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Transcriptional regulation plays a crucial role in determining cell fate and disease, yet inferring the key regulators from gene expression data remains a significant challenge. Existing methods for estimating transcription factor (TF) activity often rely on static TF-gene interaction databases and cannot adapt to changes in regulatory mechanisms across different cell types and disease conditions. Here, we present a new algorithm - Transcriptional Inference using Gene Expression and Regulatory data (TIGER) - that overcomes these limitations by flexibly modeling activation and inhibition events, up-weighting essential edges, shrinking irrelevant edges towards zero through a sparse Bayesian prior, and simultaneously estimating both TF activity levels and changes in the underlying regulatory network. When applied to yeast and cancer TF knock-out datasets, TIGER outperforms comparable methods in terms of prediction accuracy. Moreover, our application of TIGER to tissue- and cell-type-specific RNA-seq data demonstrates its ability to uncover differences in regulatory mechanisms. Collectively, our findings highlight the utility of modeling context-specific regulation when inferring transcription factor activities. [ABSTRACT FROM AUTHOR]

A widely used organophosphate flame retardant (OPFR), triphenyl phosphate (TPP), is frequently detected in various environmental media and humans. However, there is little known on the human corneal epithelium of health risk when exposed to TPP. In this study, human normal corneal epithelial cells (HCECs) were used to investigate the cell viability, morphology, apoptosis, and mitochondrial membrane potential after they were exposed to TPP, as well as their underlying molecular mechanisms. We found that TPP decreased cell viability in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of 220 μM. Furthermore, TPP significantly induced HCEC apoptosis, decreased mitochondrial membrane potential in a dose-dependent manner, and changed the mRNA levels of the apoptosis biomarker genes (Cyt c, Caspase-9, Caspase-3, Bcl-2, and Bax). The results showed that TPP induced cytotoxicity in HCECs, eventually leading to apoptosis and changes in mitochondrial membrane potential. In addition, the caspase-dependent mitochondrial pathways may be involved in TPP-induced HCEC apoptosis. This study provides a reference for the human corneal toxicity of TPP, indicating that the risks of OPFR to human health cannot be ignored. [ABSTRACT FROM AUTHOR]

The MYB transcription factor gene family is among the most extensive superfamilies of transcription factors in plants and is involved in various essential functions, such as plant growth, defense, and pigment formation. Salvia nemorosa is a perennial herb belonging to the Lamiaceae family, and S. nemorosa has various colors and high ornamental value. However, there is little known about its genome-wide MYB gene family and response to flower color formation. In this study, 142 SnMYB genes (MYB genes of S. nemorosa) were totally identified, and phylogenetic relationships, conserved motifs, gene structures, and expression profiles during flower development stages were analyzed. A phylogenetic analysis indicated that MYB proteins in S. nemorosa could be categorized into 24 subgroups, as supported by the conserved motif compositions and gene structures. Furthermore, according to their similarity with AtMYB genes associated with the control of anthocyanin production, ten SnMYB genes related to anthocyanin biosynthesis were speculated and chosen for further qRT-PCR analyses. The results indicated that five SnMYB genes (SnMYB75, SnMYB90, SnMYB6, SnMYB82, and SnMYB12) were expressed significantly differently in flower development stages. In conclusion, our study establishes the groundwork for understanding the anthocyanin biosynthesis of the SnMYB gene family and has the potential to enhance the breeding of S. nemorosa. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) play pivotal roles in improving plant stress tolerance. To investigate the mechanism of BR regulation of salt tolerance in kiwifruit, we used 'Hongyang' kiwifruit as the test material. We exposed the plants to 150 mmol/L NaCl stress and irrigated them with exogenous BR (2,4-epibrassinolide). The phenotypic analysis showed that salt stress significantly inhibited photosynthesis in kiwifruit, leading to a significant increase in the H2O2 content of leaves and roots and a significant increase in Na+/K+, resulting in oxidative damage and an ion imbalance. BR treatment resulted in enhanced photosynthesis, reduced H2O2 content, and reduced Na+/K+ in leaves, alleviating the salt stress injury. Furthermore, transcriptome enrichment analysis showed that the differentially expressed genes (DEGs) related to BR treatment are involved in pathways such as starch and sucrose metabolism, pentose and glucuronate interconversions, and plant hormone signal transduction, among others. Among the DEGs involved in plant hormone signal transduction, those with the highest expression were involved in abscisic acid signal transduction. Moreover, there was a significant increase in the expression of the AcHKT1 gene, which regulates ion transduction, and the antioxidant enzyme AcFSD2 gene, which is a key gene for improving salt tolerance. The data suggest that BRs can improve salt tolerance by regulating ion homeostasis and reducing oxidative stress. [ABSTRACT FROM AUTHOR]

Purpose: Tenosynovial giant cell tumour (TGCT) is a benign hyperplastic and inflammatory disease of the joint synovium or tendon sheaths, which may be misdiagnosed due to its atypical symptoms and imaging features. We aimed to identify biomarkers with high sensitivity and specificity to aid in diagnosing TGCT. Methods: Two scRNA-seq datasets (GSE210750 and GSE152805) and two microarray datasets (GSE3698 and GSE175626) were downloaded from the Gene Expression Omnibus (GEO) database. By integrating the scRNA-seq datasets, we discovered that the osteoclasts are abundant in TGCT in contrast to the control. The single-sample gene set enrichment analysis (ssGSEA) further validated this discovery. Differentially expressed genes (DEGs) of the GSE3698 dataset were screened and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were conducted. Osteoclast-specific up-regulated genes (OCSURGs) were identified by intersecting the osteoclast marker genes in the scRNA-seq and the up-regulated DEGs in the microarray and by the least absolute shrinkage and selection operator (LASSO) regression algorithm. The expression levels of OCSURGs were validated by an external dataset GSE175626. Then, single gene GSEA, protein–protein interaction (PPI) network, and gene-drug network of OCSURGs were performed. Result: 22 seurat clusters were acquired and annotated into 10 cell types based on the scRNA-seq data. TGCT had a larger population of osteoclasts compared to the control. A total of 159 osteoclast marker genes and 104 DEGs (including 61 up-regulated genes and 43 down-regulated genes) were screened from the scRNA-seq analysis and the microarray analysis. Three OCSURGs (MMP9, SPP1, and TYROBP) were finally identified. The AUC of the ROC curve in the training and testing datasets suggested a favourable diagnostic capability. The PPI network results illustrated the protein–protein interaction of each OCSURG. Drugs that potentially target the OCSURGs were predicted by the DGIdb database. Conclusion: MMP9, SPP1, and TYROBP were identified as osteoclast-specific up-regulated genes of the tenosynovial giant cell tumour via bioinformatic analysis, which had a reasonable diagnostic efficiency and served as potential drug targets. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii (C. reinhardtii) is a single-cell green alga that can be easily genetically manipulated. With its favorable characteristics of rapid growth, low cost, non-toxicity, and the ability for post-translational protein modification, C. reinhardtii has emerged as an attractive option for the biosynthesis of various valuable products. To enhance the expression level of exogenous genes and overcome the silencing of foreign genes by C. reinhardtii, synthetic promoters such as the chimeric promoter AR have been constructed and evaluated. In this study, a synthetic promoter GA was constructed by hybridizing core fragments from the natural promoters of the acyl carrier protein gene (ACP2) and the glutamate dehydrogenase gene (GDH2). The GA promoter exhibited a significant increase (7 times) in expressing GUS, over the AR promoter as positive control. The GA promoter also displayed a strong responsiveness to blue light (BL), where the GUS expression was doubled compared to the white light (WL) condition. The ability of the GA promoter was further tested in the expression of another exogenous cadA gene, responsible for catalyzing the decarboxylation of lysine to produce cadaverine. The cadaverine yield driven by the GA promoter was increased by 1–2 times under WL and 2–3 times under BL as compared to the AR promoter. This study obtained, for the first time, a blue light-responsive GDH2 minimal fragment in C. reinhardtii, which delivered a doubling effect under BL when used alone or in hybrid. Together with the strong GA synthetic promoter, this study offered useful tools of synthetic biology to the algal biotechnology field. [ABSTRACT FROM AUTHOR]

