Mammalian Database Research Articles

Metabolic Transcriptional Activation in Ulcerative Colitis Identified Through scRNA-seq Analysis.

Ulcerative colitis is a chronic inflammatory disease affecting the colon. During chronic inflammation of epithelial cells, lipid metabolism via pro-inflammatory eicosanoids is known to modify the immune response. Starting from the Mammalian Metabolic Database, the expression of metabolic enzymes was investigated in two independent cohorts from transcriptome datasets GSE38713 and GSE11223, which analyzed ulcerative colitis tissue samples from the digestive tract. In the first cohort, 145 differentially expressed enzymes were identified as significantly regulated between ulcerative colitis tissues and normal controls. Overexpressed enzymes were selected to tune an Elastic Net model in the second cohort. Using the best parameters, the model achieved a prediction accuracy for ulcerative colitis with an area under the curve (AUC) of 0.79. Twenty-two metabolic enzymes were found to be commonly overexpressed in both independent cohorts, with decreasing Elastic Net predictive coefficients as follows: LIPG (3.98), PSAT1 (3.69), PGM3 (2.74), CD38 (2.28), BLVRA (1.99), CBR3 (1.94), NT5DC2 (1.76), PHGDH (1.71), GPX7 (1.58), CASP1 (1.56), ASRGL1 (1.4), SOD3 (1.25), CHST2 (0.965), CHST11 (0.95), KYNU (0.94), PLAG2G7 (0.92), SRM (0.87), PTGS2 (0.80), LPIN1 (0.47), ME1 (0.31), PTGDS (0.14), and ADA (0.13). Functional enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database highlighted the main implications of these enzymes in cysteine and methionine metabolism (adjusted p-value = 0.01), arachidonic acid and prostaglandin metabolism (adjusted p-value = 0.01), and carbon metabolism (adjusted p-value = 0.04). A metabolic score based on the transcriptional activation of the validated twenty-two enzymes was found to be significantly greater in Ulcerative colitis samples compared to healthy donor samples (p-value = 1.52 × 10-8). A metabolic expression score was established and reflects the implications of heterogeneous metabolic pathway deregulations in the digestive tract of patients with ulcerative colitis.

Genomic Regions Associated with Growth and Reproduction Traits in Pink-Eyed White Mink.

In mink breeding, balanced selection for growth and reproductive features is essential because these traits are contradictory. The variables of total number born (TNB), number born alive (NBA), and body weight (BW) are highly valuable in terms of their importance in mink production. A comprehensive understanding of the molecular mechanisms that drive these features could offer vital insights into their genetic compositions. In the present study, the single-nucleotide polymorphism (SNP) genotypes of 219 minks were obtained via double digest restriction-site associated DNA sequencing (ddRAD-seq). Following several rounds of screening, about 2,415,121 high-quality SNPs were selected for a genome-wide association study (GWAS). The GWAS was used to determine BW and reproductive traits in pink-eyed white mink. It was suggested that SLC26A36, STXBP5L, and RPS 29 serve as potential genes for the total number of kits born (TNB), while FSCB, PDPN, NKX 2-1, NFKB 1, NFKBIA, and GABBR1 are key genes for the number born alive (NBA). Moreover, RTTN, PRPF31, MACROD1, and KYAT1 are possible BW genes based on association results and available functional data from gene and mammalian phenotype databases. These results offer essential information about the variety of mink and theoretical principles for applying mink breeds.

Zebra-Sphinx: Modeling Sphingolipidoses in Zebrafish

Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches.

Long-Term Simulation of Microgravity Induces Changes in Gene Expression in Breast Cancer Cells.

Microgravity changes the gene expression pattern in various cell types. This study focuses on the breast cancer cell lines MCF-7 (less invasive) and MDA-MB-231 (triple-negative, highly invasive). The cells were cultured for 14 days under simulated microgravity (s-µg) conditions using a random positioning machine (RPM). We investigated cytoskeletal and extracellular matrix (ECM) factors as well as focal adhesion (FA) and the transmembrane proteins involved in different cellular signaling pathways (MAPK, PAM and VEGF). The mRNA expressions of 24 genes of interest (TUBB, ACTB, COL1A1, COL4A5, LAMA3, ITGB1, CD44, VEGF, FLK1, EGFR, SRC, FAK1, RAF1, AKT1, ERK1, MAPK14, MAP2K1, MTOR, RICTOR, VCL, PXN, CDKN1, CTNNA1 and CTNNB1) were determined by quantitative real-time PCR (qPCR) and studied using STRING interaction analysis. Histochemical staining was carried out to investigate the morphology of the adherent cells (ADs) and the multicellular spheroids (MCSs) after RPM exposure. To better understand this experimental model in the context of breast cancer patients, a weighted gene co-expression network analysis (WGCNA) was conducted to obtain the expression profiles of 35 breast cell lines from the HMS LINCS Database. The qPCR-verified genes were searched in the mammalian phenotype database and the human genome-wide association studies (GWAS) Catalog. The results demonstrated the positive association between the real metastatic microtumor environment and MCSs with respect to the extracellular matrix, cytoskeleton, morphology, different cellular signaling pathway key proteins and several other components. In summary, the microgravity-engineered three-dimensional MCS model can be utilized to study breast cancer cell behavior and to assess the therapeutic efficacies of drugs against breast cancer in the future.

