Twnbiome: a public database of the healthy Taiwanese gut microbiome.

With new advances in next generation sequencing (NGS) technology at reduced costs, research on bacterial genomes in the environment has become affordable. Compared to traditional methods, NGS provides high-throughput sequencing reads and the ability to identify many species in the microbiome that were previously unknown. Numerous bioinformatics tools and algorithms have been developed to conduct such analyses. However, in order to obtain biologically meaningful results, the researcher must select the proper tools and combine them to construct an efficient pipeline. This complex procedure may include tens of tools, each of which require correct parameter settings. Furthermore, an NGS data analysis involves multiple series of command-line tools and requires extensive computational resources, which imposes a high barrier for biologists and clinicians to conduct NGS analysis and even interpret their own data. Therefore, we established a public gut microbiome database, which we call Twnbiome, created using healthy subjects from Taiwan, with the goal of enabling microbiota research for the Taiwanese population. Twnbiome provides users with a baseline gut microbiome panel from a healthy Taiwanese cohort, which can be utilized as a reference for conducting case-control studies for a variety of diseases. It is an interactive, informative, and user-friendly database. Twnbiome additionally offers an analysis pipeline, where users can upload their data and download analyzed results. Twnbiome offers an online database which non-bioinformatics users such as clinicians and doctors can not only utilize to access a control set of data, but also analyze raw data with a few easy clicks. All results are customizable with ready-made plots and easily downloadable tables. Database URL: http://twnbiome.cgm.ntu.edu.tw/ .

High-performance deep learning pipeline predicts individuals in mixtures of DNA using sequencing data.

In this study, we proposed a deep learning (DL) model for classifying individuals from mixtures of DNA samples using 27 short tandem repeats and 94 single nucleotide polymorphisms obtained through massively parallel sequencing protocol. The model was trained/tested/validated with sequenced data from 6 individuals and then evaluated using mixtures from forensic DNA samples. The model successfully identified both the major and the minor contributors with 100% accuracy for 90 DNA mixtures, that were manually prepared by mixing sequence reads of 3 individuals at different ratios. Furthermore, the model identified 100% of the major contributors and 50-80% of the minor contributors in 20 two-sample external-mixed-samples at ratios of 1:39 and 1:9, respectively. To further demonstrate the versatility and applicability of the pipeline, we tested it on whole exome sequence data to classify subtypes of 20 breast cancer patients and achieved an area under curve of 0.85. Overall, we present, for the first time, a complete pipeline, including sequencing data processing steps and DL steps, that is applicable across different NGS platforms. We also introduced a sliding window approach, to overcome the sequence length variation problem of sequencing data, and demonstrate that it improves the model performance dramatically.

Multi-ethnic Imputation System (MI-System): A genotype imputation server for high-dimensional data.

OBJECTIVE: Genotype imputation is a commonly used technique that infers un-typed variants into a study's genotype data, allowing better identification of causal variants in disease studies. However, due to overrepresentation of Caucasian studies, there's a lack of understanding of genetic basis of health-outcomes in other ethnic populations. Therefore, facilitating imputation of missing key-predictor-variants that can potentially improve a risk health-outcome prediction model, specifically for Asian ancestry, is of utmost relevance. METHODS: We aimed to construct an imputation and analysis web-platform, that primarily facilitates, but is not limited to genotype imputation on East-Asians. The goal is to provide a collaborative imputation platform for researchers in the public domain towards rapidly and efficiently conducting accurate genotype imputation. RESULTS: We present an online genotype imputation platform, Multi-ethnic Imputation System (MI-System) (https://misystem.cgm.ntu.edu.tw/), that offers users 3 established pipelines, SHAPEIT2-IMPUTE2, SHAPEIT4-IMPUTE5, and Beagle5.1 for conducting imputation analyses. In addition to 1000 Genomes and Hapmap3, a new customized Taiwan Biobank (TWB) reference panel, specifically created for Taiwanese-Chinese ancestry is provided. MI-System further offers functions to create customized reference panels to be used for imputation, conduct quality control, split whole genome data into chromosomes, and convert genome builds. CONCLUSION: Users can upload their genotype data and perform imputation with minimum effort and resources. The utility functions further can be utilized to preprocess user uploaded data with easy clicks. MI-System potentially contributes to Asian-population genetics research, while eliminating the requirement for high performing computational resources and bioinformatics expertise. It will enable an increased pace of research and provide a knowledge-base for genetic carriers of complex diseases, therefore greatly enhancing patient-driven research. STATEMENT OF SIGNIFICANCE: Multi-ethnic Imputation System (MI-System), primarily facilitates, but is not limited to, imputation on East-Asians, through 3 established prephasing-imputation pipelines, SHAPEIT2-IMPUTE2, SHAPEIT4-IMPUTE5, and Beagle5.1, where users can upload their genotype data and perform imputation and other utility functions with minimum effort and resources. A new customized Taiwan Biobank (TWB) reference panel, specifically created for Taiwanese-Chinese ancestry is provided. Utility functions include (a) create customized reference panels, (b) conduct quality control, (c) split whole genome data into chromosomes, and (d) convert genome builds. Users can also combine 2 reference panels using the system and use combined panels as reference to conduct imputation using MI-System.

