Search

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

Aberrant expression of long non-coding RNAs (lncRNA) is associated with altered DNA methylation and histone modifications during carcinogenesis. However, identifying epigenetically dysregulated lncRNAs and characterizing their functional mechanisms in different cancer subtypes are still major challenges for cancer studies. In this study, we systematically analyzed the epigenetic alterations of lncRNAs at important regulatory elements in three breast cancer subtypes. We identified 87, 691, and 1,197 epigenetically dysregulated lncRNAs in luminal, basal, and claudin-low subtypes of breast cancer, respectively. The landscape of epigenetically dysregulated lncRNAs at enhancer elements revealed that epigenetic changes of the majority of lncRNAs occurred in a subtype-specific manner and contributed to subtype-specific biological functions. We identified six acetylation of lysine 27 on histone H3 (H3K27ac)-dysregulated lncRNAs and three DNA methylation-dysregulated lncRNAs (CTC-303L1.2, RP11-738B7.1, and SLC26A4-AS1) as prognostic biomarkers of basal subtype. These lncRNAs were involved in immune response-related biological functions. Treatment of the basal breast cancer cell line MDA-MB-468 with CREBBP/EP300 bromodomain inhibitors downregulated H3K27 acetylation levels and caused a decrease in the expression of five H3K27ac-dysregulated lncRNAs (LINC00393, KB-1836B5.1, RP1-140K8.5, AC005162.1, and AC020916.2) and inhibition of the growth of breast cancer cells. One epigenetically dysregulated lncRNA (LINC01983) and four lncRNA regulators (UCA1, RP11-221J22.2, RP11-221J22.1, and RP1-212P9.3) were identified as prognostic biomarkers of the luminal molecular subtype of breast cancer by controlling the tumor necrosis factor (TNF) signaling pathway, T helper (Th)17 cell differentiation, and T cell migration. Finally, our results highlighted a profound role of enhancer-related H3K27ac-dysregulated lncRNAs, DNA methylation-dysregulated lncRNAs, and lncRNA regulators in breast cancer subtype carcinogenesis and their potential prognostic value., Graphical Abstract, The corresponding authors and colleagues identified H3K27ac-dysregulated lncRNAs (LINC00393, KB-1836B5.1, RP1-140K8.5, AC005162.1, and AC020916.2) and DNA methylation-dysregulated lncRNAs (CTC-303L1.2, RP11-738B7.1, and SLC26A4-AS1) as prognostic biomarkers of basal subtypes. LINC01983, UCA1, RP11-221J22.2, RP11-221J22.1, and RP1-212P9.3 act as prognostic biomarkers of the luminal subtype. The inhibition of enhancer-related lncRNAs could suppress breast cancer cell growth.

Alzheimer's disease (AD) is a severe neurodegenerative disorder of the central nervous system (CNS) characterized by neuron loss and dementia. Previous abundant evidence demonstrates that the first critical step in the course of AD is the state of oxidative stress and the neuronal loss is closely related to the interaction of several signalling pathways. The neuroprotective efficacy of Rho-associated protein kinase (ROCK) inhibitor in the treatment of AD has been reported, but its exact mechanism has not been well elucidated. The purpose of this study is to investigate the therapeutic effects of Fasudil on amyloid precursor protein/presenilin-1 (APP/PS1) mice and to discover the potential underlying mechanism. Sixteen 8-month-old APP/PS1 mice were divided into model and Fasudil treatment groups and 8 wild-type mice were used as a normal control group. After the behavioural test, all mice were sacrificed for immunofluorescence and other biochemical tests. The results showed that the administration of Fasudil improved learning and memory ability, elevated the concentration of antioxidative substances and decreased lipid peroxides, as well as inhibited neuronal apoptosis by increasing the expression of B-cell lymphoma-2 (Bcl-2) (p < 0.05), reducing Bcl-2 Associated X (Bax) (p < 0.05) and cleaved caspase-3 (p < 0.05) of APP/PS1 mice. Moreover, Fasudil treatment also ameliorated the phosphorylation of p38 (p < 0.01), c-Jun N-terminal kinase (JNK) (p < 0.001) and extracellular regulated protein kinases (ERK) (p < 0.001), and accelerated the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) (p < 0.01) expression and its antioxidative downstream molecules (p < 0.05, p < 0.05, and p < 0.05, respectively). Data from the present study demonstrate that Fasudil significantly restored cognitive function, restrained oxidative stress and reduced neuronal apoptosis in the hippocampus, probably by inhibiting ROCK/MAPK and activating Nrf2 signalling pathways in APP/PS1 mice.

N-acetyl-d-neuraminic acid (NeuAc) has attracted considerable attention because of its wide-ranging applications. The use of cheap carbon sources such as glucose without the addition of any precursor in microbial NeuAc production has many advantages. In this study, improved NeuAc production was attained through the optimization of amino sugar metabolism pathway kinetics and reservation of a phosphoenolpyruvate (PEP) pool in Escherichia coli. N-acylglucosamine 2-epimerase and N-acetylneuraminate synthase from different sources and their best combinations were used to obtain optimized enzyme kinetics and expression intensity, which resulted in a significant increase in NeuAc production. Next, after a design was engineered for enabling the PEP metabolic pathway to retain the PEP pool, the production of NeuAc reached 16.7 g/L, which is the highest NeuAc production rate that has been reported from using glucose as the sole carbon source.

Background Fetal growth restriction (FGR) contributes the primary cause of perinatal mortality and morbidity with impacts on the long-term health. To determine the core gene expression network and gene signatures, which in combination with ultrasound confirmation will more effectively differentiate constitutionally normal small for gestational age and pathological FGR groups, we performed RNA sequencing for protein-coding genes, lncRNAs, and small RNAs in a case–control study of umbilical cord blood. Results Five pairs of FGR case and control umbilical cord blood samples were used for RNA sequencing and weighted gene co-expression network analysis (WGCNA). Results showed that 339 mRNAs, 295 lncRNAs, and 13 miRNAs were significantly differentially expressed between FGR cases and controls. Bioinformatics analysis indicated that these differentially expressed molecules were mainly involved in metabolism, neural, cardiac, and immune systems, and identified 18 WGCNA modules for FGR. Further quantitative verification was performed using umbilical cord blood and maternal peripheral blood from 12 pairs of FGR cases and controls. The logistic regression and receiver operating characteristic curve indicated that RP11_552M6.1, LINC01291, and Asgr1 in umbilical cord blood, while Sfrp2, miR-432-5p, and miR-1306-3p in maternal peripheral blood had potential significance for FGR. Conclusions We comprehensively profiled the whole-transcriptome landscape of human umbilical cord blood with FGR, constructed the core WGCNA modules, and delineated the critical gene signatures of FGR. These findings provide key insight into intrauterine perturbations and candidate signatures for FGR.

