Specific Gene Regions Research Articles

Gene Deregulation and Underlying Mechanisms in Spinocerebellar Ataxias With Polyglutamine Expansion.

Polyglutamine spinocerebellar ataxias (polyQ SCAs) include SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17 and constitute a group of adult onset neurodegenerative disorders caused by the expansion of a CAG repeat sequence located within the coding region of specific genes, which translates into polyglutamine tract in the corresponding proteins. PolyQ SCAs are characterized by degeneration of the cerebellum and its associated structures and lead to progressive ataxia and other diverse symptoms. In recent years, gene and epigenetic deregulations have been shown to play a critical role in the pathogenesis of polyQ SCAs. Here, we provide an overview of the functions of wild type and pathogenic polyQ SCA proteins in gene regulation, describe the extent and nature of gene expression changes and their pathological consequences in diseases, and discuss potential avenues to further investigate converging and distinct disease pathways and to develop therapeutic strategies.

Antisense oligonucleotide-based therapies for the treatment of osteoarthritis: Opportunities and roadblocks.

Osteoarthritis (OA) is a debilitating disease with no approved disease-modifying therapies. Among the challenges for developing treatment is achieving targeted drug delivery to affected joints. This has contributed to the failure of several drug candidates for the treatment of OA. Over the past 20years, significant advances have been made in antisense oligonucleotide (ASO) technology for achieving targeted delivery to tissues and cells both in vitro and in vivo. Since ASOs are able to bind specific gene regions and regulate protein translation, they are useful for correcting aberrant endogenous mechanisms associated with certain diseases. ASOs can be delivered locally through intra-articular injection, and can enter cells through natural cellular uptake mechanisms. Despite this, ASOs have yet to be successfully tested in clinical trials for the treatment of OA. Recent chemical modification to ASOs have further improved cellular uptake and reduced toxicity. Among these are locked nucleic acid (LNA)-based ASOs, which have shown promising results in clinical trials for diseases such as hepatitis and dyslipidemia. Recently, LNA-based ASOs have been tested both in vitro and in vivo for their therapeutic potential in OA, and some have shown promising joint-protective effects in preclinical OA animal models. In order to accelerate the testing of ASO therapies in a clinical trial setting for OA, further investigation into delivery mechanisms is required. In this review article, we discuss opportunities for viral-, particle-, biomaterial-, and chemical modification-based therapies, which are currently in preclinical testing. We also address potential roadblocks in the clinical translation of ASO-based therapies for the treatment of OA, such as the limitations associated with OA animal models and the challenges with drug toxicity. Taken together, we review what is known and what would be useful to accelerate translation of ASO-based therapies for the treatment of OA.

Identification and validation of quantitative trait loci for ascites syndrome in broiler chickens using whole genome resequencing

BackgroundAscites syndrome is a hypertensive, multifactorial, multigene trait affecting meat-type chickens imposing significant economic losses on the broiler industry. A region containing the CPQ gene has been previously identified as significantly affecting ascites phenotype. The region was discovered through whole genome resequencing focused on chicken chromosome 2. The association was confirmed through further genotyping in multiple broiler populations.ResultsThe whole genome resequencing analyses have now been extended to the current chicken genome assembly. DNA samples were pooled according to gender and phenotype and the pools subjected to next generation sequencing. Loci were identified as clusters of single nucleotide polymorphisms where frequencies of the polymorphisms differed between resistant and susceptible chickens. The chickens are an unselected line descended from a commercial elite broiler line. Regions identified were specific to one or both genders. The data identify a total of 28 regions as potential quantitative trait loci for ascites. The genes from these regions have been associated with hypertensive-related traits in human association studies. One region on chicken chromosome 28 contains the LRRTM4 gene. Additional genotyping for the LRRTM4 region demonstrates an epistatic interaction with the CPQ region for ascites phenotype.ConclusionsThe 28 regions identified were not previously identified in a multi-generational genome wide association study using 60k Single Nucleotide Polymorphism panels. This work demonstrates the utility of whole genome resequencing as a cost effective, direct, and efficient method for identifying specific gene regions affecting complex traits. The approach is applicable to any organism with a genome assembly and requires no a priori assumptions.