Transcriptome profiling reveals the developmental regulation of NaCl-treated Forcipomyia taiwana eggs.

BACKGROUND: The biting midge, Forcipomyia taiwana, is one of the most annoying blood-sucking pests in Taiwan. Current chemical control methods only target the adult, not the immature stages (egg to pupa), of F. taiwana. Discovering new or alternative tactics to enhance or replace existing methods are urgently needed to improve the effectiveness of F. taiwana control. The egg is the least understood life stage in this pest species but may offer a novel point of control as addition of NaCl to the egg environment inhibits development. Thus, the objective of this study was to use RNA profiling to better understand the developmental differences between wild-type melanized (black) and NaCl-induced un-melanized (pink), infertile F. taiwana eggs. RESULTS: After de novo assembly with Trinity, 87,415 non-redundant transcripts (Ft-nr) with an N50 of 1099 were obtained. Of these, 26,247 (30%) transcripts were predicted to have long open reading frames (ORFs, defined here as ≥300 nt) and 15,270 (17.5%) transcripts have at least one predicted functional domain. A comparison between two biological replicates each of black and pink egg samples, although limited in sample size, revealed 5898 differentially expressed genes (DEGs; 40.9% of the transcripts with long ORFs) with ≥2-fold difference. Of these, 2030 were annotated to a Gene Ontology biological process and along with gene expression patterns can be separated into 5 clusters. KEGG pathway analysis revealed that 1589 transcripts could be assigned to 18 significantly enriched pathways in 2 main categories (metabolism and environmental information processing). As expected, most (88.32%) of these DEGs were down-regulated in the pink eggs. Surprisingly, the majority of genes associated with the pigmentation GO term were up-regulated in the pink egg samples. However, the two key terminal genes of the melanin synthesis pathway, laccase2 and DCE/yellow, were significantly down-regulated, and further verified by qRT-PCR. CONCLUSION: We have assembled and annotated the first egg transcriptome for F. taiwana, a biting midge. Our results suggest that down-regulation of the laccase2 and DCE/yellow genes might be the mechanism responsible for the NaCl-induced inhibition of melanization of F. taiwana eggs.

The first complete mitochondrial genome of the sand dollar Sinaechinocyamus mai (Echinoidea: Clypeasteroida).

Morphologic and molecular data often lead to different hypotheses of phylogenetic relationships. Such incongruence has been found in the echinoderm class Echinoidea. In particular, the phylogenetic status of the order Clypeasteroida is not well resolved. Complete mitochondrial genomes are currently available for 29 echinoid species, but no clypeasteroid had been sequenced to date. DNA extracted from a single live individual of Sinaechinocyamus mai was sequenced with 10× Genomics technology. This first complete mitochondrial genome (mitogenome) for the order Clypeasteroida is 15,756 base pairs in length. Phylogenomic analysis based on 34 ingroup taxa belonging to nine orders of the class Echinoidea show congruence between our new genetic inference and published trees based on morphologic characters, but also includes some intriguing differences that imply the need for additional investigation.

Smoking and Hepatitis B Virus-Related Hepatocellular Carcinoma Risk: The Mediating Roles of Viral Load and Alanine Aminotransferase.