Summary Minimycin (MIN) is a C-nucleoside antibiotic structurally related to pseudouridine, and indigoidine is a naturally occurring blue pigment produced by diverse bacteria. Although MIN and indigoidine have been known for decades, the logic underlying the divergent biosynthesis of these interesting molecules has been obscure. Here, we report the identification of a minimal 5-gene cluster (min) essential for MIN biosynthesis. We demonstrated that a non-ribosomal peptide synthetase (MinA) governs “the switch” for the divergent biosynthesis of MIN and the cryptic indigoidine. We also demonstrated that MinCN (the N-terminal phosphatase domain of MinC), MinD (uracil phosphoribosyltransferase), and MinT (transporter) function together as the safeguard enzymes, which collaboratively constitute an unusual self-resistance system. Finally, we provided evidence that MinD, utilizing an unprecedented substrate-competition strategy for self-resistance of the producer cell, maintains competition advantage over the active molecule MIN-5′-monophosphate by increasing the UMP pool in vivo. These findings greatly expand our knowledge regarding natural product biosynthesis., Graphical Abstract, Highlights • A minimal 5-gene cluster (min) is essential for minimycin biosynthesis • Divergent biosynthesis of minimycin and indigoidine is mediated by an NRPS enzyme • A cascade of three safeguard enzymes constitutes the unusual self-resistance system • MinD functions as the key safeguard enzyme by increasing the UMP pool in vivo, Biotechnology; Microbial Biotechnology; Systems Biology

CCAAT/enhancer-binding homologous protein (CHOP), a transcriptional regulator induced by endoplasmic reticulum stress (ER stress) is a pivotal factor in the ER stress-mediated apoptosis pathway. Previous studies have shown that CHOP is involved in the formation of fibrosis in a variety of tissues and is associated with alternative macrophage activation. The role of CHOP in the pathologic effects of liver fibrosis in schistosomiasis has not been reported, and underlying mechanisms remain unclear. This study is aimed at understanding the effect of CHOP on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in vivo and clarifying its mechanism. C57BL/6 mice were infected with cercariae of S. japonicum through the abdominal skin. The liver fibrosis was examined. The level of IL-13 was observed. The expressions of CHOP, Krüppel-like factor 4 (KLF4), signal transducer and activator of transcription 6 (STAT6), phosphorylation STAT6, interleukin-13 receptor alpha 1 (IL-13Rα1), and interleukin-4 receptor alpha (IL-4Rα) were analysed. The eosinophilic granuloma and collagen deposition were found around the eggs in mice infected for 6 and 10 weeks. IL-13 in plasma and IL-13Rα1 and IL-4Rα in liver tissue were significantly increased. The phosphorylated STAT6 was enhanced while Krüppel-like factor 4 (KLF4) was decreased in liver tissue. The expression of CHOP and colocalization of CHOP and CD206 were increased. Overall, these results suggest that CHOP plays a critical role in hepatic fibrosis induced by S. japonicum, likely through promoting alternative activation of macrophages.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals., Author summary Compared with the commercial breeds, Chinese local pig breeds have lower growth rates, higher fat content and better meat quality, because they have adapted to local environment and have not been strongly selected. These differences make them as exceptional resources to identify candidate markers for the improvement of meat production traits in pig breeding. In this study, we compared the genome and skeletal muscle transcriptome differences between Chinese local Tongcheng and commercial Landrace pigs, and uncovered the genetic regulation of coding and non-coding RNAs, such as SATB2 and XLOC_036765, in skeletal muscle development and diversity. This study enhances our understanding of the genetic basis of skeletal muscle development and diversity, and provide useful molecular markers for the genetic improvement of meat production traits.

Epigenetic modifications play critical roles in modulating gene expression, yet their roles in regulatory networks in human cell lines remain poorly characterized. We integrated multiomics data to construct directed regulatory networks with nodes and edges labeled with chromatin states in human cell lines. We observed extensive association of diverse chromatin states and network motifs. The gene expression analysis showed that diverse chromatin states of coherent type-1 feedforward loop (C1-FFL) and incoherent type-1 feedforward loops (I1-FFL) contributed to the dynamic expression patterns of targets. Notably, diverse chromatin state compositions could help C1- or I1-FFL to control a large number of distinct biological functions in human cell lines, such as four different types of chromatin state compositions cooperating with K562-associated C1-FFLs controlling “regulation of cytokinesis,” “G1/S transition of mitotic cell cycle,” “DNA recombination,” and “telomere maintenance,” respectively. Remarkably, we identified six chromatin state-marked C1-FFL instances (HCFC1-NFYA-ABL1, THAP1-USF1-BRCA2, ZNF263-USF1-UBA52, MYC-ATF1-UBA52, ELK1-EGR1-CCT4, and YY1-EGR1-INO80C) could act as prognostic biomarkers of acute myelogenous leukemia though influencing cancer-related biological functions, such as cell proliferation, telomere maintenance, and DNA recombination. Our results will provide novel insight for better understanding of chromatin state-mediated gene regulation and facilitate the identification of novel diagnostic and therapeutic biomarkers of human cancers.

Aging-related cognitive ability impairments are one of the main threats to public health, and impaired hippocampal neurogenesis is a major cause of cognitive decline during aging. However, the regulation of adult neurogenesis in the hippocampus requires further study. Here, we investigated the role of microRNA-153 (miR-153), a highly conserved microRNA in mice and humans, in adult neurogenesis. During the passaging of neural stem cells (NSCs) in vitro, endogenous miR-153 expression was downregulated, with a decrease in neuronal differentiation ability. In addition, miR-153 overexpression increased the neurogenesis of NSCs. Further studies showed that miR-153 regulated neurogenesis by precisely targeting the Notch signaling pathway through inhibition of Jagged1 and Hey2 translation. In vivo analysis demonstrated that miR-153 expression was decreased in the hippocampi of aged mice with impaired cognitive ability, and that miR-153 overexpression in the hippocampus promoted neurogenesis and markedly increased the cognitive abilities of the aged mice. Overall, our findings revealed that miR-153 affected neurogenesis by regulating the Notch signaling pathway and elucidated the function of miR-153 in aging-related, hippocampus-dependent cognitive ability impairments, and neurodegenerative diseases.

Honokiol (HNK) is a small-molecule lignin extracted from Magnolia Officinalis, demonstrating high potency in promoting nonrapid eye movement (NREM) sleep by modulating the benzodiazepine site of the GABAA receptor. However, the clinical use of HNK in the treatment of insomnia is restricted by its extremely low oral bioavailability. In the present work, enhanced oral bioavailability of HNK was achieved by loading it into poly lactide–glycolide acid microparticles (HNK-MP). After oral administration, HNK-MP demonstrated 15-fold increase of AUC0–12 h in comparison to free HNK. The maximum blood concentration (Cmax) of HNK in HNK-MP-treated rats was 3.6 μg/mL at 2 h after oral administration, which was 6.5-fold of that in free HNK-treated rats. Oral administration of HNK-MP (20 mg/kg) efficiently increased NREM sleep by 60% by enhancing the transition from wakefulness to NREM sleep in rats. The biosafety of HNK-MP was assessed in vivo, and no damage occurred in the gastrointestinal tract. The present study pr...

Vancomycin (Van) is a common antibiotic that pollutes food, feed and ecological environment. In the present study, an anti-Van polyclonal antibodies (pAbs) and Eu3+-labeled goat anti-rabbit IgG monoclonal antibody based indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for ultrasensitive determination of Van, and its half-maximum inhibition concentrations (IC50) was 13.74 ng/L and the limit of detection (LOD) was 1.07 ng/L. The IC-TRFIA showed strong cross-reactivity (CRs) of 36.2% for norvancomycin, and less than 0.1% for polymyxin B, kanamycin and ampicillin. It was found that the IC-TRFIA for Van spiked in different milk powder samples determination were with good accuracy, stability and repeatability, and its recoveries are 83.0–97.9%, also with its coefficient of variations (CVs) are 3.0–12.9% in intra-assay and inter-assay. The results showed that the established IC-TRFIA was promising for ultrasensitive determination of Van in food samples. Highlights Prepared high-quality Van-KLH/BSA conjugates. Obtained high activity and purify anti-Van polyclonal antibodies. Prepared high-quality Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. Firstly established an IC-TRFIA for Van based on the anti-Van polyclonal antibodies and Eu3+-labelled goat anti-rabbit IgG monoclonal antibody. The IC-TRFIA can be used for ultrasensitive determination of Van in milk powder samples.

