to analyze ZNF582 gene expression in 152 cervical tissues. We detected ZNF582 mRNA expression in cervical tissues (including cancer and non-cancer) by real-time fluorescence quantitative PCR (qPCR).Results: Among 93 high-grade cervical lesions (CINII and above) and cervical cancer samples, 57 cases were positive for HPV16/18 infection and 36 cases were negative. ZNF582 gene methylation occurred in 9 out of 51 cases in normal cervical tissues (17.6%), 16 of 35 cases in CIN tissues (45.7%), and 50 of 68 cases in cervical cancer (73.5%). The differences in methylation rate of the three groups were statistically significant (P< 0.05). The ZNF582 methylation rate in the positive HPV16/18 infection group was 73.7%, while the negative group was 63.9%. Compared with normal tissues, ZNF582 protein was highly expressed in cervical cancer tissues, but mRNA expression was low.Conclusion: While ZNF582 protein is highly expressed in cervical cancer tissues, it was not sufficient for use as a standard for cervical cancer staging. On the other hand, ZNF582 promoter methylation had high specificity and sensitivity in detecting CINII and highly diseased cervical lesions and could be used as a diagnostic marker for cervical cancer of women.Keywords: cervical cancer, ZNF582 gene, promoter methylation, HPV16/18, gene expression

BACKGROUND Adult parasitic wasps take sugars to meet their energy needs and display different lifespans and fertility in response to different sugar sources. Pteromalus puparum is an endoparasitoid with a wide range of hosts, including many lepidopteran pests. As a potential natural enemy resource, the availability of sugar sources has profound effects for wasp applications and host populations dynamics. RESULTS We assessed the effect of feeding sucrose and honey on the lifespan of P. puparum in the range 0-40% (w/v). The results indicated a statistically significant positive effect of sucrose and honey solutions on the lifespan of P. puparum female adults. Correlation analyses confirmed a strong positive correlation between high concentrations of sugar and extended lifespan. The optimum concentration of sucrose solution for wasps was 20%, while 10% for honey. Then, we examined the expression patterns of 15 lifespan-related genes. The results showed that the relative expression levels of 14 genes were significantly correlated with the mean lifespan of sucrose-fed wasps, and six genes correlated with the mean lifespan of honey-fed wasps. In addition, the models for lifespan prediction were constructed. CONCLUSION We elaborated the quantitative effects of two sugar sources (sucrose and honey) on P. puparum lifespan, investigated the expression patterns of lifespan-related genes when fed different sugar sources, and developed round lifespan prediction models accordingly. This study provides a novel tool for studying the longevity regulating mechanisms of parasitic wasps, and may be instructive for mass-production of parasitoids as biological control agents. © 2020 Society of Chemical Industry.

Sex is a hallmark of eukaryotes but its evolution in microbial eukaryotes is poorly elucidated. Recent genomic studies revealed microbial eukaryotes possess genetic toolkit necessary for sexual reproduction. However, the mechanism of sexual development in a majority of microbial eukaryotes including amoebozoans is poorly characterized. The major hurdle in studying sex in microbial eukaryotes is lack of observational evidence, primarily due to its cryptic nature. In this study, we used a tractable fusing amoeba, Cochliopodium, to investigate sexual development using stage specific Differential Gene Expression (DGE) and cytological analyses. Both DGE and cytological results showed that most of the meiosis and sex-related genes are upregulated in Cochliopodium undergoing fusion in laboratory culture. Relative gene ontology (GO) category representations in unfused (single) and fused cells revealed functional skew of the fused transcriptome toward DNA metabolism, nucleus and ligases that are suggestive of a commitment to sexual development. While single cells GO categories were dominated by metabolic pathways and other processes indicative of vegetative phase. Our study provides strong evidence that the fused cells represent a sexual stage in Cochliopodium. Our findings have further implications in understanding the evolution and mechanism of inheritance involving multiparents in other eukaryotes with similar reproductive strategy.

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterized the transcriptional profile of cortical neurons from CdLS patients and found deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupted 3-D genome organization and transcriptional control in post-mitotic cortical mouse neurons. The genes affected belonged to similar gene ontology classes and showed significant numerical overlap with those deregulated in CdLS. Interestingly, gene expression was largely rescued by subsequent reconstitution of cohesin function. These experiments show that cohesin is continuously required for neuronal gene expression and provide a tractable approach for addressing mechanisms of neuronal dysfunction in CdLS.

