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Objective Papillary thyroid microcarcinoma (PTMC) is the most prevalent histological type of thyroid carcinoma. Despite the overall favorable prognosis of PTMC, some cases exhibit aggressive phenotypes. The identification of robust biomarkers may improve early PTMC diagnosis. In this study, we integrated high-throughput transcriptome sequencing, bioinformatic analyses and experimental validation to identify key genes associated with the malignant characteristics of PTMC. Methods Total RNA was extracted from 24 PTMC samples and 7 non-malignant thyroid tissue samples, followed by RNA sequencing. The differentially expressed genes (DEGs) were identified and used to construct co-expression networks by weighted gene co-expression network analysis (WGCNA). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and protein-protein interaction networks were constructed. Key modules and hub genes showing a strong correlation with the malignant characteristics of PTMC were identified and validated. Results The green-yellow and turquoise modules generated by WGCNA were strongly associated with the malignant characteristics of PTMC. Functional enrichment analysis revealed that genes in the green-yellow module participated in cell motility and metabolism, whereas those in the turquoise module participated in several oncogenic biological processes. Nine real hub genes (FHL1, NDRG2, NEXN, SYNM, COL1A1, FN1, LAMC2, POSTN, and TGFBI) were identified and validated at the transcriptional and translational levels. Our preliminary results indicated their diagnostic potentials in PTMC. Conclusions In this study, we identified key co-expression modules and nine malignancy-related genes with potential diagnostic value in PTMC.

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.

Voltage-dependent anion channels (VDACs) are major transport proteins localized in the outer membrane of mitochondria and play critical roles in regulating plant growth and responding to stress. In this study, a total of 26 VDAC genes in common wheat (Triticum aestivum L.) were identified. TaVDACs that contained β-barrel structures were classified into three groups with phylogenetic and sequence alignment. Additionally, the gene structure and protein conserved motif composition varied among diverse subfamilies but were relatively conserved within the same subfamily. The basic elements that were stress- and hormone-related, including TATA-box, CAAT-box, MBS, LTR, TC-rich repeats, ABRE, P-box and TATC-box, were predicted within the promoter region of TaVDAC genes. TaVDAC expression patterns differed among tissues, organs and abiotic stress conditions. Overexpression (OE) of TaVDAC1-B conferred high tolerance to salinity and less resistance to drought stress in Arabidopsis thaliana. TaVDAC1-B interacted with Nucleoredoxin-D1 (TaNRX-D1) protein. Furthermore, compared with WT lines, salinity stress further upregulated the level of AtNRX1 (homologous gene of TaNRX-D1 in Arabidopsis) expression and the activity of superoxide dismutase in TaVDAC1-B OE lines, which led to a decrease in superoxide radical accumulation; drought stress further downregulated AtNRX1 expression and superoxide dismutase activity in TaVDAC1-B OE lines, resulting in the accumulation of superoxide radicals. Our study not only presents comprehensive information for understanding the VDAC gene family in wheat but also proposes a potential mechanism in response to drought and salinity stress.

The Gα subunit is an important component of the heterotrimeric G-protein complex and an integral component of several signal transduction pathways. It plays crucial roles in the diverse processes of plant growth and development, including the response to abiotic stress, regulation of root development, involvement in stomatal movement, and participation in hormone responses, which have been well investigated in many species. However, no comprehensive analysis has identified and explored the evolution, expression pattern characteristics and heat stress response of the Gα subunit genes in Rosaceae. In this study, 52 Gα subunit genes were identified in eight Rosaceae species; these genes were divided into three subfamilies (I, II, and III) based on their phylogenetic, conserved motif, and structural characteristics. Whole genome and dispersed duplication events were found to have contributed significantly to the expansion of the Gα subunit gene family, and purifying selection to have played a key role in the evolution of Gα subunit genes. An expression analysis identified some PbrGPA genes that were highly expressed in leaf, root, and fruit, and exhibited diverse spatiotemporal expression models in pear. Under abiotic stress conditions, the mRNA transcript levels of PbrGPA genes were up-regulated in response to high temperature treatment in leaves. Furthermore, three Gα subunit genes were shown to be located in the plasma membrane and nucleus in pear. In conclusion, the study of the Gα subunit gene family will help us to better understand its evolutionary history and expression patterns, while facilitating further investigations into the function of the Gα subunit gene in response to heat stress.

Background Activation of oncogenes through promoter hypomethylation and silencing of tumor suppressor genes induced by promoter hypermethylation played essential roles in the progression of lung adenocarcinoma (LUAD). This study aimed to identify the LUAD prognostic CpG sites and the regulated genes which contributed to LUAD progression. Methods Methylation profiles from TCGA and GSE60645 were used to screen the differentially methylated CpGs. Then, the Log-rank test was adopted to identify LUAD prognosis-associated CpGs. Differential gene expression and survival analyses were further performed to suggest the roles of methylation-driven genes in LUAD prognosis. Finally, models and nomograms were constructed to predict the prognosis of LUAD. Results A total of 1891 CpGs at gene promoters were differentially methylated. Among them, 54 CpGs were significantly associated with LUAD prognosis. Nine of them showed significant correlations with the expression of four genes (CCDC181, CFTR, PPP1R16B, MYEOV). CCDC181, CFTR and PPP1R16B were aberrantly down-regulated in LUAD, while MYEOV was up-regulated. All of them were significantly associated with LUAD prognosis. The LASSO regression analysis indicated that tumor stages, cg09181792, cg16998150, cg22779330 and PPP1R16B were promising prognostic factors. The AUC (area under the curve) of the model containing the clinical predictors was 0.643. The combination of CpGs and PPP1R16B with clinical variables significantly improved the predictive efficiency with an AUC of 0.714 (P = 0.036). Conclusion This study identified four pairs of promoter CpGs and genes that were significantly associated with LUAD prognosis. The integration of CpGs methylation and gene expression showed better predictive ability for LUAD prognosis.