Aroma is an important quality parameter for breeding in rice (Oryza sativa). For example, the aromatic rice varieties basmati and jasmine rice, with a popcorn-like scent, are popular worldwide and routinely command a price premium. 2-acetyl-1-pyrroline (2AP) is a key flavor compound among over 200 volatiles identified in fragrant rice. A naturally fragrant germplasm exists in multiple plant species besides rice, which all exhibit lower activity of BETAINE ALDEHYDE DEHYDROGENASE 2 (BADH2). However, no equivalent aromatic germplasm has been described in maize (Zea mays). Here, we characterized the two maize BADH2 homologs, ZmBADH2a and ZmBADH2b. We generated zmbadh2a and zmbadh2b single mutants and the zmbadh2a-zmbadh2b double mutant by CRISPR/Cas in four inbred lines. A popcorn-like scent was only noticeable in seeds from the double mutant, but not from either single mutant or in wild type. In agreement, we only detected 2AP in fresh kernels and dried mature seeds from the double mutant, which accumulated between 0.028 and 0.723 mg/kg 2AP. These results suggest that ZmBADH2a and ZmBADH2b redundantly participate in 2AP biosynthesis in maize, and represent the creation of the world's first aromatic maize by simultaneous genome editing of the two BADH2 genes.

To examine physiological responses of garlic to salinity, 17-day-old seedlings of eight soft-neck accessions were treated with 200 mM NaCl for seven days in a hydroponic system. Several morphological and physiological traits were measured at the end of the treatment, including shoot height, shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, photosynthesis rate, and concentrations of Na+ and K+ in leaves. The principal component analysis showed that shoot dry weight and K+/Na+ ratio contribute the most to salt tolerance among the garlic accessions. As a result, salt-tolerant and sensitive accessions were grouped based on these two parameters. Furthermore, to investigate the molecular mechanisms in garlic in response to salinity, the transcriptomes of leaves and roots between salt-tolerant and salt-sensitive garlic accessions were compared. Approximately 1.5 billion read pairs were obtained from 24 libraries generated from the leaves and roots of the salt-tolerant and salt-sensitive garlic accessions. A total of 47,509 genes were identified by mapping the cleaned reads to the garlic reference genome. Statistical analysis indicated that 1282 and 1068 genes were upregulated solely in the tolerant leaves and roots, whereas 1505 and 1203 genes were downregulated exclusively in the tolerant leaves and roots after NaCl treatment, respectively. Functional categorization of these genes revealed their involvement in a variety of biological processes. Several genes important for carotenoid biosynthesis, auxin signaling, and K+ transport were strongly altered in roots by NaCl treatment and could be candidate genes for garlic salt tolerance improvement.

The presence of breeding tubercles (BTs) on the pectoral fins has been investigated as a typical male secondary sexual characteristic (SSC) that distinguish males from females in adult zebrafish. However, the earliest occurrence of these tubercles and its association with puberty onset and body growth remain unclear. In this study, using morphological, histological, and statistical analyses, we examined the first appearance of BTs and puberty onset in male zebrafish, with particular emphasis on the potential impact of body growth on them. Our results revealed that BTs distributed along the first five branched pectoral fin rays were the earliest manifestation of male secondary sexual characteristics (SSCs), which is significantly strongly correlated with body weight (R2 = 0.9609, P < 0.001), and could be used as a "gold standard" for the earliest sex distinction (below 0.1 g in weight). Using the first appearance of BTs (< 0.20 mm2 ) as a metric, we established that male puberty commenced at a body weight of approximately 0.056 ± 0.015 g or a standard length of 10.99 ± 1.051 mm (mean ± SD). In this study, we thus established a simple method that can be used to sex live zebrafish at the pubertal stage and provides the first evidence for the relationship of BTs and male puberty initiation with body growth. These findings will accordingly lay a foundation for exploring mechanisms of the SSCs and male puberty onset in zebrafish and other teleost fish. This article is protected by copyright. All rights reserved.

Myeloperoxidase (MPO), a critical enzyme in antimicrobial host-defense, has been implicated in chronic inflammatory diseases such as coronary artery disease. The design and evaluation of MPO inhibitors for the treatment of cardiovascular disease are reported herein. Starting with the MPO and triazolopyridine 3 crystal structure, novel inhibitors were designed incorporating a substituted pyrazole, which allowed for substituents to interact with hydrophobic and hydrophilic patches in the active site. SAR exploration of the substituted pyrazoles led to piperidine 17, which inhibited HOCl production from activated neutrophils with an IC50 value of 2.4 μM and had selectivity against thyroid peroxidase (TPO). Optimization of alkylation chemistry on the pyrazole nitrogen facilitated the preparation of many analogs, including macrocycles designed to bridge two hydrophobic regions of the active site. Multiple macrocyclization strategies were pursued to prepare analogs that optimally bound to the active site, leading to potent macrocyclic MPO inhibitors with TPO selectivity, such as compound 30.

Garlic (Allium sativum L.) is an economically important, monocotyledonous plant with a strong taste and odor. Drought stress adversely affects its growth, development, and yield, particularly during the bolting/bulbing stage. Herein we performed RNA-seq to assess transcriptomic changes induced by drought stress in bolting/bulbing hardneck garlic plants (Purple Glazer). We observed that drought stress significantly reduced photosynthesis rate, fresh weight, and leaf water content. Transcriptomic analysis of garlic leaves under normal conditions and drought stress led to the identification of 5215 differentially expressed genes (2748 up- and 2467 downregulated). The upregulated DEGs were primarily involved in &ldquo, biological process&rdquo, &ldquo, metabolic process&rdquo, oxidation-reduction process&rdquo, carbohydrate and lipid metabolism, and &ldquo, proteolysis&rdquo, whereas the downregulated DEGs were mainly involved in &ldquo, and metabolism of various molecules. In addition, genes encoding abscisic acid biosynthetic and catabolic enzymes, heat shock proteins, and E3 ubiquitin ligases were significantly altered by drought stress, indicating involvement in drought tolerance. A further comparison with the DEGs related to salinity stress-treated garlic revealed 867 and 305 DEGs with a similar and reverse expression alteration tendency, respectively.

Yarrowia lipolytica has been extensively used to produce essential chemicals and enzymes. As in most other eukaryotes, nonhomologous end joining (NHEJ) is the major repair pathway for DNA double-strand breaks in Y. lipolytica. Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica. Through optimization of the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with 12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, the cell cycle, and the integration sites. Thus, the targeted integration rate reached 55% through G(1) phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes in the pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers and achieves one-step targeted genome integration of the 8,417-bp DNA fragment, potentially replacing current HR-dependent genome-editing methods for Y. lipolytica. IMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica. This tool does not require the construction of homologous templates and can be used to rapidly verify genetic elements and to iteratively integrate multiple-gene pathways in Y. lipolytica. This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.

Embryonic stem (ES) cells have the property of self-renewal and multi-directional differentiation, and provide an ideal model for studying early embryo development in vitro. Wnt3, as Wnt family member 3, plays a vital role during ES cell differentiation. However, the exact regulatory mechanism of Wnt3 remains to be elucidated. MicroRNAs can directly regulate gene expression at the post-transcriptional level and play critical function in cell fate determination. Here, we found the expression level of miR-184 decreased when ES cells differentiated into cardiac mesoderm then increased during the process as differentiated into cardiomyocytes, which negatively correlated with the expression of Wnt3. Overexpression of miR-184 during the process of ES cell differentiation into cardiac mesoderm repressed cardiac mesoderm differentiation and cardiomyocyte formation. Bioinformatics prediction and mechanism studies showed that miR-184 directly bound to the 3′UTR region of Wnt3 and inhibited the expression level of Wnt3. Consistently, knockdown of Wnt3 mimicked the effects of miR-184-overexpression on ES cell differentiation into cardiac mesoderm, whereas overexpression of Wnt3 rescued the inhibition effects of miR-184 overexpression on ES cell differentiation. These findings demonstrated that miR-184 is a direct regulator of Wnt3 during the differentiation process of ES cells, further enriched the epigenetic regulatory network of ES cell differentiation into cardiac mesoderm and cardiomyocytes.