Smoking interacts with hepatitis B virus (HBV) to increase the risk of hepatocellular carcinoma (HCC), which might be explained by its role in antiviral immunity. We evaluated the potential mediating role of viral load and/or alanine aminotransferase (ALT) in the relation of smoking with HBV-associated HCC risk. Using multiple mediation analyses to analyze data from 209 HCC cases and 1,256 controls nested within a cohort of 4,841 male HBV carriers, we found that the effect of smoking on the risk of subsequent HCC was substantially mediated through viral load (percent mediated, 31.7%; P = 0.0054), and a significant mediation effect by both viral load and ALT was also evidenced. Among the 1,143 subjects with repeated measures of viral load and ALT over periods of up to 16 years, we further observed that a higher number of pack-years of smoking was associated with higher viral load, maintenance of a high viral load (>4.39 log copies/mL), more severe hepatotoxicity grade, and increased likelihood of ALT ≥80 U/L (odds ratio, 3.14; 95% confidence interval, 1.03-9.64; odds ratio, 6.06; 95% confidence interval, 1.10-33.25, respectively, for 10-19 and ≥20 pack-years versus nonsmokers) during follow-up. Furthermore, plasma interferon-γ levels were reduced in smokers compared with nonsmokers (interferon-γ-positive rate, 14.9% versus 28.7%; P < 0.0001) at baseline. Smoking was also associated with a reduced natural killer (NK) cell frequency in peripheral blood, characterized by reduced NK function through a systems immunology approach, after long-term follow-up in a subsample (n = 171). The combination of smoking and reduced NK cell frequency further increased viral load and the likelihood of ALT ≥80 U/L. Conclusion: The data highlight a role of smoking in HBV viral load, underlining the importance of smoking prevention and cessation in hepatitis B management.

anamiR: integrated analysis of MicroRNA and gene expression profiling.

BACKGROUND: With advancements in high-throughput technologies, the cost of obtaining expression profiles of both mRNA and microRNA in the same individual has substantially decreased. Integrated analysis of these profiles can help to elucidate the functional effects of RNA expression in complex diseases, such as cancer. However, fundamental discrepancies are observed in the results from microRNA-mRNA target gene prediction algorithms, and few packages can be used to analyze microRNA and mRNA expression levels simultaneously. RESULTS: To address these issues, an R package, anamiR, was developed. A total of 10 experimental/prediction databases were integrated. Two analytical functions are provided in anamiR, including the single marker test and functional gene set enrichment analysis, and several parameters can be changed by users. Here we demonstrate the potential application of the anamiR package to 2 publicly available microarray datasets. CONCLUSION: The anamiR package is effective for an integrated analysis of both RNA and microRNA profiles. By characterizing biological functions and signaling pathways, this package helps identify dysregulated genes/miRNAs from biological and medical experiments. The source code and manual of the anamiR package are freely available at https://bioconductor.org/packages/release/bioc/html/anamiR.html .

miR-338-5p inhibits cell proliferation, colony formation, migration and cisplatin resistance in esophageal squamous cancer cells by targeting FERMT2.

Esophageal cancer is one of the leading causes of cancer death in the male population of Eastern Asia. In addition, esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer among the world. Owing to the poor overall 5-year survival rate, novel effective treatment strategies are needed. MicroRNAs are important gene regulators that are dysregulated in many cancer types. In our previous study, we applied next-generation sequencing to demonstrate that miR-338-5p was downregulated in the tumor tissue of patients with versus without recurrence. In this study, we further studied the roles of miR-338-5p in ESCC. The expression of endogenous miR-338-5p was at lower levels in ESCC cells compared with normal cells. Functional assays showed that miR-338-5p reduced cell proliferation, colony formation, migration and cisplatin resistance in an ESCC cell line, CE-81T. Potential target genes of miR-338-5p were identified by microarray and prediction tools, and 31 genes were selected. Among these, Fermitin family homolog 2 (FERMT2) plays an oncogenic role in ESCC, so it was chosen for further study. Luciferase assays showed the direct binding between miR-338-5p and the 3' untranslated region of FERMT2. Silencing of FERMT2 inhibited cell proliferation, colony formation, migration and cisplatin resistance. Pathway analysis revealed that the integrin-linked protein kinase signaling pathway, in which FERMT2 participates, was significantly affected by a miR-338-5p mimic. Our results suggest that miR-338-5p may play an antioncogenic role in ESCC via repressing FERMT2.

Incorporation of deep-sea and small-sized species provides new insights into gastropods phylogeny.