Development of Malaysian Mammal Online Database

Malaysia is one of 17 mega diverse countries in the world. Despite this fact, there is still no online standalone database that focuses on Malaysian mammals. Creation of a mammalian database would allow researchers and conservationists to utilize the database for research, policy making and to answer broad scale of biodiversity questions. Human development has contribute to the depleting of mammal population and species extinction rate is faster than the effort of cataloguing them. The aim of this study is to 1) identify online database features and the type of data required for mammal database, 2) collect and populate database from existing mammal data to assist the said database and 3) to test the acceptance of the database by user of this study. The secondary data from existing database such as Global Biodiversity Information Facility (GBIF), Animal Diversity Web (ADW) and Malaysia Biodiversity Information System (MyBIS) was collected to determine the required features needed in the database. The database adhered to TDWG standard that facilitates exchange of biological information. Examination on GBIF have been made to observe their data presentation since GBIF used TDWG Standard. Rapid Prototype Model was utilized as development method and Omeka Classic was used as platform of Content Management System. The mammal data was collected online and populate into the database. The acceptance testing in form of questionnaire were distributed to verify whether the database is function properly and meet the requirement of the study. We present MYMODS, a comprehensive mammal database with 361 mammal species in Malaysia deposited in the database. The development of this database will enable the researcher and conservationist to seek and identify mammal information based on species’ scientific name, description and IUCN Red List. We believe that MYMODS will contribute to the digital knowledge that provide tools for mammal research and contributes in management of biodiversity and conservation in Malaysia. MYMODS is available at https://cercom.uthm.edu.my/mymods/

Draft genome of the lowland anoa (Bubalus depressicornis) and comparison with buffalo genome assemblies (Bovidae, Bubalina).

Genomic data for wild species of the genus Bubalus (Asian buffaloes) are still lacking while several whole genomes are currently available for domestic water buffaloes. To address this, we sequenced the genome of a wild endangered dwarf buffalo, the lowland anoa (Bubalus depressicornis), produced a draft genome assembly and made comparison to published buffalo genomes. The lowland anoa genome assembly was 2.56 Gbp long and contained 103,135 contigs, the longest contig being 337.39 kbp long. N50 and L50 values were 38.73 and 19.83 kbp, respectively, mean coverage was 44× and GC content was 41.74%. Two strategies were adopted to evaluate genome completeness: (1) determination of genomic features with de novo and homology-based predictions using annotations of chromosome-level genome assembly of the river buffalo and (2) employment of benchmarking against universal single-copy orthologs (BUSCO). Homology-based predictions identified 94.51% complete and 3.65% partial genomic features. De novo gene predictions identified 32,393 genes, representing 97.14% of the reference's annotated genes, whilst BUSCO search against the mammalian orthologs database identified 71.1% complete, 11.7% fragmented, and 17.2% missing orthologs, indicating a good level of completeness for downstream analyses. Repeat analyses indicated that the lowland anoa genome contains 42.12% of repetitive regions. The genome assembly of the lowland anoa is expected to contribute to comparative genome analyses among bovid species.