The body color of Neocaridina denticulate sinensis is a compelling phenotypic trait, in which a cascade of carotenoid metabolic processes plays an important role. The study was conducted to compare the transcriptome of cephalothoraxes among three pigmentation phenotypes (red, blue, and chocolate) of N. denticulate sinensis. The purpose of this study was to explore the candidate genes associated with different colors of N. denticulate sinensis. Nine cDNA libraries in three groups were constructed from the cephalothoraxes of shrimps. After assembly, 75022 unigenes were obtained in total with an average length of 1026 bp and N50 length of 1876 bp. There were 45977, 25284, 23605, 21913 unigenes annotated in the Nr, Swissprot, KOG, and KEGG databases, respectively. Differential expression analysis revealed that there were 829, 554, and 3194 differentially expressed genes (DEGs) in RD vs BL, RD vs CH, and BL vs CH, respectively. These DEGs may play roles in the absorption, transport, and metabolism of carotenoids. We also emphasized that electron transfer across the inner mitochondrial membrane (IMM) was a key process in pigment metabolism. In addition, a total of 6328 simple sequence repeats (SSRs) were also detected in N. denticulate sinensis. The results laid a solid foundation for further research on the molecular mechanism of integument pigmentation in the crustacean and contributed to developing more attractive aquatic animals.

Plant immunity to pathogen infections is a dynamic response that involves multiple organelles and defence signalling systems such as hypersensitive response (HR) and systemic acquired resistance (SAR). The latter requires the function of Pathogenesis-related (PR) proteins, a common plant protein family with diverse roles in plant innate immunity. Our previous proteomics study showed that a PR gene (ITC1587_Bchr9_P26466_MUSBA) was differentially regulated during a compatible banana-M. incognita interaction, substantiating the isolation of this gene in the current study. Here, we successfully isolated and characterised Pathogenesis-related-10 (PR10) gene with β-1,3-glucanase and ribonuclease (RNase) activities from two Musa acuminata cultivars (denoted as MaPR10) namely Berangan and Grand Naine (ITC1256). We found that MaPR10 cloned sequences possess glycine-rich loop domain and shared conserved motifs specific to PR10 gene group, confirming its identity as a member of this group. Interestingly, we also found a catalytic domain sequence for glycoside hydrolase family 16 (EXDXXE), unique only to MaPR10 cloned sequences. Two peptide variants closely related to the reference sequence ITC1587_Bchr9_P26466_MUSBA namely MaPR10-BeB5 and MaPR10-GNA5 were overexpressed and purified to test for their functionality. Here, we confirmed that both protein variants possess β-1,3-glucanase and ribonuclease (RNase) activities, and inhibit the growth of Aspergillus fumigatus, a human opportunistic pathogen. To our knowledge, this is the first PR10 plant proteins with such properties to be reported thus far.

Genome plasticity is a key determinant that Acinetobacter johnsonii could widely distribute in natural and clinical environments. However, little attention has been paid to figure out the changes in the genome during A. johnsonii’s evolution. Here, a comparative genomic analysis of A. johnsonii isolated from clinical and environmental sources was conducted. In this study, we found A. johnsonii has an open pan-genome and has great adaptability to different environments. Based on the results of the phylogenetic tree, ANI value and the distribution of accessory genes, we found that strains from the same habitat had a high degree of similarity. Though genes associated with the fundamental process were mostly conserved in evolution, clinical-derived isolates accumulate more genes associated with translational modification, β-lactamase and defense mechanisms, whereas environmental-derived isolates enriched more genes related to substances degradation. In addition, clinical-derived strains harbored some “strong” virulence islands and resistance islands. This study highlights the evolutionary relationship of A. johnsonii isolates from clinical and environmental sources.

Small-sized and trypanotolerant West African taurine (Bos taurus) cattle are a unique case of human-mediated process of adaptation to a challenging environment. Extensive gene flow with Sahelian zebu (B. indicus), bigger and with some resistance to tick attack, occurred for centuries and allowed the apparition of stable crossbred populations (sanga) having intermediate characteristics. Up to 237 individuals belonging to 10 different taurine, zebu and sanga cattle populations sampled in Benin, Burkina Faso and Niger were typed using the BovineHD BeadChip of Illumina to identify signatures of selection, assessed using three different Extended-Haplotype-Homozygosity-based statistics, overlapping with ancient, originated 1024 or 2048 generations ago, Homozygosity-By-Descent segments in the cattle genome. Candidate genomic regions were defined ensuring their importance within cattle type and using zebu as reference. Functional annotation analysis identified four statistically significant Annotation Clusters in taurine cattle (from ACt1 to ACt4), one (ACs1) in sanga, and another (ACz1) in zebu cattle, fitting well with expectations. ACt1 included genes primarily associated with innate immunity; ACt2 involved bitter taste receptor genes of importance to adaptation to changing environments; ACt3 included 68 genes coding ATP-binding proteins, some of them located on trypanotolerance-related QTL regions, that can partially underlie immune response and the additive mechanism of trypanotolerance; ACt4 was associated with growth and small size (NPPC gene); ACs1 included genes involved in immune response; and ACz1 is related with ectoparasite resistance. Our results provide a new set of genomic areas and candidate genes giving new insights on the genomic impact of adaptation in West African cattle.