Grain yield, which is mainly contributed by tillering capacity as well as kernel number and weight, is the most important trait to plant breeders and agronomists. Label-free quantitative proteomics was used to analyse yield-contributing organs in wheat. These were leaf sample, tiller initiation, spike initiation, ovary and three successive kernel development stages at 5, 10 and 15 days after anthesis (DAA). We identified 3182 proteins across all samples. The largest number was obtained for spike initiation (1673), while the smallest was kernel sample at 15 DAA (709). Of the 3182 proteins, 296 of them were common to all seven organs. Organ-specific proteins ranged from 148 in ovary to 561 in spike initiation. When relative protein abundances were compared to that of leaf sample, 347 and 519 proteins were identified as differentially abundant in tiller initiation and spike initiation, respectively. When compared with ovary, 81, 35 and 96 proteins were identified as differentially abundant in kernels sampled at 5, 10 and 15 DAA, respectively. Our study indicated that two Argonaute proteins were solely expressed in spike initiation. Of the four expansin proteins detected, three of them were mainly expressed during the first 10 days of kernel development after anthesis. We also detected cell wall invertases and sucrose and starch synthases mainly during the kernel development period. The manipulation of these proteins could lead to increases in tillers, kernels per spike or final grain weight, and is worth exploring in future studies., We studied the proteome profiles during the kernel development process in wheat. Using proteomics procedure, seven plant organs were quantified for protein abundance. A total of 3182 proteins were identified across all the organs. Two hundred ninety-six proteins were identified in all the organs. This study demonstrated organ-specific detection and abundance of many important wheat proteins that may contribute to tiller formation, spike initiation and kernel development. The findings from this study could pave the way for in-depth study of the different components of yield formation in wheat.

The Qinghai-Tibetan Plateau has a mean altitude exceeding 4000 m and covers about 2.5 million km². More than 60% of this area is alpine grassland. Exclosures have been widely used in this region to study the sustainable use of grassland resources. We used patterns of functional trait diversity to infer the effects of exclosures on community assembly in alpine meadows. We studied functional diversity using five traits under grazing and three enclosed (exclosure) plots (3, 8, and 18 years old) in an alpine meadow on the Qinghai-Tibetan Plateau. We quantified the strength of the community assembly processes by comparing the observed functional trait diversity with a null model that assumes random community assembly. We found evidence for deterministic assembly processes for plant communities in exclosures. The changes in CWM of the five traits from grazing land to 18-year exclosure indicated that environmental filtering occurred due to the exclosures. Multivariate functional diversity (MFDis and MPDₛₑₛ), and functional diversity of individual traits, including that of leaf area, leaf weight and aboveground biomass (FDis of leaf area, leaf weight, and aboveground biomass), increased gradually from grazing land to the 18-year exclosure, and the values of the 18-year exclosure were significantly greater than null expectation. This can be interpreted to indicate that exclosures resulted in greater competitive interaction between species. These results suggest that the effect of exclosures on community assembly is more deterministic than stochastic in this meadow.

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC50) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC10) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs.

Clarifying the regulatory mechanisms of embryonic stem cell (ESC) neural differentiation is helpful not only for understanding neural development but also for obtaining high-quality neural progenitor cells required by stem cell therapy of neurodegenerative diseases. Here, we found that long noncoding RNA 1604 (lncRNA-1604) was highly expressed in cytoplasm during neural differentiation, and knockdown of lncRNA-1604 significantly repressed neural differentiation of mouse ESCs both in vitro and in vivo. Bioinformatics prediction and mechanistic analysis revealed that lncRNA-1604 functioned as a novel competing endogenous RNA of miR-200c and regulated the core transcription factors ZEB1 and ZEB2 during neural differentiation. Furthermore, we also demonstrated the critical role of miR-200c and ZEB1/2 in mouse neural differentiation. Either introduction of miR-200c sponge or overexpression of ZEB1/2 significantly reversed the lncRNA-1604 knockdown-induced repression of mouse ESC neural differentiation. Collectively, these findings not only identified a previously unknown role of lncRNA-1604 and ZEB1/2 but also elucidated a new regulatory lncRNA-1604/miR-200c/ZEB axis in neural differentiation.

The pathogenicity of Metarhizium rileyi is a multi-faceted process that depends on many factors. This study attempts to decipher those factors of M. rileyi by investigating its pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae) larvae. Through morphogenesis analysis, we for the first time demonstrated the infection structure, appressorium, of M. rileyi that can generate a more than 4 MPa turgor pressure. The Mrpmk1 gene was found to be essential for appressorium differentiation and mycelium reemerging, ΔMrpmk1 mutant exhibited no pathogenicity towards S. litura by natural infection process. Delayed appressorium formation time, decreased appressorium formation rate and turgor pressure of ΔMrpbs2 mutant manifested itself in postponed death time and lower mortality against S. litura. Following invasion into the larval hemocoel, M. rileyi cells transformed into blastospores, which may be conducive to dispersal and propagation, moreover, the blastospore form M. rileyi may subverted phagocytic defenses. Then M. rileyi cells morphed into extended hyphal body to cope with elongated hemocytes that participated in encapsulation. In the end, M. rileyi mycelia reemerged from the larval cadaver evenly to form muscardine cadaver. Eventually, conidia were produced to complete the infection cycle. During the infection, M. rileyi triggered both cellular and humoral immunity of S. litura. Besides morphological changes, stage-specifically produced oxalic acid and F-actin arrangement may play roles in nutrient acquisition and mycelium reemerging, respectively.

Large-scale sequencing studies discovered substantial genetic variants occurring in enhancers which regulate genes via long range chromatin interactions. Importantly, such variants could affect enhancer regulation by changing transcription factor bindings or enhancer hijacking, and in turn, make an essential contribution to disease progression. To facilitate better usage of published data and exploring enhancer deregulation in various human diseases, we created DiseaseEnhancer (http://biocc.hrbmu.edu.cn/DiseaseEnhancer/), a manually curated database for disease-associated enhancers. As of July 2017, DiseaseEnhancer includes 847 disease-associated enhancers in 143 human diseases. Database features include basic enhancer information (i.e. genomic location and target genes); disease types; associated variants on the enhancer and their mediated phenotypes (i.e. gain/loss of enhancer and the alterations of transcription factor bindings). We also include a feature on our website to export any query results into a file and download the full database. DiseaseEnhancer provides a promising avenue for researchers to facilitate the understanding of enhancer deregulation in disease pathogenesis, and identify new biomarkers for disease diagnosis and therapy.

