Comprehensive genomics and expression analysis of eceriferum (CER) genes in sunflower (Helianthus annuus)

Sunflower occupies the fourth position among oilseed crops the around the world. Eceriferum (CER) is an important gene family that plays critical role in very-long-chain fatty acids elongation and biosynthesis of epicuticular waxes under both biotic and abiotic stress conditions. The aim of present study was to investigate the effect of sunflower CER genes during drought stress condition. Thus, comparative analysis was undertaken for sunflower CER genes with Arabidopsis genome to determine phylogenetic relationship, chromosomal mapping, gene structures, gene ontology and conserved motifs. Furthermore, we subjected the sunflower cultivars under drought stress and used qRT-PCR analysis to explore the expression pattern of CER genes during drought conditions. We identified thirty-seven unevenly distributed CER genes in the sunflower genome. The phylogenetic analysis revealed that CER genes were grouped into seven clades in Arabidopsis, Helianthus annuus, and Gossypium hirsutum. Expression analysis showed that genes CER10 and CER60 were upregulated in sunflower during drought conditions, indicating that these genes are activated during drought stress. The results obtained will serve to characterize the CER gene family in sunflower and exploit the role of these genes in wax biosynthesis under limited water conditions.Key messageCuticular waxes protect the plants from drought stress, so we observed the expression of wax bio synthesis genes in recently sequences genome of Helianthus annuus. We observed that expression of wax biosynthesis genes CER10 and CER60 was upregulated when the plants were subjected to drought stress.     

Morphological and molecular analyses of Leafhopper,Amrasca biguttula (Ishida) (Hemiptera: Cicadellidae) infesting eggplant (Solanum melongena L.) in Luzon Island,Philippines

Eggplant (Solanum melongena L.) is the leading vegetable grown and consumed in the Philippines, and the eggplant leafhopper is one of its most important insect pests. Nine local populations of leafhopper infesting eggplants were collected from major eggplant growing areas in Luzon Island to determine if these populations are constituted singly or by different species and to assess the level of variability among them. Morphological analysis of the adult male abdominal and genital traits and partial mitochondrial COI gene sequence identified the eggplant leafhopper populations in Luzon Island as belonging to Amrasca (=Sundapteryx) biguttula (Ishida) (Hemiptera:Cicadelliae). Observed male specimens exhibited modifications in the pregenital abdominal tergites VII and VIII that were previously reported to not be found in other Amrasca species. Principal Component Analysis of four morphometric genital traits revealed high similarity among the leafhopper populations regardless of eggplant production areas. Partial COI sequence analysis idenfitied 11 sequence variants (haplotypes), with one predominant haplotype. Overall, the leafhopper populations from Luzon Island exhibited moderate haplotype diversity and low nucleotide diversity, indicating low genetic diversity. Topologies from a maximum likelihood tree indicate all eleven haplotypes cluster in a single clade with other A. biguttula infesting cotton and potato in India, but the Philippine population forms a separate subgroup although with weak bootstrap support. Further analysis with the Generalized Mixed Yule Coalescent (GMYC) method classified the different haplotypes into a single GMYC entity.     

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.     

Expression profiles and functional characterization of common carp (Cyprinus carpio) T2Rs

Bitter taste perception is mediated by a family of G protein-coupled receptors (T2Rs) in vertebrates. Common carp (Cyprinus carpio), which has experienced an additional round of whole genome duplication during the course of evolution, has a small number of T2R genes similar to zebrafish, a closely related cyprinid fish species, and their expression pattern at the cellular level or their cognate ligands have not been elucidated yet. Here, we showed through in situ hybridization experiments, that three common carp T2R (ccT2R) genes encoding ccT2R200-1, ccT2R202-1, and ccT2R202-2, were specifically expressed in the subsets of taste receptor cells in the lips and gill rakers. ccT2R200-1 was co-expressed with genes encoding downstream signal transduction molecules, such as PLC-β2 and Gαia. Heterologous expression system revealed that each ccT2R showed narrowly, intermediately, or broadly tuned ligand specificity, as in the case of zebrafish T2Rs. However, ccT2Rs showed different ligand profiles from their orthologous zebrafish T2Rs previously reported. Finally, we identified three ccT2Rs, namely ccT2R200-1, ccT2R200-2, and ccT2R203-1, to be activated by natural bitter compounds, andrographolide and/or picrotoxinin, which elicited no response to zebrafish T2Rs, in a dose-dependent manner. These results suggest that some ccT2Rs may have evolved to function in the oral cavity as taste receptors for natural bitter compounds found in the habitats in a species-specific manner.     