To investigate the variations in gene expression in grass carp under high-temperature stress, two libraries were constructed from a high-temperature treatment group (T33) and a control group (T27) and sequenced using Illumina sequencing technology. The results showed that sequencing generated a total of 279,398,348 raw reads, approximately 40.7-51.8 M clean reads were obtained from each library, and the percentage of uniquely mapped transcripts ranged from 80.13 to 84.58%. A total of 260 differentially expressed genes (DEGs) were identified under high-temperature stress, among which 84 genes were upregulated and 176 genes were downregulated. Ten DEGs were randomly selected for quantitative RT-PCR (qRT-PCR) analysis, and the results confirmed that the transcriptome analysis was reliable. Furthermore, the DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and the results showed that most of the DEGs were involved in protein, lipid and carbohydrate metabolism. Moreover, plasma urea nitrogen (Urea) and triglyceride (TG) contents were significantly lower in the high-temperature treatment group than in the control group (P < 0.01). In summary, these results indicated that high-temperature stress could inhibit protein synthesis, decrease fatty acid synthesis, and weaken carbohydrate metabolism in juvenile grass carp.

Traditional methods to understand leukemia stem cell (LSC)'s biological characteristics include constructing LSC-like cells and mouse models by transgenic or knock-in methods. However, there are some potential pitfalls in using this method, such as retroviral insertion mutagenesis, non-physiological level gene expression, non-physiological expansion, and difficulty to construct. The mRNAsi index for each sample of the Cancer Genome Atlas (TCGA) could avoid these potential pitfalls by machine learning. In this work, we aimed to construct a network of LSC genes utilizing the mRNAsi. First, mRNAsi value was analyzed with expressions distributions, survival analysis, age, and gender in acute myeloid leukemia (AML) samples. Then, we used the weighted gene co-expression network analysis (WGCNA) to construct modules of stemness genes. The correlation of the LSC genes transcription and interplay among LSC proteins was analyzed. We performed functional and pathway enrichment analysis to annotate stemness genes. Survival analysis further identified prognostic biomarkers by clinical data of TCGA and the Gene Expression Omnibus (GEO) database. We found that the result of mRNAsi overall survival is not significant, which may be due to the heterogeneity of AML in the stage of myeloid differentiation, French-American-British (FAB) classification systems. Enrichment analysis indicated that the stemness genes were biologically clustered as a group and mainly associated with cell cycle and mitosis. Moreover, 10 key genes (SNRNP40, RFC4, RFC5, CDC6, HSPE1, PA2G4, SNAP23P, DARS2, MIS18A, and HPRT1) were screened by survival analysis with the data from TCGA and GEO. Among them, RFC4 and RFC5 were the distinguished biomarkers for their double-validated prognostic value in both databases. Additionally, the expression of RFC4 and RFC5 had the same trend as mRNAsi score in FAB subtypes. In conclusion, our result demonstrated that mRNAsi based LSC-related genes were found to have strong interactions as a cluster. These genes, especially RFC4 and RFC5, could be the therapeutic targets for inhibiting the stemness characteristics of AML. This work is also a comprehensive pipeline for future cancer stem cell studies.

Growth traits is a critical economic trait for animal husbandry. In this study, the SNPs of CTNNA3 and CAP2 genes were investigated to check whether they are associated with growth traits (body weight, body height, body length and chest circumference) in Hu sheep. The result of the association analysis indicated that the mutation in CTNNA3 (g.2018018 A G) were associated significantly with body weight, body height, body length and chest circumference (P 0.05), the mutation in CAP2 (g.8588 T C) were associated significantly with body height at 140, 160, 180 days (P 0.05), AA and CC of CTNNA3 and CAP2 were the dominant genotypes associated with growth traits in Hu sheep. Moreover, combined effect analyses indicated that the growth traits with combined genotypes AA

Toxin–antitoxin (TA) systems were initially discovered as plasmid addiction systems. Previously, our studies implied that the high stability of the IncP-7 plasmid pCAR1 in different Pseudomonas spp. hosts was due to the presence of a TA system on the plasmid. Bioinformatics approaches suggested that ORF174 and ORF175 could constitute a type II TA system, a member of the RES-Xre family, and that these two open reading frames (ORFs) constitute a single operon. As expected, the ORF175 product is a toxin, which decreases the viability of the host, P. resinovorans, while the ORF174 product functions as an antitoxin that counteracts the effect of ORF175 on cell growth. Based on these findings, we renamed ORF174 and ORF175 as prpA (antitoxin gene) and prpT (toxin gene), respectively. The prpA and prpT genes were cloned into the unstable plasmid vector pSEVA644. The recombinant vector was stably maintained in P. resinovorans and Escherichia. coli cells under nonselective conditions following 6 days of daily subculturing. The empty vector (without the prpA and prpT genes) could not be maintained, which suggested that the PrpA/T system can be used as a tool to improve the stability of otherwise unstable plasmids in P. resinovorans and E. coli strains. To our knowledge, this is the first description of a plasmid-encoded TA system, RES-Xre, that confers plasmid stability.