Bilirubin-related adverse reactions (ADR, e.g., jaundice and hyperbilirubinemia) induced by herbs rich in certain polyphenolic acids are widely reported. However, the causes and the mechanisms underlying these ADR are not well understood. The purpose of this article is to determine the mechanism by which certain polyphenolic acids inhibit UGT1A1-mediated bilirubin glucuronidation, leading to jaundice or hyperbilirubinemia. We investigated in vitro inhibitory effects on bilirubin glucuronidation of salvianolic acid A (SAA), salvianolic acid B (SAB), danshensu (DSS), protocatechuic aldehyde (PA), and rosmarinic acid (RA), as well as two Salvia miltiorrhiza injections (DSI and CDI) rich in polyphenolic acids. The results showed that average formation rates of three bilirubin glucuronides displayed a significant difference (p0.05) and the formation of monoglucuronide was favored regardless if an inhibitor was present or not. SAA, SAB, DSI, and CDI, but not DSS, PA, and RA, significantly inhibited human UGT1A1-mediated bilirubin glucuronidation via a mixed-type inhibitory mechanism. Average IC

Congenital heart disease (CHD) affects up to 1 % of live births1. Although a genetic etiology is indicated by an increased recurrence risk2,3, sporadic occurrence suggests that CHD genetics is complex4. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS5–7. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR–Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.

Several studies have identified the critical role of calcium-sensing receptors (CaSRs) in cardiac ischaemia/reperfusion injury and cardiac hypertrophy and have demonstrated that CaSRs induce myocardial apoptosis by activating MAPKs. Using acute myocardial infarction rat models, we found that a combination therapy of CaSR inhibition and embryonic stem cell (ESC) transplantation after acute myocardial infarction (AMI) leads to a dramatic reduction in the infarct size; a significant increase in the maximum rising and falling rate (+dp/dtmax and -dp/dtmax, respectively) of left ventricular pressure; a significant decrease in left ventricular end-diastolic pressure; a significant decrease in the mRNA expression level of CaSR, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, p-ERK, p-JNK and p-P38 protein together with apoptosis indexes in the C and E groups; and a significant decrease in cTnT levels as well as LDH and CK activity. These findings indicate that cardiac function could be enhanced significantly by combination therapy with CaSR inhibition and ESC transplantation; the effect was better than ESC transplantation alone, and the mechanism might be associated with a reduction in cell apoptosis via the inhibition of the MAPK pathway. Apoptosis could be reduced through CaSR, which regulates the MAPK pathway and apoptosis-related protein. Our study indicated that CaSR inhibitors have a pivotal role in the treatment of AMI.

Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL-1 and the medium inhibition of control (IC50) was 0.73 ng mL-1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were

Objective—Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. Methods— We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases which were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and Type 1 or Type 2 of human prions on diagnostic performance. Results—The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1-2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, while genetic prion diseases revealed distinct profiles for each PRNP gene mutation. Interpretation—The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and Tau proteins and together with PRNP gene sequencing, the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease—two important considerations for envisioned therapeutic interventions. This article is protected by copyright. All rights reserved.

Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258–1.407 μg/mL, the medium inhibition concentration (IC 50 ) were 0.727–0.892 μg/mL and detection limit (IC 10 ) were 0.029–0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21–5.94 × 10 7 M −1 . The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%–100.8% with a CV at 2.5%–9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.

Embryonic stem cells (ESCs) have self-renewal and multi-lineage differentiation potential and perform critical functions in development and biomedicine. Several long noncoding RNAs (lncRNAs) have been reported as key regulators of stem cell pluripotency and differentiation. However, the function and regulatory mechanism of lncRNAs during the initiation of ESC differentiation remains unclear. Here, we found that linc1557 was highly expressed in mouse ESCs and required for the initiation of ESC differentiation. Knockdown of linc1557 increased the expression and phosphorylation levels of signal transducer and activator of transcription 3 (STAT3), a key factor in the leukemia inhibitory factor (LIF)/STAT3 signaling pathway. Furthermore, we found that linc1557 directly bound to Stat3 mRNA and affected its stability. The differentially expressed transcriptome after linc1557 knockdown in ESCs was involved primarily in multicellular organism development and cell differentiation as similar to that after Stat3 knockdown. Moreover, either knockdown of Stat3 or addition of a LIF/STAT3 signaling inhibitor rescued the suppressive effects of linc1557 knockdown on the initiation of mouse ESC differentiation. These findings not only elucidated the critical function of linc1557 in the initiation of mouse ESC differentiation but also clarified that its specific mechanism as directly affecting Stat3 mRNA stability, which enhanced the understanding of the lncRNA-mediated regulatory mechanism for mRNA stability and key signaling pathways in ESC pluripotency and differentiation.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease with poor prognosis. The mechanisms underlying the role of alveolar epithelial cell (AEC) senescence in IPF remain poorly understood. We aimed to investigate if PTEN/Akt activates AEC senescence to induce pulmonary fibrosis. We investigated the association between PTEN/Akt and cellular senescence in lung tissues from IPF patients. As a result, decreased PTEN and activated Akt pathway were found in AECs in fibrotic lung tissues detected by immunohistochemistry (IHC) and immunofluorescence (IF). Increased expression levels of aging-associated markers (P21WAF1 and SA-β-gal) in AECs treated with bleomycin were found. AEC senescence was accelerated by PTEN knockdown and attenuated by PTEN overexpression. Bleomycin induced AEC senescence was reversed by Akt2 knockdown and the pharmacological inhibitors (LY294002 and MK2206) of the Akt pathway. Reducing Akt activation dramatically improved lung fibrosis in a fibrotic mice model. In addition, a co-immunoprecipitation (co-IP) assay demonstrated that PTEN physically associated with Akt. These indicated that senescent AECs modulated by the PTEN/Akt pathway promote lung fibrosis. In conclusion, our study demonstrated that as a trigger indicator in IPF, the senescence process in AECs should be a potential therapeutic target and that the PTEN/Akt pathway may be a promising candidate for intervention.

[Image: see text] Myeloperoxidase (MPO) generates reactive oxygen species that potentially contribute to many chronic inflammatory diseases. A recently reported triazolopyrimidine MPO inhibitor was optimized to improve acid stability and remove methyl guanine methyl transferase (MGMT) activity. Multiple synthetic routes were explored that allowed rapid optimization of a key benzyl ether side chain. Crystal structures of inhibitors bound to the MPO active site demonstrated alternate binding modes and guided rational design of MPO inhibitors. Thioether 36 showed significant inhibition of MPO activity in an acute mouse inflammation model after oral dosing.

The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRβ-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.

Based on advantages of microwave-assisted extraction (MAE), aqueous two-phase extraction (ATPE) and reversed micellar extraction (RME), a novel method of extraction and purification of alkaloids from Sophora flavescens Ait. was put forward by using focused microwave-assisted aqueous two-phase extraction (FMAATPE) coupled with the reversed micellar extraction (RME). The aqueous two-phase system (ATPS) consisted of ethanol and ammonia sulfate was used as extracting solvent, FMAATPE conditions such as the compositions of ATPS, and the extraction temperature, extraction time and solvent-to-materials ratio were optimized by orthogonal test through HPLC analysis. Then alkaloids extracted by FMAATPE were further purified with reverse micelles of sodium dodecyl benzene sulfonate/isooctane/ n -octanol. The relative purity of total alkaloids in the product was improved greatly by RME from 58.30% to 88.75% (w/w), and purification recovery was higher than that of solvent extraction, ion exchange resin and macroporous adsorption resin. Moreover, the reverse micelles used could be readily recycled by adding salt and water, keeping better extraction performance with about 80% (w/w) of recovery. Thus, the proposed method is easy to adjust, operate and enlarge for better application prospect; the purity of alkaloids obtained meets the requirements of the compound drug preparation.

Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development., Summary: Outflow tract malalignment and multiple birth defects observed in the Prickle1 mutant may arise from cell polarity perturbation, which may involve disruptions in Wnt signaling and of cilia function.