Rhodopirellula aestuarii sp. nov., a novel member of the genus Rhodopirellula isolated from brackish sediments collected in the Tagus River estuary,Portugal

Bacteria within the phylum Planctomycetota are biologically relevant due to unique characteristics among prokaryotes. Members of the genus Rhodopirellula can be abundant in marine habitats, however, only six species are currently validly described. In this study, we expand the explored genus diversity by formally describing a novel species. The pink-coloured strain ICT_H3.1^T was isolated from brackish sediments collected in the Tagus estuary (Portugal) and a 16S rRNA gene sequence-based analysis placed this strain into the genus Rhodopirellula (family Pirellulaceae). The closest type strain is Rhodopirellula rubra LF2^T, suggested by a similarity of 98.4% of the 16S rRNA gene sequence. Strain ICT_H3.1^T is heterotrophic, aerobic and able to grow under microaerobic conditions. The strain grows between 15 and 37 °C, over a range of pH 6.5 to 11.0 and from 1 to 8% (w/v) NaCl. Several nitrogen and carbon sources were utilized by the novel isolate. Cells have an elongated pear-shape with 2.0 ± 0.3 × 0.9 ± 0.2 µm in size. Cells of strain ICT_H3.1^T cluster in rosettes through a holdfast structure and divide by budding. Younger cells are motile. Ultrathin cell sections show cytoplasmic membrane invaginations and polar fimbriae. The genome size is 9,072,081 base pairs with a DNA G + C content of 56.1 mol%. Genomic, physiological and morphological comparison of strain ICT_H3.1^T with its relatives suggest that it belongs to a novel species within the genus Rhodopirellula. Hence, we propose the name Rhodopirellula aestuarii sp. nov., represented by ICT_H3.1^T (=CECT30431^T = LMG32464^T) as the type strain of this novel species.16S rRNA gene accession number: GenBank = OK001858.Genome accession number: The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAMQBK000000000. The version described in this paper is version JAMQBK010000000.     

Comprehensive analysis of gene expression profiles to identify differential prognostic factors of primary and metastatic breast cancer

Breast cancer accounts for nearly half of all cancer-related deaths in women worldwide. However, the molecular mechanisms that lead to tumour development and progression remain poorly understood and there is a need to identify candidate genes associated with primary and metastatic breast cancer progression and prognosis. In this study, candidate genes associated with prognosis of primary and metastatic breast cancer were explored through a novel bioinformatics approach. Primary and metastatic breast cancer tissues and adjacent normal breast tissues were evaluated to identify biomarkers characteristic of primary and metastatic breast cancer. The Cancer Genome Atlas-breast invasive carcinoma (TCGA-BRCA) dataset (ID: HS-01619) was downloaded using the mRNASeq platform. Genevestigator 8.3.2 was used to analyse TCGA-BRCA gene expression profiles between the sample groups and identify the differentially-expressed genes (DEGs) in each group. For each group, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were used to determine the function of DEGs. Networks of protein–protein interactions were constructed to identify the top hub genes with the highest degree of interaction. Additionally, the top hub genes were validated based on overall survival and immunohistochemistry using The Human Protein Atlas. Of the top 20 hub genes identified, four (KRT14, KIT, RAD51, and TTK) were considered as prognostic risk factors based on overall survival. KRT14 and KIT expression levels were upregulated while those of RAD51 and TTK were downregulated in patients with breast cancer. The four proposed candidate hub genes might aid in further understanding the molecular changes that distinguish primary breast tumours from metastatic tumours as well as help in developing novel therapeutics. Furthermore, they may serve as effective prognostic risk markers based on the strong correlation between their expression and patient overall survival.     

Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.     