The WD40 transcription factor family is a superfamily found in all eukaryotes that plays important roles in regulating growth and development. To our knowledge, to date, WD40 superfamily genes have been identified and characterized in several plant species, but little information is available on the WD40 superfamily genes in peach. In this study, we identified 220 members of the WD40 superfamily in the peach genome, and these members were further classified into five subfamilies based on phylogenetic comparison with those in Arabidopsis. The members within each subfamily had conserved motifs and gene structures. The WD40 genes were unevenly distributed on chromosomes 1 to 8 of the peach genome. Additionally, 58 pairs of paralog WD40 members were found on eight chromosomes in peach, and 242 pairs of orthologous WD40 genes in peach and Arabidopsis were matched. The 54 selected putative WD40 genes in peach had diverse expression patterns in red-fleshed and white-fleshed peach fruits at five developmental stages. Prupe.6G211800.1 was located only on the cytomembrane, while Prupe.1G428200.1 and Prupe.I003200.1 were located on both the cytomembrane and in the nucleus; Prupe.1G558700.1 was densely localized around the nuclear rim but relatively faintly localized in the nucleoplasm; Prupe.5G116300.1 was located in the nucleus and cytomembrane with strong signals but showed weak signals in the cytoplasm; and Prupe.8G212400.1 and Prupe.1G053600.1 were located mainly in the nuclear envelope and cytomembrane but relatively faintly in the nucleoplasm. This study provides a foundation for the further functional verification of WD40 genes in peach.

The glycerol-3-phosphate dehydrogenase (GPD) gene family plays a major role in glycerol synthesis and adaptation to abiotic stresses. Few studies on GPD family genes from the halotolerant algae Dunaliella salina are available. In this study, seven DsaGPD genes were identified by mining D. salina sequencing data. Among them, DsaGPD5 contained the canonical NAD+-GPD protein domain, called si-GPD. In comparison, DsaGPD1-4 not only contained the canonical NAD+-GPD domain but also a unique domain, the haloacid dehalogenase (HAD)-like superfamily domain, in their N-terminal region, called bi-GPD. DsaGPD6, 7 contained the FAD+-GPD domain. In the transient expression system, DsaGPD1, 3, 4 were found in the cytosol of Arabidopsis thaliana protoplast, DsaGPD2, 5 in the chloroplast, and DsaGPD6, 7 in the mitochondria. MEME analysis showed that six conserved motifs were present in both si-GPDs and bi-GPDs, whereas seven highly conserved motifs were only present in bi-GPDs. The quantitative real-time PCR results showed significant induction of the DsaGPD genes under abiotic stresses, indicating their tolerance-related role in D. salina. DsaGPD2 and DsaGPD5 may be the osmoregulator form and glyceride form in the chloroplast, respectively. The evolutionary forces acting on si-GPDs and bi-GPDs were different in the same organism: bi-GPDs were under purifying selection, while si-GPDs were mainly under positive selection. Furthermore, evolution of the N_HAD domain and C_GPD domain in bi-GPDs is highly correlated. In summary, this study characterizes DsaGPD gene family members and provides useful information for elucidating the salt tolerance mechanism in D. salina.

Cotton fiber initiation is the first step in fiber development, and it determines the yield. Here, genome-wide transcriptome profiling of Gossypium arboreum was performed to determine the molecular basis of cotton fiber initiation. A comparison of the transcriptomes of fiber-bearing ovules at −0.5, 0, 0.5, 1, 1.5, 2, 2.5 and 3 d post-anthesis detected 12,049 differentially expressed genes that mainly participated in ribosome, carbon metabolism and amino acid biosynthesis pathways. Genes encoding alcohol dehydrogenase 1 and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, involving in fatty acid degradation and flavonoid biosynthesis, were enriched. Furthermore, 1049 differentially expressed transcription factors were identified. Among these, 17 were trihelix family transcription factors, which play important roles in plant development and responses to biotic and abiotic stresses. In total, 52 full-length trihelix genes, named as GaGTs, were identified in G. arboreum and located in 12 of the 13 cotton chromosomes. Transcriptomic data and a quantitative real-time PCR analysis indicated that several GaGTs were significantly induced during fiber initiation in G. arboreum. Thus, the genome-wide comprehensive analysis of gene expression in G. arboreum fiber initiation will serve as a useful resource for unraveling the functions of specific genes. The phylogenetic relationships and expression analyses of the G. arboreum trihelix genes established a solid foundation for future comprehensive functional analyses of the GaGTs.