The use of phylogeny with uneven or limited taxon sampling may bias our interpretation of organismal evolution, for instance, the origin(s) of the deep-sea animals. The Mollusca is the second most speciose phylum, in which the Gastropoda forms the largest group. However, the currently proposed hypotheses of gastropod phylogeny are mainly based on part of their taxonomic diversity, notably on the large-sized and shallow-water species. In this study, we aimed at correcting this bias by reconstructing the phylogeny with new mitogenomes of deep-sea gastropods including Anatoma sp., Bathysciadiidae sp., Bayerotrochus teramachii, Calliotropis micraulax, Coccocrater sp., Cocculina subcompressa, Lepetodrilus guaymasensis, Peltospira smaragdina, Perotrochus caledonicus, Pseudococculinidae sp., and Shinkailepas briandi. This dataset provided the first reports of the mitogenomes for the Cocculiniformia, three vetigastropod superfamilies: Pleurotomarioidea, Lepetelloidea, and Scissurelloidea, and the neritimorph family Phenacolepadidae. The addition of deep-sea representatives also allowed us to evaluate the evolution of habitat use in gastropods. Our results showed a strongly supported sister-group relationship between the deep-sea lineages Cocculiniformia and Neomphalina. Within the Vetigastropoda, the Pleurotomarioidea was revealed as the sister-group of the remaining vetigastropods. Although this clade was presently restricted to the deep sea, fossil records showed that it has only recently invaded this habitat, thus suggesting that shallow waters was the ancestral habitat for the Vetigastropoda. The deep-sea Lepetelloidea and Lepetodriloidea formed a well-supported clade, with the Scissurelloidea sister to it, suggesting an early transition from shallow water to deep sea in this lineage. In addition, the switch between different chemosynthetic habitats was also observed in deep-sea gastropod lineages, notably in Neomphalina and Lepetelloidea. In both cases, the biogenic substrates appeared as the putative ancestral habitat, confirming the previously proposed hypothesis of a wooden-step to deep-sea vents scenario of evolution of habitat use for these taxa.

Using proteomic profiling to characterize protein signatures of different thymoma subtypes.

BACKGROUND: Histology is a traditional way to classify subtypes of thymoma, because of low cost and convenience. Yet, due to the diverse morphology of thymoma, this method increases the complexity of histopathologic classification, and requires experienced experts to perform correct diagnosis. Therefore, in this study, we developed an alternative method by identifying protein biomarkers in order to assist clinical practitioners to make right classification of thymoma subtypes. METHODS: In total, 204 differentially expressed proteins in three subtypes of thymoma, AB, B2, and B3, were identified using mass spectrometry. Pathway analysis showed that the differentially expressed proteins in the three subtypes were involved in activation-related, signaling transduction-related and complement system-related pathways. To predict the subtypes of thymoma using the identified protein signatures, a support vector machine algorithm was used. Leave-one-out cross validation methods and receiver operating characteristic analysis were used to evaluate the predictive performance. RESULTS: The mean accuracy rates were > 80% and areas under the curve were ≧0.93 across these three subtypes. Especially, subtype B3 had the highest accuracy rate (96%) and subtype AB had the greatest area under the curve (0.99). One of the differentially expressed proteins COL17A2 was further validated using immunohistochemistry. CONCLUSIONS: In summary, we identified specific protein signatures for accurately classifying subtypes of thymoma, which could facilitate accurate diagnosis of thymoma patients.

O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

Disseminated castration-resistant prostate cancer (CRPC) is a common disease in men that is characterized by limited survival and resistance to androgen-deprivation therapy. The increase in human epidermal growth factor receptor 2 (HER2) signaling contributes to androgen receptor activity in a subset of patients with CRPC; however, enigmatically, HER2-targeted therapies have demonstrated a lack of efficacy in patients with CRPC. Aberrant glycosylation is a hallmark of cancer and involves key processes that support cancer progression. Using transcriptomic analysis of prostate cancer data sets, histopathologic examination of clinical specimens, and in vivo experiments of xenograft models, we reveal in this study a coordinated increase in glycan-binding protein, galectin-4, specific glycosyltransferases of core 1 synthase, glycoprotein- N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) and ST3 beta-galactoside α-2,3-sialyltransferase 1 (ST3GAL1), and resulting mucin-type O-glycans during the progression of CRPC. Furthermore, galectin-4 engaged with C1GALT1-dependent O-glycans to promote castration resistance and metastasis by activating receptor tyrosine kinase signaling and cancer cell stemness properties mediated by SRY-box 9 (SOX9). This galectin-glycan interaction up-regulated the MYC-dependent expression of C1GALT1 and ST3GAL1, which altered cellular mucin-type O-glycosylation to allow for galectin-4 binding. In clinical prostate cancer, high-level expression of C1GALT1 and galectin-4 together predict poor overall survival compared with low-level expression of C1GALT1 and galectin-4. In summary, MYC regulates abnormal O-glycosylation, thus priming cells for binding to galectin-4 and downstream signaling, which promotes castration resistance and metastasis.-Tzeng, S.-F., Tsai, C.-H., Chao, T.-K., Chou, Y.-C., Yang, Y.-C., Tsai, M.-H., Cha, T.-L., Hsiao, P.-W. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

The chondroprotective effect of diosmin on human articular chondrocytes under oxidative stress.