Identification of two cassava receptor-like cytoplasmic kinase genes related to disease resistance via genome-wide and functional analysis

Receptor-like cytoplasmic kinases (RLCKs) play important roles in various developmental processes and stress responses in plants. Whereas, the detailed information of this family in cassava has not clear yet. In this study, A total of 322 MeRLCK genes were identified in the cassava genome, and they could be divided into twelve clades (Clades I-XII) according to their phylogenetic relationships. Most RLCK members in the same clade have similar characteristics and motif compositions. Over half of the RLCKs possess cis-elements in their promoters that respond to ABA, MeJA, defense reactions, and stress. Under Xpm11 infection, the expression levels of four genes show significant changes, suggesting their involvement in Xpm11 resistance. Two RLCK (MeRLCK11 and MeRLCK84) genes potentially involved in resistance to cassava bacterial blight were identified through VIGS experiments. This work laid the foundation for studying the function of the cassava RLCK genes, especially the genes related to pathogen resistance.     

Mendelian randomization analysis identified genes potentially pleiotropically associated with periodontitis

ObjectiveTo prioritize genes that were pleiotropically or potentially causally associated with periodontitis.MethodsWe applied the summary data-based Mendelian randomization (SMR) method integrating genome-wide association study (GWAS) for periodontitis and expression quantitative trait loci (eQTL) data to identify genes that were pleiotropically associated with periodontitis. We performed separate SMR analysis using CAGE eQTL data and GTEx eQTL data. SMR analysis were done for participants of European and East Asian ancestries, separately.ResultsWe identified multiple genes showing pleiotropic association with periodontitis in participants of European ancestry and participants of East Asian ancestry. PDCD2 (corresponding probe: ILMN_1758915) was the top hit showing pleotropic association with periodontitis in the participants of European ancestry using CAGE eQTL data, and BX093763 (corresponding probe: ILMN_1899903) and AC104135.3 (corresponding probe: ENSG00000204792.2) were the top hits in the participants of East Asian ancestry using CAGE eQTL data and GTEx eQTL data, respectively.ConclusionWe identified multiple genes that may be involved in the pathogenesis of periodontitis in participants of European ancestry and participants of East Asian ancestry. Our findings provided important leads to a better understanding of the mechanisms underlying periodontitis and revealed potential therapeutic targets for the effective treatment of periodontitis.     

Exonic variants in multiple myeloma patients associated with relapsed/ refractory and response to bortezomib regimens

Novel treatment in multiple myeloma represented by proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies have produced a deep response. However, relapses are possible, and all classes of drugs are refractory to patients. Next-generation sequencing has improved our understanding of the multiple myeloma genome related to drug resistance and has discovered many genomic variants. Therefore, this study was conducted to investigate new variants associated with drug resistance in MM patients who relapsed and refractory to bortezomib regimen and daratumumab treatment using next-generation sequencing for whole-exome sequencing. Peripheral blood samples were collected in EDTA tubes from six patients; four were in relapsed and refractory to bortezomib regimens and daratumumab; two patients responded to bortezomib regimens. Whole-exome sequencing was performed by the MGI-DNBSEQ-G400 instrument. We identified 21 variants in multiple myeloma patients. Seventeen variants were found in relapsed and refractory multiple myeloma in 11 genes (GNAQ, PMS1, CREB1, NSUNS2, PIK3CG, ROS1, PMS2, FIT4, KDM5A, STK11 and ZFHX3). And four variants were identified in two patients with response to bortezomib regimens in 4 genes (RAF1, CREB1, ZFHX3 and INSR). We have observed several genetic variants in many genes that may have been associated with the poor prognosis and poor response to treatment in these patients. These values should be further confirmed in large sample studies using the RNA-seq technique to identify genome expression.     