Recent studies have shown that Nogo-A and the Nogo-A receptor affect β-amyloid metabolism and the downstream Rho GTP enzyme signaling pathway, which may affect the levels of β-amyloid and tau. Nogo-A may play a key role in the pathogenesis of Alzheimer's disease. However, the underlying molecular mechanisms of Fasudil treatment in Alzheimer's disease are not yet clear. Our results have found that Fasudil treatment for two months substantially ameliorated behavioral deficits, diminished β-amyloid plaque and tau protein pathology, and alleviated neuronal apoptosis in APP/PS1 transgenic mice. More importantly, two well-established markers for synaptic function, growth-associated protein 43 and synaptophysin, were upregulated after Fasudil treatment. Finally, the levels of Nogo-A, Nogo-A receptor complex NgR/p75NTR/LINGO-1 and the downstream Rho/Rho kinase signaling pathway were significantly reduced. These findings suggest that Fasudil exerts its neuroprotective function in Alzheimer's disease by inhibiting the Nogo-A/NgR1/RhoA signaling pathway.

Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 107 CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed in Escherichia coli (E.coli) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 μg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC50) of 0.471 μg/L and a limit of detection (LOD) of 0.044 μg/L, which is below the maximum residue limit standard (MRLs) of 1.0 μg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2–106.3% with CVs of 2.62–10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.