Excessive oxidative stress, which can amplify inflammatory responses, is involved in the pathologic progression of knee osteoarthritis. Diosmin is known to possess a variety of biological functions such as antiinflammatory and antioxidant activities. We therefore demonstrated the chondroprotective potentials of diosmin on human articular chondrocytes under oxidative stress. The cytotoxicity of diosmin (5, 10, 50, and 100 µM) to chondrocytes was first evaluated. Subsequently, the cells were treated with diosmin (5 and 10 µM) after hydrogen peroxide (H2 O2 ) exposure. We found that the cytotoxicity of diosmin occurred in a dose-dependent manner (10, 50, and 100 µM), and low-dose diosmin (5 µM) slightly impaired cell viability. Diosmin supplementations (5 and 10 µM) did not show beneficial effects on mitochondrial activity, cytotoxicity, proliferation, and survival and the cell senescence was ameliorated in H2 O2 -exposed chondrocytes. On the other hand, diosmin down-regulated the mRNA levels of iNOS, COX-2, IL-1ß, COL1A1, MMP-3, and MMP-9; up-regulated TIMP-1 and SOX9; and improved COL2A1 in chondrocytes under oxidative stresses. Furthermore, diosmin also regulated glutathione reductase and glutathione peroxidase of H2 O2 -exposed chondrocytes. In conclusion, diosmin displayed a remarkable antiinflammatory effect compared with the antioxidant capacity on human chondrocytes. Diosmin can maintain the homeostasis of extracellular matrix of articular cartilage.

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Different effects of long noncoding RNA NDRG1-OT1 fragments on NDRG1 transcription in breast cancer cells under hypoxia.

Hypoxia plays a crucial role in the aggressiveness of solid tumors by driving multiple signaling pathways. Recently, long non-coding RNA (lncRNA) has been reported to promote or inhibit tumor aggressiveness by regulating gene expression. Previous studies in our laboratory found that the lncRNA NDRG1-OT1 is significantly up-regulated under hypoxia and inhibits its target gene NDRG1 at both the mRNA and protein levels. At the protein level, NDRG1-OT1 increases NDRG1 degradation via ubiquitin-mediated proteolysis. However, the repressive mechanism of NDRG1 at the RNA level is still unknown. Therefore, the purpose of this study was to study how NDRG1-OT1 transcriptionally regulates its target gene NDRG1. Luciferase reporter assays showed that NDRG1-OT1 decreased NDRG1 promoter activities. Mass spectrometry, bioinformatics tools, genetic manipulation, and immunoblotting were used to identify the interacting proteins. Surprisingly, different fragments of NDRG1-OT1 had opposite effects on NDRG1. The first quarter fragment (1-149 nt) of NDRG1-OT1 had no effect on the NDRG1 promoter; the second quarter fragment (150-263 nt) repressed NDRG1 by increasing the binding affinity of HNRNPA1; the third quarter fragment (264-392 nt) improved NDRG1 promoter activity by recruiting HIF-1α; the fourth quarter fragment (393-508 nt) down-regulated NDRG1 promoter activity via down-regulation of KHSRP under hypoxia. In summary, we have found a novel mechanism by which different fragments of the same lncRNA can cause opposite effects within the same target gene.

iGC-an integrated analysis package of gene expression and copy number alteration.

BACKGROUND: With the advancement in high-throughput technologies, researchers can simultaneously investigate gene expression and copy number alteration (CNA) data from individual patients at a lower cost. Traditional analysis methods analyze each type of data individually and integrate their results using Venn diagrams. Challenges arise, however, when the results are irreproducible and inconsistent across multiple platforms. To address these issues, one possible approach is to concurrently analyze both gene expression profiling and CNAs in the same individual. RESULTS: We have developed an open-source R/Bioconductor package (iGC). Multiple input formats are supported and users can define their own criteria for identifying differentially expressed genes driven by CNAs. The analysis of two real microarray datasets demonstrated that the CNA-driven genes identified by the iGC package showed significantly higher Pearson correlation coefficients with their gene expression levels and copy numbers than those genes located in a genomic region with CNA. Compared with the Venn diagram approach, the iGC package showed better performance. CONCLUSION: The iGC package is effective and useful for identifying CNA-driven genes. By simultaneously considering both comparative genomic and transcriptomic data, it can provide better understanding of biological and medical questions. The iGC package's source code and manual are freely available at https://www.bioconductor.org/packages/release/bioc/html/iGC.html .