Alterations in the global gene expression profile are considered to contribute to the various physiological and pathological changes during the course of ageing. Genes that code for the molecular components of the innate system are alter markedly as ageing occurs; and this may define the susceptibility of very young and very old individuals to reproductive tract infections. The expression pattern of genes that code for beta-defensins (effectors of innate immune response) in male reproductive tract tissues of different stages of ageing is not yet reported. Further, the induction of beta-defensins during endotoxin challenge and whether epigenetic modulators can influence the expression of these genes in different stages of ageing are not reported. We analyzed the basal mRNA levels of beta-defensins and defensin-like proteins (Sperm Associated Antigen 11 (SPAG11) family members), their induction during endotoxin challenge and modulation by epigenetic modifiers (Trichostatin A and Azacytidine) in the caput, cauda, testis, prostate and seminal vesicle of rats that represent early stage to late stages of life (20 day to 730 day old). We observed differential basal gene expression pattern in the male reproductive tract tissues and the induction by LPS was not consistent neither among the age groups not the tissues analyzed. Trichostatin A and Azacytidine also influenced antimicrobial gene expression and the pattern was not consistent in different tissues obtained from different age groups. Results of this study demonstrate that antimicrobial gene expression varies to a great extent during ageing and is strongly influenced by endotoxins and epigenetic modulators.

Investigation of sex determination system in Eriocheir sinensis is important because of sex-dimorphism in its growth traits. However, little information about the sex-related genes in embryonic development stages were exposed. To obtain more information of sex determination in Chinese mitten crab, we performed the transcriptome analysis in embryonic development stage (fertilized egg stage, cleavage stage, blastula stage, gastrula stage and heartbeat stage) of Chinese mitten crab using nextgeneration sequencing technology. Thirty-one of 32,088 annotated unigenes were identified as sex-related genes including 16 genes involved in primary sex determination in model organisms and 8 genes of SOX family and 7 genes of DMRT gene family. Heatmap based on the RPKM value in five embryonic development stages indicated that these genes were clustered into two branches. Analysis of the differentially expressed 12 genes, including 3 genes of SOX family, 3 genes of DMRT gene family and 6 genes involved in primary sex determination in model organisms, showed significantly difference between the first three periods (fertilized egg stage-cleavage stage-blastula stage) and the last two periods (gastrula stage-heartbeat stage) and all 12 genes were up-regulated after blastula stage. In conclusion, we inferred that sex determination might be initiated after blastula stage in E. sinensis. Transcriptome analysis from embryonic development stage could provide a background information for further investigation in sex determination of Eriocheir sinensis.

Background Autosomal recessive non-syndromic hearing loss (ARNSHL) is a highly heterogeneous genetic disease. PDZD7 is a new ARNSHL associated gene. Until now, nine PDZD7 biallelic mutation families with ARNSHL have been reported. Here we report a case of Chinese patient with ARNSHL linked to novel mutations in PDZD7 genes. Method The pathogenic mutations were detected by whole exome sequencing for hereditary deafness-related genes of both the proband and his parents. We used kinship detection, mutational hazard prediction, genotype-phenotype correlation analysis and variation screening for potential pathogenic mutations. Re-sequencing was used to confirm the mutations by Sanger sequence. Real time quantitative PCR (RT-qPCR) was used to analyze the PDZD7 gene expression. Population-based screening for variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations of PDZD7 gene. Results We determined three variants of the PDZD7 gene that contributed to the deafness of the patient (PDZD7 c.192G > A, p. Met64Ile; c.1648C > T p. Gln550* and c.2341_2352delCGCAGCCGCAGCp. Arg781_Ser 784del). Pathogenic analysis in accordance with the ACMG/AMP Standards and Guidelines identified two novel mutations as Likely Pathogenic. The expression level of PDZD7 gene in the patient was decreased compared to the normal control (P Conclusion Three mutations in PDZD7 gene linked to ARNSHL were identified in a Chinese pedigree. The findings expand not only our knowledge of genetic causes of ARNSHL, but also PDZD7 genes mutation spectrum of the disease. They will aid personalized genetic counseling, molecular diagnostics and clinical management of this condition.

GATA5 is a member of the GATA transcription factor family, which serves essential roles in varieties of cellular functions and biological processes. In this study, we have accomplished the molecular cloning, bioinformatic analysis and preliminary function study of C. semilaevis GATA5. The full-length cDNA nucleotide sequence is 1955 bp, with a coding sequence of 1167 bp, which encodes a polypeptide of 388 amino acids. Homology, phylogenetic, gene structure and synteny analysis showed that C. semilaevis GATA5 was highly conserved among vertebrates. Tissue distribution pattern exhibited that C. semilaevis GATA5 was significantly expressed in heart, intestine, liver, kidney and gonad, with a sexual dimorphic feature observed in testis and ovary. Embryonic development expression profiles showed that C. semilaevis GATA5 transcripts increased at the blastula stage, and peaked at the heat-beating period. Strong signals were detected at spermatids of male testis and stage III oocytes of female ovary by ISH. The expression of C. semilaevis GATA5 was regulated by 17α-MT and E2 after hormone stimulation to the ovary. Together, all the results pointed out that GATA5 might play a vital role during gonadal maturation and the reproductive cycle of C. semilaevis. This study lays the foundation for further researches on the sex control breeding in tongue sole.