Knock-in of the duck retinoic acid-inducible gene I (RIG-I) into the Mx gene in DF-1 cells enables both stable and immune response-dependent RIG-I expression

Waterfowls, such as ducks, are natural hosts of avian influenza virus (AIV) and can genetically limit the pathogenicity. On the other hand, some AIV strains cause severe pathogenicity in chickens. It is suggested that differences in the pathogenicity of AIV infection between waterfowls and chickens are related to the expression of retinoic acid-inducible gene I (RIG-I), a pattern recognition receptor that chickens evolutionally lack. Here, we knocked-in the duck RIG-I bearing the T2A peptide sequence at the 3′ region of the Mx, an interferon-stimulated gene (ISG), in chicken embryo fibroblast cells (DF-1) using the precise integration into target chromosome (PITCh) system to control the duck RIG-I expression in chickens. The expression patterns of the duck RIG-I were then analyzed using qPCR. The knocked-in DF-1 cells expressed RIG-I via the stimulation of IFN-β and poly(I:C) in a dose-dependent manner. Moreover, poly(I:C) stimulation in the knocked-in DF-1 cells upregulated RIG-I-like receptor (RLR) family signaling pathway-related genes IFN-β, OASL, and IRF7. The IFN-β-dependent expression of RIG-I and upregulation of IFN-β in the poly(I:C) stimulation demonstrated a positive-feedback loop via RIG-I, usually evident in ducks. Overall, this novel strategy established RIG-I-dependent immune response in chickens without overexpression of RIG-I and disruption of the host genes.     

Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae Species,comparative analysis and phylogenetic relationships

This current study presents, for the first time, the complete chloroplast genome of two Cleomaceae species: Dipterygium glaucum and Cleome chrysantha in order to evaluate the evolutionary relationship. The cp genome is 158,576 bp in length with 35.74% GC content in D. glaucum and 158,111 bp with 35.96% GC in C. chrysantha. Inverted repeats IR 26,209 bp, 26,251 bp each, LSC of 87,738 bp, 87,184 bp and SSC of 18,420 bp, 18,425 bp respectively. There are 136 genes in the genome, which includes 80 protein coding genes, 31 tRNA genes and four rRNA genes were observed in both chloroplast genomes. 117 genes are unique while the remaining 19 genes are duplicated in IR regions. The analysis of repeats shows that the cp genome includes all types of repeats with more frequent occurrences of palindromic; Also, this analysis indicates that the total number of simple sequence repeats (SSR) were 323 in D. glaucum, and 313 in C. chrysantha, of which the majority of the SSRs in these plastid genomes were mononucleotide repeats A/T which are located in the intergenic spacer. Moreover, the comparative analysis of the four cp sequences revealed four hotspot genes (atpF, rpoC2, rps19, and ycf1), these variable regions could be used as molecular makers for the species authentication as well as resources for inferring phylogenetic relationships of the species. All the relationships in the phylogenetic tree are with high support, this indicate that the complete chloroplast genome is a useful data for inferring phylogenetic relationship within the Cleomaceae and other families. The simple sequence repeats identified will be useful for identification, genetic diversity, and other evolutionary studies of the species. This study reported the first cp genome of the genus Dipterygium and Cleome. The finding of this study will be beneficial for biological disciplines such as evolutionary and genetic diversity studies of the species within the core Cleomaceae.     

The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p < 0.05) difference after transfection. The expression of BAK, FAS, and BAX genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant (p < 0.05) increases. Although, the increase of SURVIVIN and P53 expressions in transformed cells were not statistically significant (p > 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.     

Migratory birds as the vehicle of transmission of multi drug resistant extended spectrum β lactamase producing Escherichia fergusonii,an emerging zoonotic pathogen

The acquisition of multi-drug resistance (MDR) genes by pathogenic bacterial bugs and their dispersal to different food webs has become a silent pandemic. The multiplied use of different antibacterial therapeutics during COVID-19 pandemic has accelerated the process among emerging pathogens. Wild migratory birds play an important role in the spread of MDR pathogens and MDR gene flow due to the consumption of contaminated food and water. Escherichia fergusonii is an emerging pathogen of family Enterobacteriaceae and commonly causes disease in human and animals. The present study focused on the isolation of E. fergusonii from blood, saliva, and intestine of selected migratory birds of the Hazara Division. The sensitivity of isolated strains was assessed against ten different antibiotics. The isolation frequency of E. fergusonii was 69%. In blood samples, a high rate of resistance was observed against ceftriaxone (80%) followed by ampicillin (76%) whereas, in oral and intestinal samples, ceftriaxone resistant strains were 56% and 57% while ampicillin resistance was 49% and 52% respectively. The overall ceftriaxone and ampicillin-resistant cases in all three sample sources were 71% and 65% respectively. In comparison to oral and intestinal samples, high numbers of ceftriaxone-resistant strains were isolated from the blood of mallard while ampicillin-resistant strains were observed in blood samples of cattle egrets. 16S rRNA-based confirmed strains of E. fergusonii were processed for detection of CTX-M and TEM-1 gene through Polymerase chain reaction (PCR) after DNA extraction. Hundred percent ceftriaxone resistant isolates possessed CTX-M and all ampicillin-resistant strains harbored TEM-1 genes. Amplified products were sequenced by using the Sanger sequencing method and the resulted sequences were checked for similarity in the nucleotide Database through the BLAST program. TEM-1 gene showed 99% and the CTX-M gene showed 98% similar sequences in the Database. The 16S rRNA sequence and nucleotide sequences for TEM-1 and CTX-M genes were submitted to Gene Bank with accession numbers LC521304, LC521306, LC521307 respectively. We posit to combat MDR gene flow among the bacterial pathogens across different geographical locations, regular surveillance of new zoonotic pathogens must be conducted.     