DNMT3L promotes quiescence in postnatal spermatogonial progenitor cells.

The ability of adult stem cells to reside in a quiescent state is crucial for preventing premature exhaustion of the stem cell pool. However, the intrinsic epigenetic factors that regulate spermatogonial stem cell quiescence are largely unknown. Here, we investigate in mice how DNA methyltransferase 3-like (DNMT3L), an epigenetic regulator important for interpreting chromatin context and facilitating de novo DNA methylation, sustains the long-term male germ cell pool. We demonstrated that stem cell-enriched THY1(+) spermatogonial stem/progenitor cells (SPCs) constituted a DNMT3L-expressing population in postnatal testes. DNMT3L influenced the stability of promyelocytic leukemia zinc finger (PLZF), potentially by downregulating Cdk2/CDK2 expression, which sequestered CDK2-mediated PLZF degradation. Reduced PLZF in Dnmt3l KO THY1(+) cells released its antagonist, Sal-like protein 4A (SALL4A), which is associated with overactivated ERK and AKT signaling cascades. Furthermore, DNMT3L was required to suppress the cell proliferation-promoting factor SALL4B in THY1(+) SPCs and to prevent premature stem cell exhaustion. Our results indicate that DNMT3L is required to delicately balance the cycling and quiescence of SPCs. These findings reveal a novel role for DNMT3L in modulating postnatal SPC cell fate decisions.

Identification of Potential Plasma Biomarkers for Nonalcoholic Fatty Liver Disease by Integrating Transcriptomics and Proteomics in Laying Hens.

Background: Prevalent worldwide obesity is associated with increased incidence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. The identification of noninvasive biomarkers for NAFLD is of recent interest. Because primary de novo lipogenesis occurs in chicken liver as in human liver, adult chickens with age-associated steatosis resembling human NAFLD is an appealing animal model.Objective: The objective of this study was to screen potential biomarkers in the chicken model for NAFLD by transcriptomic and proteomic analysis.Methods: Hy-Line W-36 laying hens were fed standard feed from 25 to 45 wk of age to induce fatty liver. They were killed every 4 wk, and liver and plasma were collected at each time point to assess fatty liver development and for transcriptomic and proteomic analysis. Next, selected biomarkers were confirmed in additional experiments by providing supplements of the hepatoprotective nutrients betaine [300, 600, or 900 parts per million (ppm) in vivo; 2 mM in vitro] or docosahexaenoic acid (DHA; 1% in vivo; 100 µM in vitro) to 30-wk-old Hy-Line W-36 laying hens for 4 mo and to Hy-Line W-36 chicken primary hepatocytes with oleic acid-induced steatosis. Liver or hepatocyte lipid contents and the expression of biomarkers were then examined.Results: Plasma acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL), and glutathione S-transferase (GST) concentrations are well-established biomarkers for NAFLD. Selected biomarkers had significant positive associations with hepatic lipid deposition (P < 0.001). Betaine (900 ppm in vivo; 2 mM in vitro) and DHA (1% in vivo; 100 µM in vitro) supplementation both resulted in lower steatosis accompanied by the reduced expression of selected biomarkers in vivo and in vitro (P < 0.05).Conclusion: This study used adult laying hens to identify biomarkers for NAFLD and indicated that AACS, DPP4, GLUL, and GST could be considered to be potential diagnostic indicators for NAFLD in the future.

Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer.

Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7(R)) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7(R) tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7(R) cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib resistance and a downstream target of the AKT/mTOR/HIF-1α signaling pathway. In addition, increased galectin-1 expression in HCC patients' serum was associated with poor tumor control and low response rate. We also found that a high serum galectin-1 level was an independent factor associated with poor progression-free survival and overall survival. In conclusion, these results suggest that galectin-1 is a possible biomarker for predicting the response of HCC patients to treatment with sorafenib. As such, it may assist in the stratification of HCC and help direct personalized therapy.

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

BACKGROUND: Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. RESULTS: We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. CONCLUSION: We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.