Short communication: Milk microbiota profiling on water buffalo with full-length 16S rRNA using nanopore sequencing

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.

Phylogenetic analysis of Hepatozoon spp. (Apicomplexa: Hepatozoidae) infecting Philodryas patagoniensis (Serpentes: Dipsadidae) in Uruguay.

The purpose of this study was to perform a phylogenetic analysis of Hepatozoon spp. infecting Philodryas patagoniensis in Uruguay. Twenty-five road-killed specimens of P. patagoniensis from ten departments were obtained. Samples of blood and/or heart tissue were taken. Polymerase chain reaction (PCR) assay was carried out amplifying a specific target region of the 18S rRNA gene of Hepatozoon spp. Eighteen out of twenty-five samples were positive to Hepatozoon spp., which gave an overall prevalence of 72%. Phylogenetic analyses with the obtained sequences were carried out to determine the relationship with closely related species found in the region. The results revealed that samples were split into two clades with a high bootstrap support. Clade I was formed with Hepatozoon spp. sequences obtained in this study from P. patagoniensis, Hepatozoon cuestensis from Crotalus durissus terrificus and Hepatozoon musa from Philodryas nattereri, and Hepatozoon spp. retrieved from Cerdocyon thous, Hemidactylus mabouia, and Phyllopezus pollicaris from Brazil, respectively. Clade II was grouped with Hepatozoon cevapii and Hepatozoon massardii, both species described for C. d. terrificus from Brazil. This is the first report of Hepatozoon spp. in snakes from Uruguay.

RelA Governs a Network of Islet-Specific Metabolic Genes Necessary for Beta-Cell Function

NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role NF-κB plays in normal metabolism. Here, to define this role in the context of beta cell function and metabolic control, we deleted the canonical NF-κB transcription factor RelA/p65 specifically in pancreatic beta cells to generate βP65KO mice. RelA deficiency resulted in complete loss of stimulus dependent inflammatory gene upregulation, consistent with its known role to govern inflammation. However, RelA deletion also rendered mice glucose intolerant due to a functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βP65KO islets failed to secrete insulin ex vivo in response to a glucose challenge, and were unable to restore metabolic control when transplanted into secondary diabetic recipients. Maintenance of glucose tolerance required RelA, but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knock-out of the essential activator of NF-κB, NEMO, or beta cell overexpression of the negative NF-κB regulator, A20, did not cause severe glucose intolerance. Thus, basal RelA activity performs an essential and islet-intrinsic role to maintain normal glucose homeostasis. Genome wide bioinformatic mapping revealed the presence of RelA binding sites in the promoter regions of specific metabolic genes, and in the majority (~70%) of islet enhancer hubs that are responsible for shaping beta cell type-specific gene expression programmes. Indeed, islet specific metabolic genes Slc2a2 and Capn9, identified within the large network of islet enhancer hub genes showed dysregulated expression in βP65KO islets. These data demonstrate an unappreciated role for RelA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism.

Rapid, one-step DNA extraction for the identification of German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) using DNA sequence of mitochondrial ribosomal RNA gene (16S rRNA gene)