Objective: Daweizi (DWZ) is a famous indigenous pig breed in China and characterized by tender meat and high fat percentage. However, the expression profiles and functions of transcripts in DWZ pigs is still in infancy. The object of this study was to depict the transcript profiles in DWZ pigs and screen the potential pathway influence adipogenesis and fat deposition, Methods: Histological analysis of backfat tissue was firstly performed between DWZ and lean-type Yorkshire pigs, and then RNA sequencing technology was utilized to explore miRNAs, lncRNAs and mRNAs profiles in backfat tissue. 18 differentially expressed (DE) transcripts were randomly selected for quantitative real-time polymerase chain reaction (QPCR) to validate the reliability of the sequencing results. Finally, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were conducted to investigate the potential pathways influence adipocyte differentiation, adipogenesis and lipid metabolism, and a schematic model was further proposed. Results: A total of 1,625 differentially expressed transcripts were identified in DWZ pigs, including 27 upregulated and 45 downregulated miRNAs, 64 upregulated and 119 downregulated lncRNA, 814 upregulated and 556 downregulated mRNAs. QPCR analysis exhibited strong consistency with the sequencing data. GO and KEGG analysis elucidated that the differentially expressed transcripts were mainly associated with cell growth and death, signal transduction, peroxisome proliferator-activated receptors (PPAR), AMP-activated protein kinase (AMPK), PI3K-Akt, adipocytokine and foxo signaling pathways, all of which are strongly involved in cell development, lipid metabolism and adipogenesis. Further analysis indicated that the BGIR9823_87926/miR-194a-5p/AQP7 network may be effective in the process of adipocyte differentiation or adipogenesis. Conclusion: Our study provides comprehensive insights into the regulatory network of backfat deposition and lipid metabolism in pigs from the point of view of miRNAs, lncRNAs and mRNAs. [ABSTRACT FROM AUTHOR]

Background: Anthocyanins are widely applied as a marker for haploid identification after haploid induction in maize. However, the factors affecting anthocyanin biosynthesis in immature embryos and the genes regulating this process remain unclear. Results: In this study, we analyzed the influence of genetic background of the male and female parents, embryo age and light exposure on anthocyanin accumulation in embryos. The results showed that light exposure was the most crucial factor enhancing the pigmentation of immature embryos. The identification accuracy of haploid embryos reached 96.4% after light exposure, but was only 11.0% following dark treatment. The total anthocyanin content was 7-fold higher in immature embryos cultured for 24 h under light conditions compared to embryos cultured in the dark. Transcriptome analysis revealed that the differentially expressed genes between immature embryos cultured for 24 h in dark and light chambers were significantly enriched in the pathways of flavonoid, flavone, flavonol and anthocyanin biosynthesis. Among the genes involved in anthocyanin biosynthesis, five up-regulated genes were identified: F3H, DFR, ANS, F3′H and the MYB transcription factor-encoding gene C1. The expression patterns of 14 selected genes were confirmed using quantitative reverse transcription-polymerase chain reaction. Conclusion: Light is the most important factor facilitating anthocyanin accumulation in immature embryos. After 24 h of exposure to light, the expression levels of the structural genes F3H, DFR, ANS, F3′H and transcription factor gene C1 were significantly up-regulated. This study provides new insight into the factors and key genes regulating anthocyanin biosynthesis in immature embryos, and supports improved efficiency of immature haploid embryo selection during doubled haploid breeding of maize. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is a typical endocrine-disrupting chemical (EDC) used worldwide. Considering its adverse effects, BPA has been banned or strictly restricted in some nations, and many analogs have been introduced to the market. In this study, we selected three representative substitutes, BPS, BPF, and BPAF, along with BPA, to assess the developmental and reproductive effects on Daphnia magna. The F0 generation was exposed to bisphenols (BPs) at an environmentally relevant concentration (100 μg/L) for 21 d; then the embryo spawn at day 21 was collected. Behavior traits, the activity of antioxidant enzymes, and gene transcription were evaluated at three developmental stages (days 7, 14, and 21). Notably, body length, heart rate, and thoracic limb beating were significantly decreased, and D. magna behaved more sluggishly in the exposed group. Moreover, exposure to BPs significantly increased the antioxidant enzymatic activities, which indicated that BPs activated the antioxidant defense system. Additionally, gene expression indicated intergenerational effects in larvae, particularly in the BPAF group. In conclusion, BPA analogs such as BPF and BPAF showed similar or stronger reproductive and developmental toxicity than BPA in D. magna. These findings collectively deepen our understanding of the toxicity of BPA analogs and provide empirical evidence for screening safe alternatives to BPA. [ABSTRACT FROM AUTHOR]

Styrax tonkinensis, whose seeds are rich in unsaturated fatty acids (UFAs), is a high oil value tree species, and the seed oil has perfect biodiesel properties. Therefore, the elucidation of the effect of 24-epibrassinolide (EBL) on fatty acid (FA) concentration and the expression of FA biosynthesis-related genes is critical for deeply studying the seed oil in S. tonkinensis. In this study, we aimed to investigate the changing trend of FA concentration and composition and identify candidate genes involved in FA biosynthesis under EBL treatment using transcriptome sequencing and GC-MS. The results showed that 5 μmol/L of EBL (EBL5) boosted the accumulation of FA and had the hugest effect on FA concentration at 70 days after flowering (DAF). A total of 20 FAs were identified; among them, palmitic acid, oleic acid, linoleic acid, and linolenic acid were the main components. In total, 117,904 unigenes were detected, and the average length was 1120 bp. Among them, 1205 unigenes were assigned to 'lipid translations and metabolism' in COG categories, while 290 unigenes were assigned to 'biosynthesis of unsaturated fatty acid' in KEGG categories. Twelve important genes related to FA biosynthesis were identified, and their expression levels were confirmed by quantitative real-time PCR. KAR, KASIII, and accA, encoding FA biosynthesis-related enzymes, all expressed the highest at 70 DAF, which was coincident with a rapid rise in FA concentration during seed development. FAD2 and FATB conduced to UFA and saturated fatty acids (SFA) accumulation, respectively. EBL5 induced the expression of FA biosynthesis-related genes. The concentration of FA was increased after EBL5 application, and EBL5 also enhanced the enzyme activity by promoting the expression of genes related to FA biosynthesis. Our research could provide a reference for understanding the FA biosynthesis of S. tonkinensis seeds at physiological and molecular levels. [ABSTRACT FROM AUTHOR]

Simple Summary: The DKK gene family plays an important role in immune mechanisms and embryonic development, but studies on Pelodiscus sinensis have not been reported. In this study, for the first time in the context of P. sinensis, we analyzed the relevant features of the DKK gene family using bioinformatics methods and constructed transcriptional profiles and tissue expression profiles. The DKK1 and DKK3 genes were found to be highly expressed in the ovaries, while DKKL1 and DKK4 were significantly differentiated in the testes. In this study, we preliminarily investigated the function of the DKK gene family in the context of P. sinensis, and this study lays the foundation for further systematic research on the molecular mechanism of sex differentiation in P. sinensis. The DKK family is a canonical small family of WNT antagonists. Though recent studies have suggested that the DKK gene family may be involved in sex differentiation in Pelodiscus sinensis, there are still a lot of things about the DKK gene family that we do not know. In this study, we used bioinformatics methods to identify members of the DKK gene family in P. sinensis and analyzed their phylogeny, covariance, gene structure, structural domains, promoter conserved sites, signal peptides, gonadal transcription factors, transcriptional profiles, and tissue expression profiles. Additionally, qRT-PCR results were utilized for the validation and preliminary investigation of the function of the DKK gene family in P. sinensis. The results showed that the DKK gene family is divided into six subfamilies, distributed on six different chromosomal scaffolds containing different gene structures and conserved motifs with the same structural domains, and all of the members were secreted proteins. Our transcriptional profiling and embryonic expression analysis showed that DKKL1 and DKK4 were significantly expressed in the testes, whereas DKK1 and DKK3 were significantly upregulated in the ovaries. This suggests a potential function in sex differentiation in P. sinensis. Our results may provide a basic theoretical basis for the sex differentiation process in P. sinensis. [ABSTRACT FROM AUTHOR]