Publisher's version (útgefin grein)., Background: Genome-wide association studies conducted on QRS duration, an electrocardiographic measurement associated with heart failure and sudden cardiac death, have led to novel biological insights into cardiac function. However, the variants identified fall predominantly in non-coding regions and their underlying mechanisms remain unclear. Results: Here, we identify putative functional coding variation associated with changes in the QRS interval duration by combining Illumina HumanExome BeadChip genotype data from 77,898 participants of European ancestry and 7695 of African descent in our discovery cohort, followed by replication in 111,874 individuals of European ancestry from the UK Biobank and deCODE cohorts. We identify ten novel loci, seven within coding regions, including ADAMTS6, significantly associated with QRS duration in gene-based analyses. ADAMTS6 encodes a secreted metalloprotease of currently unknown function. In vitro validation analysis shows that the QRS-associated variants lead to impaired ADAMTS6 secretion and loss-of function analysis in mice demonstrates a previously unappreciated role for ADAMTS6 in connexin 43 gap junction expression, which is essential for myocardial conduction. Conclusions: Our approach identifies novel coding and non-coding variants underlying ventricular depolarization and provides a possible mechanism for the ADAMTS6-associated conduction changes., Funding This work was funded by a grant to YJ from the British Heart Foundation (PG/12/38/29615). AGES: This study has been funded by NIH contracts N01-AG-1-2100 and 271201200022C, the NIA Intramural Research Program, Hjartavernd (the Icelandic Heart Association), and the Althingi (the Icelandic Parliament). The study is approved by the Icelandic National Bioethics Committee, VSN: 00–063. The researchers are indebted to the participants for their willingness to participate in the study. ARIC: The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C), R01HL087641, R01HL59367, and R01HL086694; National Human Genome Research Institute contract U01HG004402; and National Institutes of Health contract HHSN268200625226C. The authors thank the staff and participants of the ARIC study for their important contributions. Infrastructure was partly supported by Grant Number UL1RR025005, a component of the National Institutes of Health and NIH Roadmap for Medical Research. Funding support for “Building on GWAS for NHLBI-diseases: the U.S. CHARGE consortium” was provided by the NIH through the American Recovery and Reinvestment Act of 2009 (ARRA) (5RC2HL102419). BRIGHT: The Exome Chip genotyping was funded by Wellcome Trust Strategic Awards (083948 and 085475). This work was also supported by the Medical Research Council of Great Britain (Grant no. G9521010D); and by the British Heart Foundation (Grant no. PG/02/128). AFD was supported by the British Heart Foundation (Grant nos. RG/07/005/23633 and SP/08/005/25115); and by the European Union Ingenious HyperCare Consortium: Integrated Genomics, Clinical Research, and Care in Hypertension (grant no. LSHM-C7–2006-037093). The BRIGHT study is extremely grateful to all the patients who participated in the study and the BRIGHT nursing team. We would also like to thank the Barts Genome Centre staff for their assistance with this project. CHS: This Cardiovascular Health Study (CHS) research was supported by NHLBI contracts HHSN268201800001C, HHSN268201200036C, HHSN268200800007C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086; and NHLBI grants R01HL068986, U01HL080295, R01HL087652, R01HL105756, R01HL103612, R01HL120393, and U01HL130114 with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided through R01AG023629 from the National Institute on Aging (NIA). A full list of principal CHS investigators and institutions can be found at CHS-NHLBI.org. The provision of genotyping data was supported in part by the National Center for Advancing Translational Sciences, CTSI grant UL1TR001881, and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ERF: The ERF study as a part of EUROSPAN (European Special Populations Research Network) was supported by European Commission FP6 STRP grant number 018947 (LSHG-CT-2006-01947) and also received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013)/grant agreement HEALTH-F4–2007-201413 by the European Commission under the programme “Quality of Life and Management of the Living Resources” of 5th Framework Programme (no. QLG2-CT-2002-01254). The ERF study was further supported by ENGAGE consortium and CMSB. High-throughput analysis of the ERF data was supported by joint grant from Netherlands Organization for Scientific Research and the Russian Foundation for Basic Research (NWO-RFBR 047.017.043). We are grateful to all study participants and their relatives, general practitioners, and neurologists for their contributions to the ERF study and to P Veraart for her help in genealogy, J Vergeer for the supervision of the laboratory work, and P Snijders for his help in data collection. FHS: The Framingham Heart Study (FHS) research reported in this article was supported by a grant from the National Heart, Lung, and Blood Institute (NHLBI), HL120393. Generation Scotland: Generation Scotland received core support from the Chief Scientist Office of the Scottish Government Health Directorates (CZD/16/6) and the Scottish Funding Council (HR03006). Genotyping of the Generation Scotland and Scottish Family Health Study samples was carried out by the Genetics Core Laboratory at the Clinical Research Facility, Edinburgh, Scotland and was funded by the UK’s Medical Research Council. GOCHA: The Genetics of Cerebral Hemorrhage with Anticoagulation was carried out as a collaborative study supported by grants R01NS073344, R01NS059727, and 5K23NS059774 from the NIH–National Institute of Neurological Disorders and Stroke (NIH-NINDS). GRAPHIC: The GRAPHIC Study was funded by the British Heart Foundation (BHF/RG/2000004). NJS and CPN are supported by the British Heart Foundation and is a NIHR Senior Investigator. This work falls under the portfolio of research supported by the NIHR Leicester Cardiovascular Biomedical Research. INGI-FVG: This study has been funded by Regione FVG (L.26.2008). INTER99: The Inter99 was initiated by Torben Jørgensen (PI), Knut Borch-Johnsen (co-PI), Hans Ibsen and Troels F. Thomsen. The steering committee comprises the former two and Charlotta Pisinger. The study was financially supported by research grants from the Danish Research Council, the Danish Centre for Health Technology Assessment, Novo Nordisk Inc., Research Foundation of Copenhagen County, Ministry of Internal Affairs and Health, the Danish Heart Foundation, the Danish Pharmaceutical Association, the Augustinus Foundation, the Ib Henriksen Foundation, the Becket Foundation, and the Danish Diabetes Association. The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent Research Center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation (www.metabol.ku.dk). JHS: We thank the Jackson Heart Study (JHS) participants and staff for their contributions to this work. The JHS is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. Dr. Wilson is supported by U54GM115428 from the National Institute of General Medical Sciences. KORA: The KORA study was initiated and financed by the Helmholtz Zentrum München – German Research Center for Environmental Health, which is funded by the German Federal Ministry of Education and Research (BMBF) and by the State of Bavaria. Furthermore, KORA research was supported within the Munich Center of Health Sciences (MC-Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. Korcula: This work was funded by the Medical Research Council UK, The Croatian Ministry of Science, Education and Sports (grant 216–1080315-0302), the Croatian Science Foundation (grant 8875), the Centre of Excellence in Personalized health care, and the Centre of Competencies for Integrative Treatment, Prevention and Rehabilitation using TMS. LifeLines: The LifeLines Cohort Study and generation and management of GWAS genotype data for the LifeLines Cohort Study are supported by The Netherlands Organization of Scientific Research NWO (grant 175.010.2007.006), the Economic Structure Enhancing Fund (FES) of the Dutch government, the Ministry of Economic Affairs, the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the Northern Netherlands Collaboration of Provinces (SNN), the Province of Groningen, University Medical Center Groningen, the University of Groningen, Dutch Kidney Foundation, and Dutch Diabetes Research Foundation. Niek Verweij is supported by NWO-VENI (016.186.125) and Marie Sklodowska-Curie GF (call: H2020-MSCA-IF-2014, Project ID: 661395). UHP: Folkert W. Asselbergs is supported by UCL Hospitals NIHR Biomedical Research Centre. Ilonca Vaartjes is supported by a Dutch Heart Foundation grant DHF project “Facts and Figures.” MGH-CAMP: Dr. Patrick Ellinor is funded by NIH grants (2R01HL092577, 1R01HL128914, R01HL104156, and K24HL105780) and American Heart Association Established Investigator Award 13EIA14220013 (Ellinor). Dr. Steve Lubitz is funded by NIH grants K23HL114724 and a Doris Duke Charitable Foundation Clinical Scientist Development Award 2014105. NEO: The authors of the NEO study thank all individuals who participated in the Netherlands Epidemiology in Obesity study, all participating general practitioners for inviting eligible participants, and all research nurses for collection of the data. We thank the NEO study group, Pat van Beelen, Petra Noordijk, and Ingeborg de Jonge for the coordination, lab, and data management of the NEO study. We also thank Arie Maan for the analyses of the electrocardiograms. The genotyping in the NEO study was supported by the Centre National de Génotypage (Paris, France), headed by Jean-Francois Deleuze. The NEO study is supported by the participating Departments, the Division and the Board of Directors of the Leiden University Medical Center, and by the Leiden University, Research Profile Area Vascular and Regenerative Medicine. Dennis Mook-Kanamori is supported by Dutch Science Organization (ZonMW-VENI Grant 916.14.023). RS-I: The generation and management of the Illumina Exome Chip v1.0 array data for the Rotterdam Study (RS-I) was executed by the Human Genotyping Facility of the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands. The Exome chip array dataset was funded by the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO)-sponsored Netherlands Consortium for Healthy Aging (NCHA; project nr. 050–060-810); the Netherlands Organization for Scientific Research (NWO; project number 184021007); and by the Rainbow Project (RP10; Netherlands Exome Chip Project) of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL; www.bbmri.nl). We thank Ms. Mila Jhamai, Ms. Sarah Higgins, and Mr. Marijn Verkerk for their help in creating the exome chip database, and Carolina Medina-Gomez, MSc, Lennard Karsten, MSc, and Linda Broer PhD for QC and variant calling. Variants were called using the best practice protocol developed by Grove et al. as part of the CHARGE consortium exome chip central calling effort. The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Research Institute for Diseases in the Elderly (RIDE), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam. The authors are grateful to the study participants, the staff from the Rotterdam Study, and the participating general practitioners and pharmacists. The work of Bruno H. Stricker is supported by grants from the Netherlands Organization for Health Research and Development (ZonMw) (Priority Medicines Elderly 113102005 to ME and DoelmatigheidsOnderzoek 80–82500–98-10208 to BHS). The work of Mark Eijgelsheim is supported by grants from the Netherlands Organization for Health Research and Development (ZonMw) (Priority Medicines Elderly 113102005 to ME and DoelmatigheidsOnderzoek 80–82500–98-10208 to BHS). SHIP: SHIP is supported by the BMBF (grants 01ZZ9603, 01ZZ0103, and 01ZZ0403) and the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG]; grant GR 1912/5–1). SHIP and SHIP-TREND are part of the Community Medicine Research net (CMR) of the Ernst-Moritz-Arndt University Greifswald (EMAU) which is funded by the BMBF as well as the Ministry for Education, Science and Culture and the Ministry of Labor, Equal Opportunities, and Social Affairs of the Federal State of Mecklenburg-West Pomerania. The CMR encompasses several research projects that share data from SHIP. The EMAU is a member of the Center of Knowledge Interchange (CKI) program of the Siemens AG. SNP typing of SHIP and SHIP-TREND using the Illumina Infinium HumanExome BeadChip (version v1.0) was supported by the BMBF (grant 03Z1CN22). We thank all SHIP and SHIP-TREND participants and staff members as well as the genotyping staff involved in the generation of the SNP data. TWINSUK: TwinsUK is funded by the Wellcome Trust, Medical Research Council, European Union, the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility and Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London. UKBB: This research has been conducted using the UK Biobank Resource (application 8256 - Understanding genetic influences in the response of the cardiac electrical system to exercise) and is supported by Medical Research Council grant MR/N025083/1. We also wish to acknowledge the support of the NIHR Cardiovascular Biomedical Research Unit at Barts and Queen Mary University of London, UK. PD Lambiase acknowledges support from the UCLH Biomedicine NIHR. MO is supported by an IEF 2013 Marie Curie fellowship. JR acknowledges support from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA grant agreement no. 608765. YFS: The Young Finns Study has been financially supported by the Academy of Finland: grants 286284, 134309 (Eye), 126925, 121584, 124282, 129378 (Salve), 117787 (Gendi), and 41071 (Skidi); the Social Insurance Institution of Finland; Competitive State Research Financing of the Expert Responsibility area of Kuopio, Tampere and Turku University Hospitals (grant X51001); Juho Vainio Foundation; Paavo Nurmi Foundation; Finnish Foundation for Cardiovascular Research; Finnish Cultural Foundation; Tampere Tuberculosis Foundation; Emil Aaltonen Foundation; Yrjö Jahnsson Foundation; Signe and Ane Gyllenberg Foundation; and Diabetes Research Foundation of Finnish Diabetes Association. The expert technical assistance in the statistical analyses by Irina Lisinen is gratefully acknowledged. Cell culture and biochemistry: Funding was provided by the National Institutes of Health (Program of Excellence in Glycoscience award HL107147 to SSA and F32AR063548 to TJM) and the David and Lindsay Morgenthaler Postdoctoral Fellowship (to TJM) and by the Allen Distinguished Investigator Program, through support made by The Paul G. Allen Frontiers Group and the American Heart Association (to SSA). Mutant mouse model: Adamts6 mutant mice were generated and further propagated and analyzed by funding provided by NIH grants HL098180 and HL132024 (to CWL) and by the Allen Distinguished Investigator Program, through support made by The Paul G. Allen Frontiers Group and the American Heart Association (to SSA).

Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7μg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05μg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation

RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of 12 subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, and thus the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, that directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. An interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 subcomplex is disrupted, leading to defects in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 subcomplex and subsequently the biogenesis of RNAPII.