In this study, DNA sequencing was used to study species identification and phylogeny of German cockroach that has medical importance, So, the comparisons can be made with specific region of mitochondrial ribosomal RNA gene (16S rRNA gene) of two collected Blattella germanica the first from central city of Mashhad (GM), Iran, and the second from city of Tehran (GT), Iran, and other individuals of B. germanica from different areas, that already have been recorded in the BLAST database, on the basis of the Single Nucleotide Polymorphisms (SNPs), nucleotide substitutions, and DNA and putative amino acid sequence data. Also, a phylogeny for these cockroaches based on the DNA sequence of the 16S rRNA genes is offered. Our results indicated that the nucleotide sequence of the B. germanica (GM) matched with sequences from other subspecies, except the (thymine–adenine) SNPs at position 15 and 19 of the 376-bp, and the nucleotide sequence of B. germanica (GT), also, matched with sequences from other subspecies, except (adenine-guanine) SNP at position 360-bp, and (thymine-guanine) SNP at position 368-bp of a portion of the 16s rRNA gene. Therefore, the B. germanica (GM), B. germanica (GT), and B. germanica isolates in BLAST database were different among them. Furthermore, we found that the forward (LR-J-13017) primer and reverse (LR-N-13398) primer were very impressive to distinguish among cockroaches belong sub-order Dictyoptera especially German cockroach. As well, these primers were produced a small DNA fragment of the 16S rRNA gene that includes all SNPs required for B. germanica isolates identification.

A Fully Automated Gridding Technique for Real Composite cDNA Microarray Images

Genome-wide screening using microarrays of DNA will be of great use in the early diagnosis of diseases such as cancer and HIV. It also makes use of gene discovery, pharmacogenomics, toxicogenomics, and nutrigenomics for other applications. A DNA microarray image lays out an orderly arranged specific gene regions called spots. Microarray image analysis consists primarily of preprocessing, spot area gridding, spot segmentation, and intensity extraction. The first two phases are focused on this work: preprocessing and gridding. The experiment is conducted on real composite cDNA microarray images. A composite microarray image is formed by suitably stacking a red channel image and a green channel image acquired from a microarray experiment either in the RGB domain or in the GRB domain. The blue channel is kept as zero. In order to reduce the challenging problems of microarray images, an efficient preprocessing algorithm is proposed here for these composite images. We have developed a fully automated gridding algorithm integrating global subgrid gridding and local gridding of spots. This technique extracts the structural information namely inter-subgrid spacing, inter-spot spacing and spot center position to achieve efficient gridding. The traits of a microarray image are evaluated using three parameters namely Mean square error, Naturalness quality image evaluator and degree of contrast. The accuracy of the experimental results indicates that this combined preprocessing and gridding technique performs better than existing competitive methods in SIB, GEO, SMD and DeRisi datasets which are most commonly used by the research community for microarray image analysis techniques.

Microbial contamination in food, food- handlers’ hands and surfaces and evaluation of contamination sources by the similarity between isolates

Food can be contaminated with surfaces and food workers during chopping, shredding and serving. Pathogenic microorganisms are transmitted by direct contact with food or indirectly with airborne particles. The aim of this study was to determine the prevalence and the relationship between pathogenic microorganisms isolated from food, kitchen equipment and foodhandler’s hands. A total of 212 microbiological samples were collected from surfaces, foods and food handlers’ hands at different six canteens inside of the Afyon Kocatepe University campus during the period 2017-2018. Following biochemical tests, identification of Staphylococcus species were performed from a specific region of 23S rRNA gene. The genetic relationships between totally 25 Staphylococcus spp. and Proteus mirabilis isolates were determined. S.epidermidis was detected in two samples from knife handle (5%), nine samples from hands (20%) and in one of food sample (1%), too. S.aureus was found to be existed in one sample from hands (2.2%), two samples of soujouk (2%) and in one sample pancakes (3%) obtained from the university canteens. Besides from one of food sample (1%) S.sciuri, from one of hand sample (2.2%) S.haemolyticus and one of the food samples (1%) S.saprophyticus bacteria were identified. As a result, foods, food preparation surfaces and foodhandler’s hands were contaminated with microorganisms in canteens and they were similar/same to each other. Also, considering the number of isolates, the highest of contamination is the hands of food-handler.

New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless.