Despite its history as a developmental and evolutionary model organism, gene expression analysis in the large milkweed bug, Oncopeltus fasciatus, has rarely been explored using quantitative real-time PCR. The strength of this method depends greatly on the endogenous controls used for normalization, which are lacking for the milkweed bug system. Here, to fill in this gap in our knowledge, we validated the stability of a set of ten candidate reference genes identified from the O. fasciatus transcriptome, and did so upon exposure to a dietary toxin, a cardiac glycoside, and across four different exposure periods. To increase robustness against gDNA contaminants, genome resources were used to design intron-bridging primers. A comprehensive stability validation by the Bestkeeper, Normfinder, geNorm and comparative ΔCt methods identified ef1a and tubulin as the most stable genes across treatments and time points, whereas 18S rRNA was the most unstable. However, accounting for the temporal scale indicated that time point confined normalizers might enable higher quantification accuracy for treatment comparison. Overall this study demonstrates: (i) a robust RT-qPCR primer design approach is possible for non-model organisms where genome annotation is often incomplete, and (ii) the importance of detailed reference gene stability exploration in multifactorial experimental designs.

Small Tail Han Sheep are an excellent local sheep breed in China, and their outstanding reproductive performance is one of their very important biological characteristics. Clarifying the ovary development process of these ewes should provide a theoretical basis for improving their reproductive efficiency. In this study, we identified the differentially expressed (DE) microRNAs (miRNAs) in 2-, 6-, and 12-month-old small-tail Han sheep ovaries by constructing and analyzing the miRNA expression profiles. These findings clarify the molecular mechanisms regulating the excellent reproductive performance of small-tail Han ewes. We used RNA-Seq technology and bioinformatic to analyze these profiles. Eleven, 13, and 19 DE miRNAs were identified in the 2- vs 6-, 6- vs 12-, and 2- vs 12-month-old ovaries, respectively. In total, 54, 37, and 198 predicted target genes of these DE miRNAs were identified in these three groups, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that in the 2- vs 6-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 102 GO terms and seven signaling pathways; in the 6- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 52 GO terms and three signaling pathways; and in the 2- vs 12-month-old ovaries, the target genes of DE known sheep miRNAs were involved in 88 GO terms and six signaling pathways. Three miRNA-target regulatory networks were constructed based on these DE miRNA-target interactions. Nine miRNAs were selected to confirm to the accuracy of the miRNA sequencing data with qRT-PCR. The site at which oar-miR-432 binds RPS6KA1 was determined with a dual-luciferase system. This is the first integrated analysis the expression profiles of miRNAs and their targets during ovarian development in small-tail Han sheep. These data clarify the molecular regulatory mechanisms underlying sheep ovarian development and identify biomarkers that influence the reproductive performance of small-tail Han ewes.

Purpose In order to identify the molecular characteristics and improve the efficacy of early diagnosis of mucinous epithelial ovarian cancer (mEOC), here, the transcriptome profiling by weighted gene co-expression network analysis (WGCNA) has been proposed as an effective method. Methods The gene expression dataset GSE26193 was reanalyzed with a systematical approach, WGCNA. mEOC-related gene co-expression modules were detected and the functional enrichments of these modules were performed at GO and KEGG terms. Ten hub genes in the mEOC-related modules were validated using two independent datasets GSE44104 and GSE30274. Results 11 co-expressed gene modules were identified by WGCNA based on 4917 genes and 99 epithelial ovarian cancer samples. The turquoise module was found to be significantly associated with the subtype of mEOC. KEGG pathway enrichment analysis showed genes in the turquoise module significantly enriched in metabolism of xenobiotics by cytochrome P450 and steroid hormone biosynthesis. Ten hub genes (LIPH, BCAS1, FUT3, ZG16B, PTPRH, SLC4A4, MUC13, TFF1, HNF4G and TFF2) in the turquoise module were validated to be highly expressed in mEOC using two independent gene expression datasets GSE44104 and GSE30274. Conclusion Our work proposed an applicable framework of molecular characteristics for patients with mEOC, which may help us to obtain a precise and comprehensive understanding on the molecular complexities of mEOC. The hub genes identified in our study, as potential specific biomarkers of mEOC, may be applied in the early diagnosis of mEOC in the future.

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.

The genic male sterility (MS) plays a major role in melon hybrids production, it could reduce the cost of pollination and increase the yield and quality. However, the molecular mechanism underlying genetic male sterility is yet poorly understood. The morphological differences of flower buds of melon were observed showed that the flower buds were tetrad when they were 1 mm stage and monocyte microspore when they were 2 mm stage. Electron microscopy showed that there was significant difference between MS lines and MF (male fertility) lines. In order to detect the global expression of the genes during the melon anther development and association with MS, 12 DEGs (differentially expressed genes) libraries were constructed from the anther of MS and MF in the bud stage with 1 and 2 mm diameter, respectively. A total of 765 DEGs expressed in anther during different developmental stage (MS 1 mm vs. MS 2 mm), 148 and 309 DEGs were found to be related to MS as compared to MF (MS 1 mm vs. MF 1 mm, and MS 2 mm vs. MF 2 mm) at a false discovery rate FDR