Construction of miRNA-mRNA network for the identification of key biological markers and their associated pathways in IgA nephropathy by employing the integrated bioinformatics analysis

BackgroundAbout half-century ago, Immunoglobulin A nephropathy (IgAN) was discovered as a complicated disease with frequent clinical symptoms. Until now, exact mechanism underlying the pathogenesis of IgAN is poorly known. Therefore, current study was aimed to understand the molecular mechanism of IgAN by identifying the key miRNAs and their targeted hub genes. The key miRNAs might contribute to the diagnosis and therapy of IgAN, and could turn out to be a new star in the field of IgAN.MethodsThe microarray datasets were downloaded from Gene Expresssion Omnibus (GEO) database and analyzed using R package (LIMMA) in order to obtain differential expressed genes (DEGs). Then, the hub genes were identified using cytoHubba plugin of cytoscpae tool and other bioinformatics approaches including protein-protein interaction (PPI) network analysis, module analysis, and miRNA-hub gene network construction was also performed.ResultsA total of 348 DEGs were identified, of which 107 were upregulated genes and 241 were downregulated genes. Subsequently, the 12 overlapped genes were predicted from cytoHubba, and considered as hub genes. Moreover, a network among miRNA-hub genes was created to explore the correlation between the hub genes and their targeted miRNAs. Network construction ultimately lead to the identification of nine gene named FN1, EGR1, FOS, JUN, SERPINE1, MMP2, ATF3, MYC, and IL1B and one novel key miRNA namely, has-miR-144-3p as biomarker for diagnosis and therapy of IgAN.ConclusionThis study updates the information and yield a new perspective in context of understanding the pathogenesis and development of IgAN. In future, key miRNAs might be capable of improving the personalized detection and therapies for IgAN. In vivo and in vitro investigation of miRNAs and pathway interaction is essential to delineate the specific roles of the novel miRNAs, which may help to further reveal the mechanisms underlying IgAN.     

Whole exome sequencing,in silico and functional studies confirm the association of the GJB2 mutation p.Cys169Tyr with deafness and suggest a role for the TMEM59 gene in the hearing process

The development of next generation sequencing techniques has facilitated the detection of mutations at an unprecedented rate. These efficient tools have been particularly beneficial for extremely heterogeneous disorders such as autosomal recessive non-syndromic hearing loss, the most common form of genetic deafness. GJB2 mutations are the most common cause of hereditary hearing loss. Amongst them the NM_004004.5: c.506G > A (p.Cys169Tyr) mutation has been associated with varying severity of hearing loss with unclear segregation patterns. In this study, we report a large consanguineous Emirati family with severe to profound hearing loss fully segregating the GJB2 missense mutation p.Cys169Tyr. Whole exome sequencing (WES), in silico, splicing and expression analyses ruled out the implication of any other variants and confirmed the implication of the p.Cys169Tyr mutation in this deafness family. We also show preliminary murine expression analysis that suggests a link between the TMEM59 gene and the hearing process. The present study improves our understanding of the molecular pathogenesis of hearing loss. It also emphasizes the significance of combining next generation sequencing approaches and segregation analyses especially in the diagnosis of disorders characterized by complex genetic heterogeneity.