Drosophila melanogaster has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS (TIM), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, τ) is influenced by temperature in some of these mutants, whereas τ is temperature-independent in other mutant lines as in wild-type flies. This, so-called “temperature compensation” ability is compromised in the mutant timeless allele “ritsu” (timrit), and, as we show here, also in the timblind allele, mapping to the same region of TIM. To test if this region of TIM is indeed important for temperature compensation, we generated a collection of new mutants and mapped functional protein domains involved in the regulation of τ and in general clock function. We developed a protocol for targeted mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Our protocol is a fast and cost-efficient systematic approach for functional analysis of protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with a strong temperature compensation defect map to one specific region of TIM. Although the exact mechanism of how these mutations affect TIM function is as yet unknown, our in silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of TIM. Immunostaining for PER was performed on two TIM mutants that display longer τ at 25°C and complete arrhythmicity at 28°C. Consistently with the behavioral phenotype, PER immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.

Transcription factor Znf2 coordinates with the chromatin remodeling SWI/SNF complex to regulate cryptococcal cellular differentiation

Cellular differentiation is instructed by developmental regulators in coordination with chromatin remodeling complexes. Much information about their coordination comes from studies in the model ascomycetous yeasts. It is not clear, however, what kind of information that can be extrapolated to species of other phyla in Kingdom Fungi. In the basidiomycete Cryptococcus neoformans, the transcription factor Znf2 controls yeast-to-hypha differentiation. Through a forward genetic screen, we identified the basidiomycete-specific factor Brf1. We discovered Brf1 works together with Snf5 in the SWI/SNF chromatin remodeling complex in concert with existent Znf2 to execute cellular differentiation. We demonstrated that SWI/SNF assists Znf2 in opening the promoter regions of hyphal specific genes, including the ZNF2 gene itself. This complex also supports Znf2 to fully associate with its target regions. Importantly, our findings revealed key differences in composition and biological function of the SWI/SNF complex in the two major phyla of Kingdom Fungi.

Use of nanotechnology for infectious disease diagnostics: application in drug resistant tuberculosis

BackgroundThe increased transmission of multidrug-resistant (MDR) tuberculosis (TB) poses a challenge to tuberculosis prevention and control in Sri Lanka. Isoniazid (INH) is a key element of the first line anti tuberculosis treatment regimen. Resistance to INH may lead to development of MDR TB. Therefore, early detection of INH resistance is important to curb spread of resistance. Due to the limited availability of rapid molecular methods for detection of drug resistance in Sri Lanka, this study was aimed at developing a simple and rapid gold nanoparticle (AuNP) based lateral flow strip for the simultaneous detection of the most common INH resistance mutation (katG S315 T, 78.6%) and Mycobacterium tuberculosis (MTb).MethodsLateral flow strip was designed on an inert plastic backing layer containing a sample pad, nitrocellulose membrane and an absorption pad. Biotin labeled 4 capture probes which separately conjugated with streptavidin were immobilized on the nitrocellulose. The test sample was prepared by multiplex PCR using primers to amplify codon 315 region of the katG gene and MTb specific IS6110 region. The two detection probes complementary to the 5′ end of each amplified fragment was conjugated with gold nanoparticles (20 nm) and coupled with the above amplified PCR products were applied on the sample pad. The hybridization of the amplified target regions to the respective capture probes takes place when the sample moves towards the absorption pad. Positive hybridization is indicated by red colour lines.ResultsThe three immobilized capture probes on the strip (for the detection of TB, katG wild type and mutation) were 100 and 96.6% specific and 100 and 92.1% sensitive respectively.ConclusionThe AuNP based lateral flow assay was capable of differentiating the specific mutation and the wild type along with MTb identification within 3 h.