3-Methylbutanal is a key volatile compound that imparts a nutty flavor to Cheddar cheese. Lactococcus lactis has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese. However, the mechanism of action and genetic diversity of this bacterium for 3-methylbutanal biosynthesis remains unclear. In this study, we investigated the association between the L. lactis genotype and phenotype in the biosynthesis of 3-methylbutanal via both direct and indirect pathways. Fourteen strains of L. lactis were screened for the capacity to produce 3-methylbutanal, and strain 408 (>140 μ M) produced the highest among all tested strains, which exhibited both α-keto acid decarboxylase and α-ketoacid dehydrogenase activities. Furthermore, the results of a sodium meta-arsenite inhibition experiment showed that the 3-methylbutanal–producing capacities of each strain declined to various degrees. The kdcA gene, which encodes the direct pathway component α-ketoacid decarboxylase, was detected in 4 of the 14 strains, of which only strain 408 contained the full-length gene. We then characterized the genes associated with the indirect pathway by detecting the expression levels of the pdh gene cluster, ack , and pta , which were expressed at relatively higher levels in a high-yield strain than in a low-yield strain. As a result, these L. lactis strains were divided into 3 categories according to gene diversity, gene expression, and 3-methylbutanal production. The results of this study refine our knowledge of the genetic determinants of 3-methylbutanal biosynthesis in L. lactis and explain the effect of both synthesis pathways on 3-methylbutanal production. [Display omitted] [ABSTRACT FROM AUTHOR]

The rapid development of single-cell RNA sequencing (scRNA-Seq) technology provides strong technical support for accurate and efficient analyzing single-cell gene expression data. However, the analysis of scRNA-Seq is accompanied by many obstacles, including dropout events and the curse of dimensionality. Here, we propose the scGMAI, which is a new single-cell Gaussian mixture clustering method based on autoencoder networks and the fast independent component analysis (FastICA). Specifically, scGMAI utilizes autoencoder networks to reconstruct gene expression values from scRNA-Seq data and FastICA is used to reduce the dimensions of reconstructed data. The integration of these computational techniques in scGMAI leads to outperforming results compared to existing tools, including Seurat, in clustering cells from 17 public scRNA-Seq datasets. In summary, scGMAI is an effective tool for accurately clustering and identifying cell types from scRNA-Seq data and shows the great potential of its applicative power in scRNA-Seq data analysis. The source code is available at https://github.com/QUST-AIBBDRC/scGMAI/. [ABSTRACT FROM AUTHOR]

As a kind of endocrine-disrupting chemicals, BPA may affect the human placenta. Due to consumer unease about BPA, many manufacturers are using alternatives to BPA, such as BPS. However, some reports suggest that BPS may produce similar results to BPA. To understand how BPA/BPS leads to reduced synthesis of placental estradiol (E2), we conducted studies using a human choriocarcinoma cell (JEG-3) model for research. In this study. Elisa assay revealed that both BPA/BPS exposures decreased E2 synthesis in JEG-3 cells. The results of RT-PCR showed that both BPA and BPS could reduce the mRNA expression of CYP19A1, a key enzyme for E2 synthesis in JEG-3 cells. In addition, Western blot assay showed that BPA/BPS-induced ER-stress PERK/eIF2α/ATF4 signaling protein expression was increased. The expression of ROS in cells after exposure to BPA/BPS was detected using the 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) method. The results of this experiment showed that BPA/BPS significantly induced an inhibition of ROS in JEG-3 cells. The present study concluded that, firstly, BPS exposure induced almost the same effect as BPA in reducing E2 synthesis in JEG-3 cells. Second, BPA/BPS exposure may reduce E2 synthesis in JEG-3 cells by increasing ROS levels and thus activating endoplasmic reticulum stress. [Display omitted] • BPS, a substitute for BPA, causes almost the same effect as BPA in reducing E2 in JEG-3 cells. • BPA and BPS activate ER stress. • Inhibitors reversed BPA or BPS-induced estradiol decline in JEG-3 cells. [ABSTRACT FROM AUTHOR]

Sulfate widely co-exists with polychlorinated biphenyls (PCBs) at various concentrations in the subsurface environment. Previous studies have suggested that sulfate often hampers microbial degradation of aliphatic chlorinated solvents such as chloroethenes. However, the impact of sulfate on microbial reductive dechlorination of aromatic PCBs and the underlying mechanisms have received limited attention. Likewise, strategies to mitigate such inhibition remain scarce. Here we found that the mechanisms and mitigation strategies of sulfate inhibition on PCB dechlorination were substrate-dependent. Under electron donor-limiting conditions, even a low concentration of sulfate (2 mM) resulted in a decreased PCB dechlorination rate by 88.7% in a co-culture comprising Dehalococcoides mccartyi CG1 and the sulfate-reducing bacterium Desulfovibrio desulfuricans F1, an inhibition which was attributed to the competition for electron donor between sulfate reduction and PCB dechlorination. As expected, re-amendment of 5 mM lactate effectively re-initiated PCB dechlorination. However, in the presence of a higher concentration of sulfate (5 mM), the PCB dechlorination rate in the co-culture was 77.7% lower than in the control, even with excessive electron donor supply. This inhibition was linked to high concentration of sulfide (∼5 mM) produced from sulfate reduction, as suggested by high availability of electron donor, recovery of dechlorination activity after removal of sulfide, and negligible influence of sulfate on PCB dechlorination in the axenic culture of D. mccartyi CG1. Indeed, sulfide (>5 mM) was found to directly suppress expression of PCB-dechlorinating reductive dehalogenase gene. The highest transcriptional level of pcbA1 was 2.9 ± 0.3 transcripts·cell−1 in the presence of ∼5 mM sulfide, which was increased to 37.4 ± 5.0 transcripts·cell−1 when sulfide was removed. Under this scenario, introduction of ferrous salts (5 mM) efficiently alleviated sulfide inhibition on PCB dechlorination. Interestingly, the augmentation of methanogens in the co-culture was also effective in mitigating sulfide inhibition on PCB dechlorination, offering a new approach to protect Dehalococcoides under sulfide stress. Collectively, these findings deepen our understanding of the influence of sulfate on microbial reductive dechlorination of PCBs and contribute to developing appropriate strategies based on geochemical conditions to alleviate sulfate inhibition during bioremediation of PCB-contaminated sites. [Display omitted] • Sulfate inhibited microbial reductive dechlorination of PCBs via different mechanisms; • Fatty acid supplementation alleviated electron donor competition when SO 4 2− ≤ 2 mM; • Sulfide lowered rate and extent of PCB dechlorination when SO 4 2− ≥ 5 mM; • Amendment of ferrous salts mitigated the inhibition of PCB dechlorination caused by sulfide; • Aggregates of methanogens increased the tolerance of D. mccartyi to the sulfide. [ABSTRACT FROM AUTHOR]

Motivation Genome-wide clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 screen has been widely used to interrogate gene functions. However, the rules to design better libraries beg further refinement. Results We found single guide RNA (sgRNA) outliers are characterized by higher G-nucleotide counts, especially in regions distal from the PAM motif and are associated with stronger off-target activities. Furthermore, using non-targeting sgRNAs as negative controls lead to strong bias, which can be mitigated by using sgRNAs targeting multiple 'safe harbor' regions. Custom-designed screens confirmed our findings and further revealed that 19 nt sgRNAs consistently gave the best signal-to-noise ratio. Collectively, our analysis motivated the design of a new genome-wide CRISPR/Cas9 screen library and uncovered some intriguing properties of the CRISPR-Cas9 system. Availability and implementation The MAGeCK workflow is available open source at https://bitbucket.org/liulab/mageck%5fnest under the MIT license. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Current study aims to investigate the biological function of circular RNA (circRNA, circ_0000337) in cervical cancer (CC). Bioinformatic analyses were used to predict targets for circ_0000337 and miR-155-5p, and analyze the gene expression differences between cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tissues and normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to assess mRNA and protein expressions of circ_0000337, microRNA-155-5p (miR-155-5p) and member RAS oncogene family (RAB3B), respectively. Following the establishment of gain/loss-of-function models, CCK-8 was performed to evaluate cell proliferation. Bioinformatics analysis, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) were used to identify the interaction in circ_0000337, miR-155-5p, and RAB3B. Circ_0000337 and RAB3B were upregulated, while miR-155-5p was downregulated in CC tissues and cell lines. circ_0000337 overexpression promoted cell proliferation, circ_0000337 knock down inhibited cell proliferation by sponging miR-155-5p. RAB3B was a target of miR-155-5p which was positively regulated by circ_0000337. In the collected CC tissues, there was a negative correlation between miR-155-5p and circ_0000337 or RAB3B, and a positive correlation between circ_0000337 and RAB3B. miR-155-5p was positively, while RAB3B was negatively correlated with OS in patients with CC, and they were negatively correlated. In conclusion, circ_0000337 upregulates RAB3B by sponging miR-155-5p to promote CC cell proliferation. [ABSTRACT FROM AUTHOR]