Rationale: Clear cell renal cell carcinoma (CCRCC) is an aggressive tumor associated with a high risk of metastasis and very low survival rate. In addition, it can cause extensive blood metastasis to the lungs, bones, and other organs. Chest wall metastatic tumors from primary CCRCC are rare. Patient concerns: In this report, we present a case of metastatic chest wall tumor that originated from a CCRCC. Diagnoses: An 86-year-old man was diagnosed with chest wall tumor using chest computed tomography. After collecting tissues from the chest wall tumor via needle biopsy, the pathological examination was combined with positive immunoreaction of CD10, epithelial membrane antigen, and vimentin, and the patient was diagnosed with metastatic CCRCC in the chest wall deposits. Interventions: The patient received radiotherapy at 2.0 Gy per time for 25 times. Outcomes: Following 2 months of treatment, the chest wall tumor had shrunk by about one-third of its size. Lessons: Our patient developed a metastatic chest wall tumor that originated from a CCRCC for which right nephrectomy had been performed 10 years previously. Although as per the literature, chest wall metastasis from CCRCC is very rare, it is important to consider tumor metastasis after several years of treatment for precise diagnosis and proper treatment.

In buried seedlings, chloroplasts are arrested at the etioplast stage, but they rapidly mature upon emergence of the seedling. Etioplast-chloroplast differentiation is halted through the integration of soil-induced signals, including pressure and the absence of light, although the details on how this information converges to regulate cellular decisions remain unclear. Here, we identify an interdependent transcription module that integrates the mechanical pressure and darkness signals to control chloroplast development in Arabidopsis thaliana. Mutations of ETHYLENE-INSENSITIVE3 (EIN3), the primary transcription factor in the ethylene signaling pathway that is activated in response to mechanical pressure, cause early development of etioplasts in the dark and severe photobleaching upon light exposure. Genetic studies demonstrate that repression of etioplast differentiation by EIN3 requires PHYTOCHROME INTERACTING FACTOR3 (PIF3), a darkness-stabilized bHLH transcription factor. EIN3 and PIF3 directly interact and form an interdependent module to repress the expression of most LIGHT HARVESTING COMPLEX (LHC) genes; overexpressing even one LHC could cause premature development of etioplasts. The EIN3-PIF3 transcription module synergistically halts chloroplast development by interdependently co-occupying the promoters of LHC genes. Thus, our results define a transcriptional regulatory module and provide mechanistic insight on the concerted regulation of chloroplast development by multiple soil-induced signals.

Bisphenol A (BPA) is a suspected human endocrine disruptor which is widely used.In order to determine whether urine BPA level is associated with serum reproductive hormone levels among male adults, we carried out a cross-sectional study in China. We recruited 592 male workers and collected their urine samples for BPA measurement. We also collected blood samples and examined serum reproductive hormones. We used multiple linear regression and log-binomial model to examine associations between urine BPA level and hormone levels after controlling for age and smoking status.An increased urine BPA level was associated with increased prolactin (p0.001), estradiol (p0.001), sex hormone-binding globulin level (p=0.001), and a reduced androstenedione (p0.001) and free androgen index level (p=0.021). Males, whose urine BPA level was in the 2nd, 3rd and highest quartiles, had respectively 1.58, 1.33 and 3.09-fold increased prevalence of having a high prolactin level (P75 level). The highest quartile of BPA level was associated with 1.63 and 1.50-fold increased prevalence of having a high estradiol and elevated sex hormone-binding globulin level. Males with higher quartile of BPA level had a lower inhibin B level.High BPA exposure is associated with increased prolactin, estradiol and sex hormone-binding globulin level in males, and may contribute to male infertility.

A rapid and effective method of integrating extraction and purification for alkaloids from Sophora flavescens Ait. was developed by microwave-assisted aqueous two-phase extraction (MAATPE) based on the high efficiency of microwave-assisted extraction (MAE) and the demixing effect of aqueous two-phase extraction (ATPE). The aqueous two-phase system (ATPS), ethanol/ammonia sulfate was chosen from seven combinations of ethanol/salt systems, and its extraction properties were investigated in detail. Key factors, namely, the compositions of ATPS, solvent-to-materials ratio, and the extraction temperature were selected for optimization of the experimental conditions using response surface methodology (RSM) on the basis of the results of the single-factor experiment. The final optimized conditions were, the compositions of ATPS: ethanol 28% (w/w) and (NH 4 ) 2 SO 4 18% (w/w), solvent-to-material ratio 60:1, temperature 90 °C, extraction time 5 min, and microwave power 780 W. MAATPE was superior to MAE, the latter using a single solvent, not only in extraction yield but also in impurity content. Moreover, compared with the combination of MAE and ATPE in the two-step mode, MAATP demonstrated fewer impurities, a better yield (63.78 ± 0.45 mg/g) and a higher recovery (92.09 ± 0.14%) in the extraction and purification of alkaloids. A continuous multiphase-extraction model of MAATPE was proposed to explicate the extraction mechanism. MAATPE revealed that the interaction between microwave and ATPS cannot only cause plant cell rupture but also accelerate demixing, improving mass-transfer from solid–liquid extraction to liquid–liquid purification. MAATPE simplified procedures also contributed to the lower loss occurrence, better extraction efficiency, and reduced impurity to target constituents.

Chlorophyll biosynthesis is one of the most important cellular processes and is essential for plant photosynthesis. After germination under the soil, dark-grown seedlings are etiolated and accumulate the chlorophyll precursor protochlorophyllide (Pchlide) in cotyledons. Upon exposure to light, Pchlide is rapidly converted to chlorophyll to initiate photoautotrophic growth. In this light-regulated de-etiolation process, multiple endogenous phytohormones are also involved. Although the co-regulation of seedling greening by light and hormones has long been observed, recent studies greatly advanced our understanding of their interplay by identifying the key components connecting these pathways. The integrators, such as PHYTOCHROMEINTERACTING FACTORs (PIFs), ELONGATED HYPOCOTYL 5 (HY5), ETHYLENE INSENSTIVE 3 (EIN3) and DELLA proteins, are key transcription regulators in light or hormone signaling pathways. This review focuses on these integrators and illustrates the regulatory networks of light and hormone interactions in chlorophyll biosynthesis.

Myeloperoxidase, a mammalian peroxidase involved in the immune system as an anti-microbial first responder, can produce hypochlorous acid in response to invading pathogens. Myeloperoxidase has been implicated in several chronic pathological diseases due to the chronic production of hypochlorous acid, as well as other reactive radical species. A high throughput screen and triaging protocol was developed to identify a reversible inhibitor of myeloperoxidase toward the potential treatment of chronic diseases such as atherosclerosis. The identification and characterization of a reversible myeloperoxidase inhibitor, 7-(benzyloxy)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine is described.

DNA methylation and histone methylation (H3K27me3) have been reported as major barriers to induced pluripotent stem cell (iPSC) generation using four core transcription factors (Oct4, Sox2, Klf4, and c-Myc, termed OSKM). Here, to illustrate the possibility of deriving iPSCs via demethylation, as well as the exact effects of DNA methylation and histone modification on gene expression regulation, we performed RNA sequencing to characterize the transcriptomes of ES cells and iPSCs derived by demethylation with miR-29b or shDnmt3a, and carried out integrated analyses. Results showed that OSKM + miR-29b-iPSC was more close to ES cells than the others, and up-regulated genes typically presented with methylated CpG-dense promoters and H3K27me3-enriched regions. The differentially expressed genes caused by introduction of DNA demethylation during somatic cell reprogramming mainly focus on stem cell associated GO terms and KEGG signaling pathways, which may decrease the tumorigenesis risk of iPSCs. These findings indicated that DNA methylation and histone methylation have synergetic effects on regulating gene expression during iPSC generation, and demethylation by miR-29b is better than shDnmt3a for iPSC quality. Furthermore, integrated analyses are superior for exploration of slight differences as missed by individual analysis.