Relation between DNA ionization potentials, single base substitutions and pathogenic variants

BackgroundIt is nowadays clear that single base substitutions that occur in the human genome, of which some lead to pathogenic conditions, are non-random and influenced by their flanking nucleobase sequences. However, despite recent progress, the understanding of these "non-local" effects is still far from being achieved.ResultsTo advance this problem, we analyzed the relationship between the base mutability in specific gene regions and the electron hole transport along the DNA base stacks, as it is one of the mechanisms that have been suggested to contribute to these effects. More precisely, we studied the connection between the normalized frequency of single base substitutions and the vertical ionization potential of the base and its flanking sequence, estimated using MP2/6-31G* ab initio quantum chemistry calculations. We found a statistically significant overall anticorrelation between these two quantities: the lower the vIP value, the more probable the substitution. Moreover, the slope of the regression lines varies. It is larger for introns than for exons and untranslated regions, and for synonymous than for missense substitutions. Interestingly, the correlation appears to be more pronounced when considering the flanking sequence of the substituted base in the 3’ rather than in the 5’ direction, which corresponds to the preferred direction of charge migration. A weaker but still statistically significant correlation is found between the ionization potentials and the pathogenicity of the base substitutions. Moreover, pathogenicity is also preferentially associated with larger changes in ionization potentials upon base substitution.ConclusionsWith this analysis we gained new insights into the complex biophysical mechanisms that are at the basis of mutagenesis and pathogenicity, and supported the role of electron-hole transport in these matters.

Abstract 5210: Darolutamide impairs prostate cancer growth by altering chromatin conformation and transcriptional activity of genes involved in cell proliferation and survival

Abstract Androgen signalling is essential for early and late stage prostate cancer (PCa), as demonstrated by the efficacy of androgen receptor (AR) antagonists. The AR gene and its regulatory element are frequently amplified in castration-resistant tumors for which improved treatments are highly needed. Recent findings underscore that gene regulatory elements play an important role in tumor development due to their impact on chromatin conformation and gene regulation. Here we describe the genome-wide effects of darolutamide, a novel AR antagonist which just completed a pivotal clinical phase III trial, by analyzing AR occupancy and its impact on chromatin looping, histone H3K27 acetylation and downstream gene regulation. Genome-wide RNA-seq and ChIP-seq were used to analyze the androgen-sensitive cell lines VCaP, LAPC4 and LNCaP treated with R1881, or with R1881 and darolutamide. Binding of AR and of the pioneer factor FOXA1, as well as histone H3K27 acetylation were determined. Chromatin conformation was detected using the HiChIP method with an AR-specific antibody. Differential gene expression was analyzed by DESeq2 and Gene Set Enrichment Analyses. ChIP-seq peak identification was done by MACS2. HiChIP analysis was performed with the hichipper pipeline. Darolutamide efficiently blocked AR signalling at the level of transcriptional regulation and altered chromatin landscape interaction. Analysis of transcriptomic data showed a strong reduction of the androgen response, as well as down-regulation of proliferation and cell cycle genes by darolutamide. Concordantly, AR occupancy was decreased genome-wide compared to androgen stimulation. Importantly, no binding cluster was identified which showed increased AR occupancy after darolutamide treatment, as reported for other AR antagonists. Interestingly, we observed in darolutamide-treated samples a parallel reduction of FOXA1 occupancy at AR sites, which argues for a co-dependence between this pioneer factor and the AR. Also, we found that H3K27 acetylation, a histone mark linked to active regions, was markedly down-regulated at specific enhancer and gene regions following darolutamide treatment, further pointing to reduced activity of enhancer regions recognized by the AR. Finally, determination of chromatin looping mediated by the AR between enhancers and gene promoters showed a dense network at genes involved in prostate cancer survival. The loss of AR binding at these regions was in line with the significantly reduced transcriptional response observed following darolutamide treatment. In conclusion, we found that darolutamide strongly prevented genome-wide AR binding in multiple hormone-sensitive cellular PCa models, importantly at genes involved in cell proliferation and survival. We furthermore highlight the cross-talk between the pioneer factor FOXA1 and the AR. Citation Format: Simon J. Baumgart, Ekaterina Nevedomskaya, Ralf Lesche, Bernard Haendler. Darolutamide impairs prostate cancer growth by altering chromatin conformation and transcriptional activity of genes involved in cell proliferation and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5210.