Recent studies of somatic and germline mutations have led to the identification of a number of factors that influence point mutation rates, including CpG methylation, expression levels, replication timing, and GC content. Intriguingly, some of the effects appear to differ between soma and germline: in particular, whereas mutation rates have been reported to decrease with expression levels in tumors, no clear effect has been detected in the germline. Distinct approaches were taken to analyze the data, however, so it is hard to know whether these apparent differences are real. To enable a cleaner comparison, we considered a statistical model in which the mutation rate of a coding region is predicted by GC content, expression levels, replication timing, and two histone repressive marks. We applied this model to both a set of germline mutations identified in exomes and to exonic somatic mutations in four types of tumors. Most determinants of mutations are shared: notably, we detected an effect of expression levels on both germline and somatic mutation rates. Moreover, in all tissues considered, higher expression levels are associated with greater strand asymmetry of mutations. However, mutation rates increase with expression levels in testis (and, more tentatively, in ovary), whereas they decrease with expression levels in somatic tissues. This contrast points to differences in damage or repair rates during transcription in soma and germline. [ABSTRACT FROM AUTHOR]

Lung cancer accounts for the vast majority of cancer-related deaths worldwide, and aberrant miRNA expression is commonly observed as the disease progresses. The current study aimed to determine the role of miR-4757-3p in the development of lung cancer. The real-time PCR test was performed to determine the expression of miR-4757-3p in lung cancer cell lines. miR-4757-3p was downregulated in A549 cells. CCK8 and transwell assays demonstrated that overexpression of miR-4757-3p significantly reduced A549 cell invasion and migration. Bioinformatic analysis by the TargetScan database predicted the possible targets of miR-4757-3p. A luciferase activity test was used to determine the direct relationship between miR-4757-3p, Wnt5a, and Wnt8b. The overexpression of miR-4757-3p drastically inhibited the expression of Wnt5a and Wnt8b. Furthermore, we discovered that silencing Wnt5a and Wnt8b significantly lowered β-catenin expression and hampered invasion and migration. Finally, miR-4757-3p inhibited lung cancer cell migration and invasion by inhibiting the activation of the Wnt signaling pathway. Our study provided evidence that miR-4757-3p could be developed as an indicator or an anticancer target in the clinical application. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs), characterized by their pluripotency and capacity for self-renewal, are driven by a complex gene expression program composed of several regulatory mechanisms. These mechanisms collaborate to maintain the delicate balance of pluripotency gene expression and their disruption leads to loss of pluripotency. In this review, we provide an extensive overview of the key pillars of mESC pluripotency by elaborating on the various essential transcription factor networks and signaling pathways that directly or indirectly support this state. Furthermore, we consider the latest developments in the role of epigenetic regulation, such as noncoding RNA signaling or histone modifications. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndrome (MDS) is cytogenetically heterogeneous and retains variable risk for acute myeloid leukemia transformation. Though not yet fully understood, there is an association between genetic abnormalities and defects in gene expression. The functional role for infrequent cytogenetic alteration remains unclear. An uncommon chromosomic abnormality is the presence of the Philadelphia (Ph) chromosome. Here, we report a patient with Ph+ MDS treated with low dose Dasatinib who achieved hematologic response for 7 months. In addition, we also examined the English literature on all de novo Ph + MDS cases between 1996 and 2015 to gain insight into clinical features and outcome. [ABSTRACT FROM AUTHOR]

The size of leaves is a vital factor in the development and overall biomass of a plant, serving as a key indicator of how a plant adapts to its environment. Rhamnus heterophylla, a species known for its heteromorphic leaves of varying sizes, presents an intriguing case for studying leaf development at the molecular level. To gain insights for further studies on the underlying mechanisms, we constructed a comprehensive reference transcriptome database using both SMART sequencing and Illumina RNA-seq technologies. Our analysis of the transcriptome data identified 88,546 isoforms, featuring an N50 size of 2386 base pairs. Furthermore, we identified 2932 transcription factors from 55 gene families, along with 14,947 unigenes that underwent alternative splicing. By comparing the gene expression patterns between large and small leaves, we pinpointed 982 differentially expressed genes (DEGs). Among these DEGs, 116 genes exhibit significantly greater activity in small leaves, while 866 genes display significantly greater activity in large leaves. Functional enrichment analyses revealed the significant involvement of these DEGs in various hormone signaling pathways. Notably, we detected a significant decrease in the expression of several genes associated with auxin synthesis, such as ARFs, GRF8, and IAA27, in small leaves. This finding sheds light on their potential role in leaf size regulation in R. heterophylla, providing valuable insights into the genes underlying this mechanism. [ABSTRACT FROM AUTHOR]

Xin-Ji-Er-Kang (XJEK) inhibited cardiovascular remodeling in hypertensive mice in our previous studies. We hypothesized that XJEK may prevent isoproterenol (ISO)-induced myocardial hypertrophy (MH) in mice by ameliorating oxidative stress (OS) through a mechanism that may be related to the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1(HO-1) pathways. Forty SPF male Kunming mice were randomized into 5 groups (n = 8 mice per group): control group, MH group, MH + different doses of XJEK (7.5 g/kg/day and 10 g/kg/day), and MH + metoprolol (60 mg/kg/day). On the eighth day after drug treatment, electrocardiogram (ECG) and echocardiography were performed, the mice were sacrificed, and blood and heart tissues were collected for further analysis. XJEK administration markedly ameliorated cardiovascular remodeling (CR), as manifested by a decreased HW/BW ratio and CSA and less collagen deposition after MH. XJEK administration also improved MH, as evidenced by decreased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) levels. XJEK also suppressed the decreased superoxide dismutase (SOD) and catalase (CAT) activities and increased malondialdehyde (MDA) levels in serum of mice with MH. XJEK-induced oxidative stress may be related to potentiating Nrf2 nuclear translocation and HO-1 expression compared with the MH groups. XJEK ameliorates MH by activating the Nrf2/HO-1 signaling pathway, suggesting that XJEK is a potential treatment for MH. [ABSTRACT FROM AUTHOR]

We mined the literature for proteomics data to examine the occurrence and metastasis of prostate cancer (PCa) through a bioinformatics analysis. We divided the differentially expressed proteins (DEPs) into two groups: the group consisting of PCa and benign tissues (P&b) and the group presenting both high and low PCa metastatic tendencies (H&L). In the P&b group, we found 320 DEPs, 20 of which were reported more than three times, and DES was the most commonly reported. Among these DEPs, the expression levels of FGG, GSN, SERPINC1, TPM1, and TUBB4B have not yet been correlated with PCa. In the H&L group, we identified 353 DEPs, 13 of which were reported more than three times. Among these DEPs, MDH2 and MYH9 have not yet been correlated with PCa metastasis. We further confirmed that DES was differentially expressed between 30 cancer and 30 benign tissues. In addition, DEPs associated with protein transport, regulation of actin cytoskeleton, and the extracellular matrix (ECM)- receptor interaction pathway were prevalent in the H&L group and have not yet been studied in detail in this context. Proteins related to homeostasis, the wound-healing response, focal adhesions, and the complement and coagulation pathways were overrepresented in both groups. Our findings suggest that the repeatedly reported DEPs in the two groups may function as potential biomarkers for detecting PCa and predicting its aggressiveness. Furthermore, the implicated biological processes and signaling pathways may help elucidate the molecular mechanisms of PCa carcinogenesis and metastasis and provide new targets for clinical treatment. [ABSTRACT FROM AUTHOR]