MC4R gene is a hypothalamic satiety control mediator in which mutations cause a monogenic form of obesity. The aim of this study was to perform a genetic screening to identify variations in the entire region of MC4R gene. A total of 236 unrelated and severely obese patients (BMI ≥ 40 kg/m2) with Spanish ancestry and severe overweight familiar history have been enrolled into the study. Seven MC4R gene variants were identified in the heterozygous state in 21 patients. Coding variants p.Thr101Ile and p.Ala259Asp are new and variants p.Ser30Phe, p.Val103Ile and p.Ile251Leu were previously described. Two variants have been also observed in the promoter region of the MC4R gene; the c.-24G>A mutation, described for the first time, and the known c.-178A>C variant. Both in silico and family segregation analysis confirm the correlation between novel identified mutations in MC4R gene and obesity development. The correlation between the four variants (c.-24G>A, p.Thr101Ile, p.Ala259Asp and p.Ser30Phe) and the obesity phenotype, therefore, allows the conclusion that all of the four mutations cause a monogenic form of obesity.

Reinforcing the immunity of pregnant sows can not only improve their own health condition but also increase the survival rate and healthy status of their piglets. This study aims to find single-nucleotide polymorphism (SNP) and molecular markers that are associated with the immune traits of pregnant sows. SLA-DOB and CD4 were selected as candidate genes, and blood samples were randomly collected from pregnant Landrace sows and used to detect T-lymphocyte subsets, interferon alpha, interleukin 6, Toll-like receptor 3, serum antibody immunoglobulin G, and porcine reproductive and respiratory syndrome virus-specific antibody. Then, association analyses were conducted for the polymorphic sites of candidate genes with immune traits. We found 12 mutations in the two genes and conducted an association study with eight of them. Our results indicated that among the eight mutations, SNP1, SNP2, and SNP3 of the SLA-DOB gene and Ins9, SNP10, and SNP11 in the CD4 gene are newly discovered mutations. Except for SNP1, SNP3, and SNP11, the other five SNPs are associated with at least one immune trait tested. Especially, SNP2 and Ins9 are significantly associated with at least one of the T-lymphocyte subgroups and at least one antibody. These novel mutations have potential important effects on the polymorphic loci of the above immune traits in pregnant sows. The results suggest that the SLA-DOB and CD4 genes and their genetic mutations can be considered as important candidate genes and mutations for the immunity of pregnant sows.

Flammulina filiformis is an edible fungus that is largely cultivated and widely consumed around the world. The quantity and quality of the primordia, which gives rise to the fruiting body, affects its production efficiency. Hydrophobins are involved in the formation of the fruiting body of macrofungi. However, functional verification of the hydrophobin genes is limited to date. In this study, we used gene silencing and overexpression analyses to investigate the function of one F. filiformis hydrophobin gene (Hyd9) during the development of the fruiting body. The Hyd9-silenced transformants exhibited sparse aerial hyphae, resulting in fewer primordia and fruiting bodies. In contrast, the Hyd9 overexpression strain displayed denser aerial hyphae and more primordia. The phenotypes of these transgenic lines strongly suggested that Hyd9 plays an important role in the formation of aerial hyphal knots (the primary stage of primordia) and primordia in F. filiformis. These results will be beneficial for developing more efficient methods to induce primordia formation in F. filiformis and other commercially valuable mushrooms.

Thyroid cancer is the most common type of endocrine tumor. The TNM classification remains a standard for treatment determination and predicting prognosis in thyroid cancer. The genes modules associated with the progression of papillary thyroid carcinoma (PTC) were not clear. We applied a weighted gene co-expression network analysis (WGCNA) and differential expression analysis to systematically identified co-expressed gene modules and hub genes associated with PTC progression based on The Cancer Genome Atlas (TCGA) PTC transcriptome sequencing data. An independent validation cohort, GSE27155, was used to evaluate the preservation of gene modules. We identified two co-expressed genes modules associated with progression of PTC. Enrichment analysis indicated that the two modules were enriched in angiogenesis and extracellular matrix organization. DCN, COL1A1, COL1A2, COL5A2 and COL3A1 were hub genes in the co-expressed network. We systematically identified co-expressed gene modules and hub genes associated with PTC progression for the first time, which provided insights into the mechanisms underlying PTC progression and some potential targets for the treatment of PTC.

Molecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2∆, dss1∆, and afg3∆) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondrial DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.

The study of EGFR gene mutational profile in NSCLC patients has a special clinical significance in the selection of patients for tyrosine-kinase inhibitors therapy. From 2017, the targeted therapy started to be accessible in public sector in Morocco, thus, the implementation of techniques for the molecular characterization of EGFR mutations in the laboratories became a necessity. The aim of this study was to present targeted methods "ADx-ARMS technology and the Idylla™ system" for the identification of EGFR mutational profile, methods that can be implemented in our clinical laboratories for routine analysis instead of outsourcing analysis to other countries. We conducted this study by processing 239 cases of NSCLC patients. Using the DNA extracted from the FFPE tissue, we evaluated somatic mutations in exons 18 to 21 of the tyrosine-kinase domain of EGFR gene by HRM-PCR combined to real time PCR "ADx-ARMS technology" and Idylla™ system. These sensitive methods showed that among the positive mutant cases, the distribution of mutations was as follows: 70% of patients having a deletion in exon 19, 15% in exon 21 (L858R), 7.5% in exon 20 and 7.5% in exon 18 (G719X). All of the positive EGFR mutations cases were adenocarcinoma and 42.1% of them were smokers. These results show the need to incorporate a quick and efficient method for the identification of EGFR mutation into routine practice in our laboratories, allowing more patients to benefit from targeted therapy.