Genetic variants in male specific region (MSY) of ZNF280BY gene and their association with semen quality traits in Murrah buffalo bulls

Bull fertility is an important factor for genetic improvement; therefore prediction of fertility an early age of the bulls by using genetic markers is a goal for livestock breeding. Zinc finger protein280BY linked gene is predominantly expressed in testis and has a major role in spermatogenesis. An investigation was carried out to detect the genetic variants in the male specific region (MSY) of ZNF280BY gene and their association with semen quality traits in Murrah buffaloes. Polymerase Chain Reaction-Single Strand Conformation Polymorphism technique (PCR-SSCP) and DNA sequencing methods revealed three different SSCP band patterns (AA, BB, CC); one transition (C10375T) and an indel (insertion of T at 10460-10461). The associations of the genetic variants with semen volume, sperm concentration, Hypo Osmotic Swelling Test (HOST) in fresh and frozen semen were observed. AA and CC genotype bulls had high volume (P£0.05) of semen per ejaculation than BB genotype bulls. BB genotype bulls had high sperm concentration (106/mL) per ejaculate in comparison to AA and CC genotype bulls (P£0.05). AA and BB genotype bulls had a higher per cent of HOST (P£0.05) reacted sperms of fresh semen and per cent acrosomal integrity of frozen semen in comparison to CC genotype bulls. The observed association between SSCP variants in MSY region of ZNF280BY gene with semen quality parameters indicated the possibilities of using ZNF280BY as a candidate gene for identification of markers for semen quality traits in Murrah buffaloes.

Nutrition, epigenetics and health

Nutrition, epigenetics and health

Variation of gene expression in plants is influenced by gene architecture and structural properties of promoters.

In higher eukaryotes, gene architecture and structural properties of promoters have emerged as significant factors influencing variation in number of transcripts (expression level) and specificity of gene expression in a tissue (expression breadth), which eventually shape the phenotype. In this study, transcriptome data of different tissue types at various developmental stages of A. thaliana, O. sativa, S. bicolor and Z. mays have been used to understand the relationship between properties of gene components and its expression. Our findings indicate that in plants, among all gene architecture and structural properties of promoters, compactness of genes in terms of intron content is significantly linked to gene expression level and breadth, whereas in human an exactly opposite scenario is seen. In plants, for the first time we have carried out a quantitative estimation of effect of a particular trait on expression level and breadth, by using multiple regression analysis and it confirms that intron content of primary transcript (as %) is a powerful determinant of expression breadth. Similarly, further regression analysis revealed that among structural properties of the promoters, stability is negatively linked to expression breadth, while DNase1 sensitivity strongly governs gene expression breadth in monocots and gene expression level in dicots. In addition, promoter regions of tissue specific genes are found to be enriched with TATA box and Y-patch motifs. Finally, multi copy orthologous genes in plants are found to be longer, highly regulated and tissue specific.

Intestine specific regulation of pig cytidine-5\u2032-monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis

N-glycolylneuraminic acid (Neu5Gc), a generic form of sialic acid, is enzymatically synthesized by cytidine-5′-monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Although expression of pig CMAH gene pcmah encoding CMAH has been reported to be regulated by pathogenic infection and developmental processes, little is known about the mechanisms underlying the regulation of pcmah gene expression. The objective of this study was to determine mechanism(s) involved in intestine specific regulation of pcmah gene by identifying several cis-acting elements and nuclear transcription factors that could directly interact with these cis-acting elements. We identified intestine specific promoter region (Pi) of pcmah gene located at upstream regions of the 5′flanking region of exon 1a and found that the promoter region is responsible for the transcriptional regulation of 5′pcmah-1. Based on reporter assays using serially constructed luciferase genes with each deleted promoter, we demonstrated that the Pi promoter activity was more active in intestinal IPI-2I cells than that in kidney PK15 cells, corresponding to both mRNA expression patterns in the two cell lines. In addition, we found that Sp1 transcription factor was necessary for basal activity of Pi promoter and that Ets-1 contributed to intestine-specific activity of Pi promoter. This study helps us understand transcriptional regulation of pcmah in the intestine of pig tissues. It also allows us to consider potential roles of Neu5Gc in interaction with environmental factors present in the intestinal tissue during pathogenic infection and developmental process.