In mammals, USP18 (ubiquitin-specific protease 18) is an interferon (IFN) inducible protein and plays a role in regulation of IFN response upon viral infection. In this study, we first cloned a USP18 homologous gene from virally-infected crucian carp ( Carassius auratus ) blastula embryonic (CAB) cells, and later found in other fish species including zebrafish. All fish USP18 genes have 10 exons and 9 introns comparable to 11 exons and 10 introns in non-fish vertebrates. Expression analysis revealed that fish USP18 was significantly induced in vitro and in vivo by IFN and IFN stimuli. Using promoter-driven luciferase reporter assay system to explore the molecular mechanism underlying fish USP18 expression, fish USP18 was identified as a typical interferon (IFN)-stimulated gene (ISG). Intracellular poly(I:C)-triggered zebrafish USP18 expression was regulated through RLR-IFN pathway, which was consistent with the fact that fish USP18 gene promoter contained two typical IFN-stimulated response elements (ISREs). Further mutation assays revealed that the distant ISRE motif primarily contributed to the induction of zebrafish USP18 by fish IFN and IFN stimuli. Functionally, fish USP18 inhibited poly(I:C)- and IFN-triggered activation of a common ISRE-containing promoter, and attenuated transcriptional expression of some ISGs including Stat1 and PKZ by recombinant IFN. Finally, we found that fish USP18 protein was expressed in cytoplasm and exhibited an ability to interact with ISG15. These results indicate that fish USP18 likely exerts its function similar to mammalian homologs. [ABSTRACT FROM AUTHOR]

Background: Histomonas meleagridis is an anaerobic, intercellular parasite, which infects gallinaceous birds such as turkeys and chickens. In recent years, the reemergence of Histomoniasis has caused serious economic losses as drugs to treat the disease have been banned. At present, H. meleagridis research focuses on virulence, gene expression analysis, and the innate immunity of the host. However, there are no studies on the differentially expressed miRNAs (DEMs) associated with the host inflammatory and immune responses induced by H. meleagridis. In this research, high-throughput sequencing was used to analyze the expression profile of cecum miRNA at 10 and 15 days post-infection (DPI) in chickens infected with Chinese JSYZ-F strain H. meleagridis. Results: Compared with the controls, 94 and 127 DEMs were found in cecum of infected chickens at 10 DPI (CE vs CC) and 15 DPI (CEH vs CCH), respectively, of which 60 DEMs were shared at two-time points. Gene Ontology (GO) functional enrichment analysis of the target genes of DEMs indicated that 881 and 1027 GO terms were significantly enriched at 10 and 15 DPI, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG, www.kegg.jp/kegg/kegg1.html) pathway enrichment analysis of the target genes of DEMs demonstrated that 5 and 3 KEGG pathways were significantly enriched at 10 and 15 DPI, respectively. For previous uses, the Kanehisa laboratory have happily provided permission. The integrated analysis of miRNA–gene network revealed that the DEMs played important roles in the host inflammatory and immune responses to H. meleagridis infection by dynamically regulating expression levels of inflammation and immune-related cytokines. Conclusion: This article not only suggested that host miRNA expression was dynamically altered by H. meleagridis and host but also revealed differences in the regulation of T cell involved in host responses to different times H. meleagridis infection. [ABSTRACT FROM AUTHOR]

Motivation The Gene Network Estimation Tool (GNET) is designed to build gene regulatory networks (GRNs) from transcriptomic gene expression data with a probabilistic graphical model. The data preprocessing, model construction and visualization modules of the original GNET software were developed on different programming platforms, which were inconvenient for users to deploy and use. Results Here, we present GNET2, an improved implementation of GNET as an integrated R package. GNET2 provides more flexibility for parameter initialization and regulatory module construction based on the core iterative modeling process of the original algorithm. The data exchange interface of GNET2 is handled within an R session automatically. Given the growing demand for regulatory network reconstruction from transcriptomic data, GNET2 offers a convenient option for GRN inference on large datasets. Availability and implementation The source code of GNET2 is available at https://github.com/jianlin-cheng/GNET2. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Extracting biologically meaningful information from the continuing flood of genomic data is a major challenge in the life sciences. Codon usage bias (CUB) is a general feature of most genomes and is thought to reflect the effects of both natural selection for efficient translation and mutation bias. Here we present a mechanistically interpretable, Bayesian model (ribosome overhead costs Stochastic Evolutionary Model of Protein Production Rate [ROC SEMPPR]) to extract meaningful information from patterns of CUB within a genome. ROC SEMPPR is grounded in population genetics and allows us to separate the contributions of mutational biases and natural selection against translational inefficiency on a gene-by-gene and codon-by-codon basis. Until now, the primary disadvantage of similar approaches was the need for genome scale measurements of gene expression. Here, we demonstrate that it is possible to both extract accurate estimates of codon-specific mutation biases and translational efficiencies while simultaneously generating accurate estimates of gene expression, rather than requiring such information. We demonstrate the utility of ROC SEMPPR using the Saccharomyces cerevisiae S288c genome. When we compare our model fits with previous approaches we observe an exceptionally high agreement between estimates of both codon-specific parameters and gene expression levels (ρ>0.99 in all cases). We also observe strong agreement between our parameter estimates and those derived from alternative data sets. For example, our estimates of mutation bias and those from mutational accumulation experiments are highly correlated (ρ=0.95). Our estimates of codon-specific translational inefficiencies and tRNA copy number-based estimates of ribosome pausing time (ρ=0.64), and mRNA and ribosome profiling footprint-based estimates of gene expression (ρ=0.53−0.74) are also highly correlated, thus supporting the hypothesis that selection against translational inefficiency is an important force driving the evolution of CUB. Surprisingly, we find that for particular amino acids, codon usage in highly expressed genes can still be largely driven by mutation bias and that failing to take mutation bias into account can lead to the misidentification of an amino acid’s “optimal” codon. In conclusion, our method demonstrates that an enormous amount of biologically important information is encoded within genome scale patterns of codon usage, accessing this information does not require gene expression measurements, but instead carefully formulated biologically interpretable models. [ABSTRACT FROM AUTHOR]

Aims Growing evidences indicate that microRNAs (miRNAs) are involved in cardiac hypertrophy development. Multiple miRNAs have been identified as diagnostic and prognostic biomarkers of cardiac hypertrophy, as well as potential therapeutic tools. The present study aimed to investigate the functions and regulatory mechanisms of miR-21-3p in cardiac hypertrophy. Methods and results Decreased expression of miR-21-3p was observed in cardiac hypertrophy induced by transverse aortic constriction (TAC) and angiotensin (Ang) II infusion in mice. To further explore the role of miR-21-3p in cardiac hypertrophy, rAAV-miR-21-3p was administered intravenously in mice. Overexpression of miR-21-3p markedly suppressed TAC-induced cardiac hypertrophy and also blocked Ang II-induced cardiac hypertrophy as determined by cardiac function measurement and biomarker detection. Furthermore, western blot assays showed that histone deacetylase-8 (HDAC8) was silenced by miR-21-3p, and luciferase reporter assays showed that miR-21-3p binds to the 3′ UTR of HDAC8. Moreover, re-expression of HDAC8 attenuated miR-21-3p-mediated suppression of cardiac hypertrophy by enhancing phospho-Akt and phospho-Gsk3β expression. Conclusion Our data reveal a role of miR-21-3p in regulating HDAC8 expression and Akt/Gsk3β pathway, and suggest that modulation of miR-21-3p levels may provide a therapeutic approach for cardiac hypertrophy. [ABSTRACT FROM PUBLISHER]