Background Cholestatic liver disease (CLD) is a highly heterogeneous hepatobiliary disease with various causes. The purpose of this research was to explore the gene expression changes throughout the course of CLD revealing potential causative molecular mechanisms and therapeutic targets. Methods We established two animal models of cholestasis: 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding for 2, 4 and 6 weeks and bile duct ligation for 14 days. Using these two models, we identified differentially expressed genes (DEGs) by RNA-Seq analysis and used the newly-found knowledge of DEGs in comprehensive bioinformatic analyses to investigate key molecular events. Sequencing results were confirmed by experimental verification. Results Our study detected overlapping DEGs in the two models, of these 568 genes were upregulated and 117 genes were downregulated. Gene Ontology analysis demonstrated that the upregulated genes were associated with the biological processes of cell adhesion, cell migration and cell motility, while the metabolic processes of various substances were enriched for the downregulated genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the upregulated pathways were mainly distributed in focal adhesion, ECM-receptor interaction and amoebiasis, while downregulated pathways focused on peroxisome proliferator-activated receptor signaling pathway, metabolic pathways and primary bile acid biosynthesis. These findings were further confirmed by protein–protein interaction network modeling. Hub genes Src, Pdgfb, Col15a1, Mmp9, Egfr were selected using centralities analyses and verified by qRT-PCR. Conclusion We profiled a global mRNA landscape in CLD to promote a complete understanding of transcriptomic events of this disease, offering candidate biomarkers and therapeutic targets for the clinic.

The solute carrier 6 (SLC6) gene family, functioning as neurotransmitter transporters, plays the crucial roles in neurotransmission, cellular and organismal homeostasis. In this study, we found an expansion of SLC6 family gene in the genome of chordate invertebrate Ciona savignyi. A total of 40 candidate genes including 29 complete and 11 putative genes were identified as SLC6 family gene homologs. Phylogenetic analysis revealed that most of these Ciona SLC6 genes were highly conserved with the vertebrate ones, although gene duplication and gene losses did exist. Four genes were selected from SLC6 subfamilies to be further investigated for their functional characteristics on cell growth and migration through overexpression approach in cultured cell lines. The results showed both SLC6A7 and SLC6A17 from amino acid transporters AA1 and AA2 sub-families, respectively, significantly suppressed the cell proliferation and migration. While SLC6A1 and SLC6A4, which were classified into GABA and monoamine transporters, respectively, did not affect the cell proliferation and migration in HEK293T, HeLa, and MCF7 cells. The whole set of C. savignyi SLC6 genes identified in this study provides an important genomic resource for future biochemical, physiological, and phylogenetic studies on SLC6 gene family. Our experimental data demonstrated that Ciona amino acid transporters, such as SLC6A7 and SLC6A17, were essential for cell physiology and behaviors, indicating their crucially potential roles in the control of cell proliferation and migration during ascidian embryogenesis.

Synthetic biology with genetically engineered (GE) cyanobacteria has the potential to produce valuable products such as biofuels. However, it is also essential to assess the potential risks of synthetic biology technology before it can be widely used. In order to address key concerns posed by the application of synthetic biology to microorganisms, studies were designed to monitor the horizontal transfer of engineered genes from GE cyanobacteria Thermosynechococcus elongatus BP1 to Escherichia coli through co-incubation. The results of these experiments demonstrated that the genetically engineered DNA construct containing alcohol producing genes and kanamycin resistance can be horizontally transferred from GE T. elongatus BP1 to wild-type E. coli following two days of liquid co-culturing. The rapid and facile transfer of foreign genes, which include antibiotic resistance, between bacterial communities signifies the need to continue to deepen our understanding of the process of horizontal gene transfer, chromosomal integration as well as further biosafety-oriented research efforts. In the era of synthetic biology, the natural microbial process for sharing genetic material will also significantly impact risk assessments, containment approaches and further policy development.

In the current study, we report three cases of Asparagine Synthetase (ASNS) Deficiency from two consanguineous families. Family 1 had two early neonatal deaths due to a novel mutation in the ASNS gene c.788C > T (p.S263F) and both the children presented with microcephaly and one of them had severe intracranial haemorrhage. The proband from the second family was homozygous for c.146G > A (p.R49Q) and manifested myoclonic seizures, developmental delay, coarse hair and diffuse cortical atrophy. Molecular docking studies of both the mutations revealed alteration in the ligand binding site. Till date, 26 mutations were reported in ASNS gene in 29 affected children indicating high degree of genetic heterogeneity and high mortality. Although asparagine depletion is not of diagnostic utility, multiple linear regression model suggested that asparagine levels vary to the extent of 20.6% based on glutamine and aspartate levels and ASNS deficiency results in depletion of asparagine synthesis. ASNS deficiency should be suspected in any neonate with microcephaly and epileptic encephalopathy.