16S rRNA sequence embeddings: Meaningful numeric feature representations of nucleotide sequences that are convenient for downstream analyses.

Advances in high-throughput sequencing have increased the availability of microbiome sequencing data that can be exploited to characterize microbiome community structure in situ. We explore using word and sentence embedding approaches for nucleotide sequences since they may be a suitable numerical representation for downstream machine learning applications (especially deep learning). This work involves first encoding (“embedding”) each sequence into a dense, low-dimensional, numeric vector space. Here, we use Skip-Gram word2vec to embed k-mers, obtained from 16S rRNA amplicon surveys, and then leverage an existing sentence embedding technique to embed all sequences belonging to specific body sites or samples. We demonstrate that these representations are meaningful, and hence the embedding space can be exploited as a form of feature extraction for exploratory analysis. We show that sequence embeddings preserve relevant information about the sequencing data such as k-mer context, sequence taxonomy, and sample class. Specifically, the sequence embedding space resolved differences among phyla, as well as differences among genera within the same family. Distances between sequence embeddings had similar qualities to distances between alignment identities, and embedding multiple sequences can be thought of as generating a consensus sequence. In addition, embeddings are versatile features that can be used for many downstream tasks, such as taxonomic and sample classification. Using sample embeddings for body site classification resulted in negligible performance loss compared to using OTU abundance data, and clustering embeddings yielded high fidelity species clusters. Lastly, the k-mer embedding space captured distinct k-mer profiles that mapped to specific regions of the 16S rRNA gene and corresponded with particular body sites. Together, our results show that embedding sequences results in meaningful representations that can be used for exploratory analyses or for downstream machine learning applications that require numeric data. Moreover, because the embeddings are trained in an unsupervised manner, unlabeled data can be embedded and used to bolster supervised machine learning tasks.

Identification of potential blood biomarkers for Parkinson\u2019s disease by gene expression and DNA methylation data integration analysis

BackgroundBlood-based gene expression or epigenetic biomarkers of Parkinson’s disease (PD) are highly desirable. However, accuracy and specificity need to be improved, and methods for the integration of gene expression with epigenetic data need to be developed in order to make this feasible.MethodsWhole blood gene expression data and DNA methylation data were downloaded from Gene Expression Omnibus (GEO) database. A linear model was used to identify significantly differentially expressed genes (DEGs) and differentially methylated genes (DMGs) according to specific gene regions 5′—C—phosphate—G—3′ (CpGs) or all gene regions CpGs in PD. Gene set enrichment analysis was then applied to DEGs and DMGs. Subsequently, data integration analysis was performed to identify robust PD-associated blood biomarkers. Finally, the random forest algorithm and a leave-one-out cross validation method were performed to construct classifiers based on gene expression data integrated with methylation data.ResultsEighty-five (85) significantly hypo-methylated and upregulated genes in PD patients compared to healthy controls were identified. The dominant hypo-methylated regions of these genes were significantly different. Some genes had a single dominant hypo-methylated region, while others had multiple dominant hypo-methylated regions. One gene expression classifier and two gene methylation classifiers based on all or dominant methylation-altered region CpGs were constructed. All have a good prediction power for PD.ConclusionsGene expression and methylation data integration analysis identified a blood-based 53-gene signature, which could be applied as a biomarker for PD.