Background/Aims: Angiotensin II (Ang II) plays a critical role in regulating vascular inflammatory diseases, such as atherosclerosis and hypertension. Early growth response-1 (Egr-1) is an immediate early gene that acts as a master switch for the inflammatory response. However, the role of Ang II in regulating Egr-1 expression in macrophages, and the effect of peroxisome proliferators-activated receptor-γ (PPAR-γ) ligand 15d-PGJ2 in this process remain to be investigated. Methods and Results: We showed that Ang II significantly up-regulated the expression of Egr-1 in RAW264.7 macrophages, and this effect was markedly attenuated by Eprosartan (an AT1R blocker), SP600125 (a JNK-specific inhibitor) and PD98059 (an ERK-specific inhibitor). Moreover, treatment of macrophages with 15d-PGJ2, a natural PPAR-γ ligand, significantly reduced Ang II-induced expression of Egr-1 and its inflammatory gene targets (IL-1β, TNF-α, TGF-β, MCP-1 and ICAM-1) through PPAR-γ activation and ROS formation. In addition, 15d-PGJ2 treatment markedly inhibited Ang II-induced macrophage migration and proliferation. Conclusions: This study for the first time demonstrates that Ang II can induce the expression of Egr-1 via AT1R/JNK and ERK signaling pathways. Activation of PPAR-γ by 15d-PGJ2 suppresses Egr-1-mediated proinflammatory response, and may be a novel therapeutic strategy for treatment of vascular inflammatory diseases. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are vital for innate immunity, and they were expressed in various immune cells, tissues and organs. Moreover, TLRs specific expression pattern in different cells, tissue and organs have been confirmed to have correlation with the ability to resistance to pathogenic challenges. The present study aimed to determine the expression profiles and levels of TLR2, 3, 4, 5, 6, 8 and 10 in the lung, trachea, intestine, stomach, liver, spleen, uterine horn and body, cervix, ovary, oviduct and hypothalamus of female rabbits, and whether the expression level of TLR2 and 4 in the ovary, oviduct, uterine horn and body, and cervix were affected by lipopolyasaccharide (LPS). The tissues of the lung, trachea, intestine, stomach, liver, spleen, uterine horn and body, cervix, ovary, oviduct and hypothalamus were collected from four rabbits which didn’t be treated as 0 h. 16 rabbits in LPS group were injected with LPS (according to 0.5 mg/kg body weight) and 16 rabbits in control group were injected with saline (LPS carrier), hereafter the tissues of the uterine horn and body, cervix, ovary and oviduct from 32 rabbits were collected after 1.5, 3, 6, and 12 h ( n = 4 each group) postinjection. The expression profiles of TLRs were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), and expression level of TLRs were examined using quantitative real-time PCR (qRT-PCR). The results shown: TLR2, 3, 4, 5, 6, 8 and 10 were expressed in lung, trachea, intestine, stomach, liver, spleen, uterine horn and body, cervix, ovary, oviduct and hypothalamus of female rabbits, but their expression level had great difference in the same organs, and each TLR has different expression level in the different organs. After LPS-stimulation, the expression of TLR2 in the uterine body and horn was significantly higher than that in control group by 3 h and 12 h of postinjection ( P < 0.05) respectively. The expression of TLR4 in ovary and uterine body was significantly higher than that in control group by 3 and 12 h of postinjection ( P < 0.05), and the expression of TLR4 in uterine body was greater than that in control by 3 h postinjection ( P < 0.05). The results suggested LPS upregulated the expression of TLR2 and 4 in uterine body and horn, and the expression of TLR4 in ovary. [ABSTRACT FROM AUTHOR]

Simple Summary: Bladder cancer (BLCA) is one of the most prevalent urinary cancers and novel non-invasive BLCA tumor biomarkers with high sensitivity and specificity are needed to support early diagnosis. Exosomal long non-coding RNAs (lncRNAs) are promising diagnostic biomarkers for BLCA. Our study suggests that the combined evaluation of lncRNA RMRP levels in urinary and plasma exosomes is an excellent potential non-invasive diagnostic biomarker for BLCA patients. Additionally, targeting the RMRP/miR-206/G6PD axis is a promising strategy for BLCA treatment. Bladder cancer (BLCA) is one of the cancers that is highly sensitive to specific non-invasive tumor biomarkers that facilitate early diagnosis. Exosome-derived long non-coding RNAs (lncRNAs) hold promise as diagnostic biomarkers for BLCA. In this study, we employed RNA-sequencing to compare the expression patterns of lncRNAs in urine exosomes from three BLCA patients and three healthy individuals. RMRP displayed the most significant differential expression. Elevated RMRP expression levels were observed in urinary and plasma exosomes from BLCA patients compared with those from healthy individuals. RMRP exhibited significant associations with certain BLCA patient clinicopathological features, including tumor stage, poor prognosis, and tumor grade. Combined diagnosis using RMRP in urine and plasma exosomes demonstrated a superior diagnostic performance with receiver operating characteristic curve analysis. RMRP was found to be related to BLCA tumor progression and the cell migration and invasion processes via the miR-206/G6PD axis both in vitro and in vivo. Mechanistically, RMRP serves as an miR-206 sponge, as suggested by dual-luciferase reporter assays and RNA immunoprecipitation. Our study suggests that the combined diagnosis of RMRP in urinary and plasma exosomes can serve as an excellent non-invasive diagnostic biomarker for BLCA patients. Additionally, targeting the RMRP/miR-206/G6PD axis holds promise as a therapeutic strategy for BLCA. [ABSTRACT FROM AUTHOR]

SUMMARY: The non‐essential supernumerary maize (Zea mays) B chromosome (B) has recently been shown to contain active genes and to be capable of impacting gene expression of the A chromosomes. However, the effect of the B chromosome on gene expression is still unclear. In addition, it is unknown whether the accumulation of the B chromosome has a cumulative effect on gene expression. To examine these questions, the global expression of genes, microRNAs (miRNAs), and transposable elements (TEs) of leaf tissue of maize W22 plants with 0–7 copies of the B chromosome was studied. All experimental genotypes with B chromosomes displayed a trend of upregulated gene expression for a subset of A‐located genes compared to the control. Over 3000 A‐located genes are significantly differentially expressed in all experimental genotypes with the B chromosome relative to the control. Modulations of these genes are largely determined by the presence rather than the copy number of the B chromosome. By contrast, the expression of most B‐located genes is positively correlated with B copy number, showing a proportional gene dosage effect. The B chromosome also causes increased expression of A‐located miRNAs. Differentially expressed miRNAs potentially regulate their targets in a cascade of effects. Furthermore, the varied copy number of the B chromosome leads to the differential expression of A‐located and B‐located TEs. The findings provide novel insights into the function and properties of the B chromosome. [ABSTRACT FROM AUTHOR]

MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferatoractivated receptor γ (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/β-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of β-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/β-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/β-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy, a common complication of diabetes, is the leading cause of blindness globally. Müller cells are key players in diabetes-associated retinal inflammation and dysfunction. However, the pathological changes of Müller cells in response to high glucose (HG) and the underlying mechanism remain unclear. The aim of the present study was to investigate the key role of heat shock protein 47 (HSP47) in HG-induced unfolded protein and inflammatory responses. Primary mouse Müller cells were starved in serum-free DMEM overnight and then treated with HG (30 mM) for 0, 6, 12 or 24 h. It was observed that HG (30 mM) significantly induced the protein expression of HSP47, inositol-requiring transmembrane kinase and endonuclease-1α (IRE1α) and spliced X-box-binding protein 1 (XBP1s) in primary mouse Müller cells compared with the untreated group. In addition, the immunoprecipitation results revealed that HSP47 directly interacted with IRE1α, and this interaction was significantly enhanced by HG exposure for 12 or 24 h compared with the untreated group. Furthermore, small interfering RNA-mediated silencing of HSP47 significantly suppressed HG-induced activation of the IRE1α/XBP1s/hypoxia inducible factor-1 subunit α (HIF-1α) pathway and upregulation of the mRNA expression levels of the inflammatory cytokines vascular endothelial growth factor, platelet-derived growth factor subunit B, inducible nitric oxide synthase and angiopoietin-2 in Müller cells. Furthermore, overexpression of IRE1α or HIF-1α partially attenuated HSP47-siRNA-mediated inhibition of inflammatory cytokine expression in Müller cells. Collectively, these results indicated that HG may induce HSP47 expression and promote the inflammatory response through enhancing the interaction between HSP47 and IRE1α, and activating the IRE1α/XBP1s/HIF-1α pathway in retinal Müller cells. [ABSTRACT FROM AUTHOR]

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,β-unsaturated β-diketone, α,β-unsaturated ketone and β′-hydroxy-α,β-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC50 values of all the synthesized derivatives, most α,β-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas β′-hydroxy-α,β-unsaturated ketones and α,β-unsaturated β-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,β-unsaturated ketone complex for help in drug design. [ABSTRACT FROM AUTHOR]

The expression of the transcription factor GATA3 in FOXP3+ regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells. [ABSTRACT FROM AUTHOR]