The present study aimed to investigate the toxic effects of different amyloidogenic light-chains (LCs) on cardiomyocytes, and demonstrate the differentially expressed genes (DEGs) and signaling pathways that participate in this process. Cultured cardiomyocytes were treated with recombinant κ LC peptide (AL-09) or with serum from a patient diagnosed with multiple myeloma (λ LC) with cardiac involvement. The 6xHis peptide or serum from healthy patients was used as peptide control or serum control, respectively. Cell viability was determined using CCK-8 assay and apoptosis was analyzed by flow cytometry. The DEGs were detected by RNA sequencing (RNA-Seq), followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Changes in gene expression levels were confirmed by reverse transcription-quantitative PCR. The cell viability in the AL-09 peptide-treated (0.2 mg/ml) and patient serum-treated (1:10 dilution) cardiomyocytes decreased to 42 and -72% of the corresponding control groups. The extent of cell apoptosis increased in AL-09-treated cardiomyocytes compared with the control group. RNA-Seq showed 256 DEGs co-existed in the two paired groups, including 127 upregulated and 88 downregulated genes. The KEGG pathways for upregulated expressed genes included the ‘TGF-β signaling pathway’, the ‘Hedgehog signaling pathway’, the ‘ErbB signaling pathway’ and ‘lysine degradation’. The higher mRNA expression of bone morphogenetic protein (Bmp) 4, Bmp6, prostaglandin G/H synthase (Ptgs)1, Ptgs2, epiregulin, Tgfa and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 were confirmed. The KEGG pathways of downregulated expressed genes included genes involved with the ‘p53 signaling pathway’ and the ‘cell cycle’. The mRNA expression levels of E3 ubiquitin-protein ligase CCNB1IP1 showed significant downregulation in the AL-09 peptide group compared with those in the 6xHis peptide group. In conclusion, cardiomyocytes treated with amyloidogenic λ and κ LCs presented with decreased cell viability compared with controls. Cell apoptosis increased in κ LC-treated cells compared with controls. The gene expression profiles associated with transforming growth factor-β-bone morphogenetic protein, the receptor tyrosine-protein kinase erbB-2 signaling pathways, prostaglandins, collagen production, the p53 signaling pathway and the cell cycle were altered in light-chain-treated cardiomyocytes.

ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

Ophiocordyceps sinensisis a highly valued fungus that has been used as traditional Asian medicine. This fungus is one of the most important sources of income for the nomadic populations of the Tibetan Plateau. With global warming and excessive collection, the wildO. sinensisresources declined dramatically. The cultivation ofO. sinensishasn’t been fully operational due to the unclear genetic basis of the fruiting body development. Here, our study conducted pairwise comparisons between transcriptomes acquired from different growth stages ofO. sinensisincluding asexual mycelium (CM), developing fruiting body (DF) and mature fruiting body (FB). All RNA-Seq reads were aligned to the genome ofO. sinensisCO18 prior to comparative analyses. Cluster analysis showed that the expression profiles of FB and DF were highly similar compared to CM. Alternative splicing analysis (AS) revealed that the stage-specific splicing genes may have important functions in the development of fruiting body. Functional enrichment analyses showed that differentially expressed genes (DEGs) were enriched in protein synthesis and baseline metabolism during fruiting body development, indicating that more protein and energy might be required for fruiting body development. In addition, some fruiting body development-associated genes impacted by ecological factors were up-regulated in FB samples, such as the nucleoside diphosphate kinase gene (ndk), β subunit of the fatty acid synthase gene (cel-2) and the superoxide dismutase gene (sod). Moreover, the expression levels of several cytoskeletons genes were significantly altered during all these growth stages, suggesting that these genes play crucial roles in both vegetative growth and the fruiting body development. Quantitative PCR (qPCR) was used to validate the gene expression profile and the results supported the accuracy of the RNA-Seq and DEGs analysis. Our study offers a novel perspective to understand the underlying growth stage-specific molecular differences and the biology ofO. sinensisfruiting body development.

The photoreceptors UV RESISTANCE LOCUS 8 (UVR8) and CRYPTOCHROMES 1 and 2 (CRYs) play major roles in the perception of UV-B (280–315 nm) and UV-A/blue radiation (315–500 nm), respectively. However, it is poorly understood how they function in sunlight. The roles of UVR8 and CRYs were assessed in a factorial experiment with Arabidopsis thaliana wild-type and photoreceptor mutants exposed to sunlight for 6 h or 12 h under five types of filters with cut-offs in UV and blue-light regions. Transcriptome-wide responses triggered by UV-B and UV-A wavelengths shorter than 350 nm (UV-Asw) required UVR8 whereas those induced by blue and UV-A wavelengths longer than 350 nm (UV-Alw) required CRYs. UVR8 modulated gene expression in response to blue light while lack of CRYs drastically enhanced gene expression in response to UV-B and UV-Asw. These results agree with our estimates of photons absorbed by these photoreceptors in sunlight and with in vitro monomerization of UVR8 by wavelengths up to 335 nm. Motif enrichment analysis predicted complex signaling downstream of UVR8 and CRYs. Our results highlight that it is important to use UV waveband definitions specific to plants’ photomorphogenesis as is routinely done in the visible region., Academy of Finland, Grant/Award Numbers: 252548, 307335; Doctoral Program in Plant Sciences, University of Helsinki; Knowledge Foundation, Grant/Award Number: 20130164; Örebro Universitet; Suomen Kulttuurirahasto; Svenska Forskningsrådet Formas, Grant/Award Number: 942-2015-516