Background: Using fatty acids (FAs) exclusively for ATP generation was reported to contribute to the development of diabetic cardiomyopathy. We studied the role of substrate metabolism related genes in the heart of the diabetes to find out a novel therapeutic target for diabetic cardiomyopathy. Methods and Results: By microarray analysis of metabolic gene expression, acyl-CoA thioesterase 1 (acot1) was clearly upregulated in the myocardia of db/db mice, compared with normal control C57BL/Ks. Therefore, gain-of-function and loss-of-function approaches were employed in db/db mice to investigate the functions of ACOT1 in oxidative stress, mitochondrial dysfunction and heart function. We found that in the hearts of db/db mice which overexpressed ACOT1, H2O2 and malondialdehyde (MDA) were reduced, the activities of ATPases in mitochondria associated with mitochondrial function were promoted, the expression of uncoupling protein 3 (UCP3) contributing to oxygen wastage for noncontractile purposes was decreased, and cardiac dysfunction was attenuated, as determined by both hemodynamic and echocardiographic detections. Consistently, ACOT1 deficiency had opposite effects, which accelerated the cardiac damage induced by diabetes. Notably, by real-time PCR, we found that overexpression of ACOT1 in diabetic heart repressed the peroxisome proliferator-activated receptor alpha/PPARγ coactivator 1α (PPARα/PGC1α) signaling, as shown by decreased expression of PGC1α and the downstream genes involved in FAs use. Conclusion: Our results demonstrated that ACOT1 played a crucial protective role in diabetic heart via PPARα/PGC1α signaling. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3' untranslated region (3'UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and Results: Using bioinformatics analysis, we found potential target sites for miRNA let-7b in 39UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. Conclusions: Our results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes. [ABSTRACT FROM AUTHOR]

There is concern that ingestion of dietary phytoestrogens may increase risk of estrogen receptor alpha (ERα)-positive breast cancer. The prenylflavone icaritin, a phytoestrogen consumed in East Asian societies for its perceived beneficial effects on bone health, stimulated the growth of breast cancer (MCF-7) cells at low concentrations. Although acting like an estrogenic ligand, icaritin exerted an unexpected suppressive effect on estrogen-stimulated breast cancer cell proliferation and gene expression at higher concentrations. Like estradiol, icaritin could dose-dependently destabilize ERα protein. However, destabilization of ERα by the estradiol/icaritin combination was profound and greater than that observed for either compound alone. Microarray gene expression analyses implicated aryl hydrocarbon receptor (AhR) signaling for this suppressive effect of icaritin. Indeed, icaritin was an AhR agonist that competitively reduced specific binding of a potent AhR agonist and increased expression of the AhR-regulated gene CYP1A1. When AhR was knocked down by small interfering RNA, the suppressive effect of icaritin on estradiol-stimulated breast cancer cell growth and gene expression was abolished, and ERα protein stability was partially restored. Similarly in an athymic nude mouse model, icaritin restricted estradiol-stimulated breast cancer xenograft growth and strongly reduced ERα protein levels. Overall, our data support the feasibility for the development of dual agonists like icaritin, which are estrogenic but yet, through activating AhR-signaling, can destabilize ERα protein to restrict ERα-positive breast cancer cell growth. [ABSTRACT FROM PUBLISHER]

AbstractInterleukin (IL)-4 and IL-13 are two key cytokines released from activated T helper type 2 (Th2) cells and strongly associated with asthma and allergic disease. We applied silencing of the IL-4 and IL-13 gene expression by RNA interference delivered by a lentiviral vector to evaluate the therapeutic role of IL-4 and IL13 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with ovalbumin (OVA), and one treatment of IL-4 and IL-13 shRNA lentiviral vector (Lenti-si-IL-4 and Lenti-si-IL-13) was instilled intratracheally 48 hr before challenge. After three challenges of OVA antigen, mice were assessed for airway inflammation and hyperresponsiveness. With infection of Lenti-si-IL-4 and Lenti-si-IL-13 in EL-4 cells, both RNA and protein expressions of IL-4 and IL-13 were obviously abrogated. Furthermore, intratracheal instillation of Lenti-si-IL-4 and Lenti-si-IL-13 in OVA-immunized mice resulted in a strong inhibition of local IL-4 and IL-13 cytokine release. Treatment with Lenti-si-IL-4 and Lenti-si-IL-13 successfully alleviated OVA-induced airway eosinophilia and Th2 cell cytokine release. Finally, to determine airway hyperresponsiveness by enhanced pause and pulmonary resistance in noninvasive and invasive body plethysmography, we found that administration of Lenti-si-IL-4 and Lenti-si-IL-13 markedly decreased airway hyperresponsiveness in OVA-immunized mice. These results suggest that inhibition of IL-4 and IL-13 gene expression by shRNA lentiviral vector markedly inhibits antigen-induced airway inflammation and hyperresponsiveness in mice. [ABSTRACT FROM AUTHOR]

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain, which play a central role in the early innate host defense against infection. In the present study, we report three novel avian cathelicidin orthologs cloned from a constructed spleen cDNA library of Coturnix coturnix, using a nested-PCR-based cloning strategy. Three coding sequences containing ORFs of 447, 465 and 456 bp encode three mature antimicrobial peptides (named Cc-CATH1, 2 and 3) of 26, 32 and 29 amino acid residues, respectively. Phylogenetic analysis indicated that precursors of Cc-CATHs are significantly conserved with known avian cathelicidins. Synthetic Cc-CATH2 and 3 displayed broad and potent antimicrobial activity against most of the 41 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, with minimum inhibitory concentration values in the range 0.3-2.5 μ for most strains with or without the presence of 100 m NaCl. Cc-CATH2 and 3 showed considerable reduction of cytotoxic activity compared to other avian cathelicidins, with average IC values of 20.18 and 17.16 μ, respectively. They also exerted a negligible hemolytic activity against human erythrocytes, lysing only 3.6% of erythrocytes at a dose up to 100 μg·mL. As expected, the recombinant Cc-CATH2 (rCc-CATH2) also showed potent bactericidal activity. All these features of Cc-CATHs encourage further studies aiming to estimate their therapeutic potential as drug leads, as well as coping with current widespread antibiotic resistance, especially the new prevalent and dangerous 'superbug' that is resistant to almost all antibiotics. Three novel avian cathelicidin orthologues were cloned encoding three antimicrobial peptides of 26, 32, 29 amino acid residues length. Synthetic Cc-CATH2 and 3 displayed broad and potent antimicrobial activity. Cc-CATH2 and 3 showed much reduced cytotoxicity, and a negligible hemolytic activity, lysing only 3.6% of erythrocytes at doses up to 100 μg·mL. Recombinant Cc-CATH2 showed potent bactericidal activity as expected. [ABSTRACT FROM AUTHOR]

GATA-3 is the key transcriptional factor for Th2 commitment in T cells and is strongly associated with asthma and allergic disease. We studied the silencing of the GATA-3 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of GATA-3 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with OVA and instilled intratracheally (IT) with GATA-3 shRNAs lentiviral vector (Lenti-si-GATA-3) once, 48 hours before challenge. After three challenges with the OVA antigen, the mice were assessed for airway hyperresponsiveness (AHR) and inflammation. With infection of Lenti-si-GATA-3 in EL-4 cells, GATA-3 gene expression was abrogated and downstream Th2 cytokines, such as interleukin-4 (IL-4) and IL-5, were also significantly inhibited. IT delivery of Lenti-si-GATA-3 in OVA-immunized mice resulted in a strong inhibition of local GATA-3 gene expression. Treatment with Lenti-si-GATA-3 successfully alleviated OVA-induced airway eosinophilia and Th2 cytokine release. While evaluating AHR by means of enhanced pause (Penh) and pulmonary resistance (RL) using body plethysmography, it was found that the administration of Lenti-si-GATA-3 had significantly decreased AHR in OVA-immunized mice. These results suggest that inhibition of GATA-3 gene expression by shRNAs lentiviral vectors strongly attenuates antigen-induced airway inflammation and hyper-responsiveness in mice.Molecular Therapy (2007) 16 1, 60–65. doi:10.1038/sj.mt.6300309 [ABSTRACT FROM AUTHOR]

Parathyroid hormone-related protein (PTHrP) regulates proliferation and differentiation of osteoblastic cells via binding to the parathyroid hormone receptor (PTH-1R). The cAMP-dependent protein kinase A pathway governs the majority of these effects, but recent evidence also implicates the MAPK pathway. MC3T3-E1 subclone 4 cells (MC4) were treated with the MAPK inhibitor U0126 and PTHrP. In differentiated MC4 cells, osteocalcin and bone sialoprotein gene expression were both down-regulated by PTHrP and also by inhibition of the MAPK pathway. PTHrP-mediated down-regulation of PTH-1R mRNA and up-regulation of c-fos mRNA were MAPK-independent, whereas PTHrP stimulation of fra-2 and interleukin-6 (IL-6) mRNA was MAPK-dependent. Luciferase promoter assays revealed that regulation of IL-6 involved the cAMP-dependent protein kinase A and MAPK pathways with a potential minor role of the protein kinase C pathway, and a promoter region containing an activator protein-1 site was necessary for PTHrP-induced IL-6 gene transcription. An alternative pathway, through cAMP/Epac/Rap1/MAPK, mediated ERK phosphorylation but was not sufficient for IL-6 promoter activation. Phosphorylation of the transcription factor CREB was also necessary but not sufficient for PTHrP-mediated IL-6 promoter activity. Most interesting, a bidirectional effect was found with PTHrP increasing phosphorylated ERK in undifferentiated MC4 cells but decreasing phosphorylated ERK in differentiated cells. These data indicate that inactivation of the MAPK pathway shows differential regulation of PTHrP-stimulated activator protein-1 members, blocks PTHrPstimulated IL-6, and synergistically down-regulates certain osteoblastic markers associated with differentiation. These novel findings indicate that the MAPK pathway plays a selective but important role in the actions of PTHrP. [ABSTRACT FROM AUTHOR]

In this study, C57BL/6J mice with high-fat diet- (HFD-) induced hyperlipidemia were treated with total Liriope spicata var. prolifera polysaccharides (TLSP: 200, 400, and 800 mg/kg body weight), simvastatin (3 mg/kg body weight), or saline for 8 weeks, respectively. The results showed that TLSP had strong lipid-lowering and hepatoprotective effects on C57BL/6J mice with HFD-induced hyperlipidemia. TLSP administration significantly reduced serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels and downregulated the expressions of peroxisome proliferator-activated receptor (PPAR)γ and fatty acid synthase (FAS) in the adipose and liver tissues of the mice. TLSP exerted hypolipidemic and hepatoprotective effects by activating lipid/bile acid metabolism via the FXH-SHP/CYP7A1 and SEBP-1c/FAC/ACC signaling pathways. Thus, TLPS is a promising natural polymer with hepatoprotective and hypolipidemic properties. [ABSTRACT FROM AUTHOR]

Objective : Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a crucial factor for the survival of neuron. The role of NMNAT2 in damage following traumatic brain injury (TBI) remains unknown. This study was designed to investigate the role of NMNAT2 in TBI-induced neuronal degeneration and neurological deficits in rats. Methods : The TBI model was established in Sprague-Dawley rats by a weight-dropping method. Real-time polymerase chain reaction, western blot, immunofluorescence, Fluoro-Jade C staining, and neurological score analyses were carried out. Results : NMNAT2 mRNA and protein levels were increased in the injured-side cortex at 6 hours and peaked 12 hours after TBI. Knocking down NMNAT2 with an injection of small interfering RNA in lateral ventricle significantly exacerbated neuronal degeneration and neurological deficits after TBI, which were accompanied by increased expression of BCL-2-associated X protein (Bax). Conclusion : NMNAT2 expression is increased and NMNAT2 exhibits neuroprotective activity in the early stages after TBI, and Bax signaling pathway may be involved in the process. Thus, NMNAT2 is likely to be an important target to prevent secondary damage following TBI [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastoma (GBM) is the most lethal adult primary brain tumor, and has no cure. This study investigated the membrane protein sortilin as a prognosis biomarker for glioblastoma (GBM). We found that sortilin is overexpressed in GBM tumors and can be detected in the blood of GBM patients. In addition, in cell cultures, targeting sortilin resulted in the inhibition of GBM cell invasion. These data highlight the value of sortilin as a potential clinical biomarker and therapeutic target for GBM and warrant further translational investigation. Glioblastoma (GBM) is a devastating brain cancer with no effective treatment, and there is an urgent need for developing innovative biomarkers as well as therapeutic targets for better management of the disease. The membrane protein sortilin has recently been shown to participate in tumor cell invasiveness in several cancers, but its involvement and clinical relevance in GBM is unclear. In the present study, we explored the expression of sortilin and its potential as a clinical biomarker and therapeutic target for GBM. Sortilin expression was investigated by immunohistochemistry and digital quantification in a series of 71 clinical cases of invasive GBM vs. 20 non-invasive gliomas. Sortilin was overexpressed in GBM and, importantly, higher expression levels were associated with worse patient survival, pointing to sortilin tissue expression as a potential prognostic biomarker for GBM. Sortilin was also detectable in the plasma of GBM patients by enzyme-linked immunosorbent assay (ELISA), but no differences were observed between sortilin levels in the blood of GBM vs. glioma patients. In vitro, sortilin was detected in 11 brain-cancer-patient-derived cell lines at the anticipated molecular weight of 100 kDa. Interestingly, targeting sortilin with the orally bioavailable small molecule inhibitor AF38469 resulted in decreased GBM invasiveness, but cancer cell proliferation was not affected, showing that sortilin is targetable in GBM. Together, these data suggest the clinical relevance for sortilin in GBM and support further investigation of GBM as a clinical biomarker and therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Lung adenocarcinoma (LUAD) has a high incidence and recurrence rate. N6-methyladenosine (m6A) modification of RNA has become a promising epigenetic marker in tumors. The dysregulation of both RNA m6A levels and m6A regulator expression levels reportedly affects essential biological processes in various tumors. Long non-coding RNAs (lncRNAs), a subgroup of RNAs over 200 nucleotides in length that do not code for protein, can be modified and regulated by m6A, but the relevant profile in LUAD remains unclear. Results: The m6A levels of total RNA were decreased in LUAD tumor tissues and cells. Multiple m6A regulators were abnormally expressed at both the RNA and protein levels, and were related in expression patterns and functionally synergistic. Our microarray revealed 2846 m6A-modified lncRNA transcripts as well as its molecular features, 143 of which were differentially m6A-modified and manifested a negative correlation between expression levels and m6A modification levels. More than half of the differentially m6A-modified lncRNAs associated with dysregulated expression. The 6-MRlncRNA risk signature was a reliable indicator for assessing survival time of LUAD patients. The competitive endogenous regulatory network suggested a potential m6A-induced pathogenicity in LUAD. Conclusions: These data have demonstrated that differential RNA m6A modification and m6A regulator expression levels were identified in LUAD patients. In addition, this study provides evidence increasing the understanding of molecular features, prognostic values, and regulatory functionalities of m6A-modified lncRNAs in LUAD. [ABSTRACT FROM AUTHOR]

In recent years, many experiments have proved that microRNAs (miRNAs) play a variety of important regulatory roles in cells, and their abnormal expression can lead to the emergence of specific diseases. Therefore, it is greatly valuable to do research on the association between miRNAs and diseases, which can effectively help prevent and treat miRNA-related diseases. At present, effective computational methods still need to be developed to better identify potential miRNA–disease associations. Inspired by graph convolutional networks, in this study, we propose a new method based on Attention aware Multi-view similarity networks and Hypergraph learning for MiRNA-Disease Associations identification (AMHMDA). First, we construct multiple similarity networks for miRNAs and diseases, and exploit the graph convolutional networks fusion attention mechanism to obtain the important information from different views. Then, in order to obtain high-quality links and richer nodes information, we introduce a kind of virtual nodes called hypernodes to construct heterogeneous hypergraph of miRNAs and diseases. Finally, we employ the attention mechanism to fuse the outputs of graph convolutional networks, predicting miRNA–disease associations. To verify the effectiveness of this method, we carry out a series of experiments on the Human MicroRNA Disease Database (HMDD v3.2). The experimental results show that AMHMDA has good performance compared with other methods. In addition, the case study results also fully demonstrate the reliable predictive performance of AMHMDA. [ABSTRACT FROM AUTHOR]

Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes. [ABSTRACT FROM AUTHOR]