Large-scale association study on daily weight gain in pigs reveals overlap of genetic factors for growth in humans

BackgroundImputation from genotyping array to whole-genome sequence variants using resequencing of representative reference populations enhances our ability to map genetic factors affecting complex phenotypes in livestock species. The accumulation of knowledge about gene function in human and laboratory animals can provide substantial advantage for genomic research in livestock species.ResultsIn this study, 201,388 pigs from three commercial Danish breeds genotyped with low to medium (8.5k to 70k) SNP arrays were imputed to whole genome sequence variants using a two-step approach. Both imputation steps achieved high accuracies, and in total this yielded 26,447,434 markers on 18 autosomes. The average estimated imputation accuracy of markers with minor allele frequency ≥ 0.05 was 0.94. To overcome the memory consumption of running genome-wide association study (GWAS) for each breed, we performed within-breed subpopulation GWAS then within-breed meta-analysis for average daily weight gain (ADG), followed by a multi-breed meta-analysis of GWAS summary statistics. We identified 15 quantitative trait loci (QTL). Our post-GWAS analysis strategy to prioritize of candidate genes including information like gene ontology, mammalian phenotype database, differential expression gene analysis of high and low feed efficiency pig and human GWAS catalog for height, obesity, and body mass index, we proposed MRAP2, LEPROT, PMAIP1, ENSSSCG00000036234, BMP2, ELFN1, LIG4 and FAM155A as the candidate genes with biological support for ADG in pigs.ConclusionOur post-GWAS analysis strategy helped to identify candidate genes not just by distance to the lead SNP but also by multiple sources of biological evidence. Besides, the identified QTL overlap with genes which are known for their association with human growth-related traits. The GWAS with this large data set showed the power to map the genetic factors associated with ADG in pigs and have added to our understanding of the genetics of growth across mammalian species.

Analysis of elimination half-lives in MamTKDB 1.0 related to bioaccumulation: Requirement of repeated administration and blood plasma values underrepresent tissues

When building the novel public mammalian toxicokinetic database (MamTKDB) we collected and included 3927 elimination half-lives (elimt1/2) for 1407 xenobiotics in various species (rat, human, mouse, dog, monkey, rabbit, cattle, pig, sheep, guinea pig, hamster, horse and goat) with specification of compartment (whole body, organ/tissue, cell type, medium) studied. Here we describe and analyse the collected data in MamTKDB 1.0. Most elimt1/2 are for humans and rats and their data differ in some ways: whereas the rat data are mainly for pesticides, the human data are mainly for pharmaceuticals and environmental contaminants. There are also differences in types of compartments studied and in metabolites followed: human elimt1/2 are mainly whole body based (i.e. based on blood plasma or excretion), animal data are additionally for various organs/tissues, cells or media. Contrary to human studies, animal studies regularly administrate radiolabeled (e.g. 14C) substances and distribution of both parent and eventual metabolites are followed, measuring the radioactivity. In rats, substances had been given through single, preconditioning or repeated administration. Single administration studies dominated, but repeated studies generally had longer elimt1/2 than single or preconditioning studies for which elimt1/2 were similar. Repeated administration studies should better ascertain steady state conditions throughout the body, a process involving time-dependent tissue loading, and the data show that for most substances, repeated studies are required to address bioaccumulation potential. About 65% of the substances in MamTKDB 1.0 fulfilled the octanol–water and octanol–air partitioning-based screening criteria (log Kow > 2 and log Koa > 5) for further bioaccumulation assessment and/or testing, and most of the substances with long elimt1/2 in both humans and rats fulfill these criteria. Of note, however, there are also many chemicals with log Kow > 2 with intermediate or short elimt1/2. Per- and polyfluoroalkyl substances (PFAS) stand out in that they often have log Koa < 5. Rats are poor toxicokinetic test models for perfluoroalkyl acids (PFAAs) for which pigs (and possibly mice) elimt1/2 data resemble those of humans better. Perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonic acids (PFSAs) of similar molecular weight had similar elimt1/2 in the species tested. For polychlorinated biphenyls (PCBs), elimt1/2 increases with the degree of chlorination in humans. In relation to other compartments, blood plasma/serum had among the shortest elimt1/2 in rats and often underrepresent elimt1/2 in tissues. Rat data were divided into 38 compartment (tissue or media) types out of which 20 had sufficient data for correlational tests. In general, there was a strong degree of correlation of rat elimt1/2 in-between most compartments, but there were also exceptions. Surprisingly, the correlation between brain and white fat was relatively weak. Interestingly, several substances or their metabolites bound to haemoglobin in red blood cells. MamTKDB 1.0 allows investigation on how certain chemical characteristics influence elimt1/2 and is a promising database for assessment of bioaccumulation potential.

Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis.

Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

Accurate, automatic annotation of peptidases with hotpep-protease

Peptidases are essential for intracellular protein processing, signaling and homeostasis, physiological processes and for digestion of food. Moreover, peptidases are important biotechnological enzymes used in processes such as industrial food processing, leather manufacturing and the washing industry. Identification of peptidases is a crucial step in their characterization but peptidase annotation is not a trivial task due to their large sequence diversity.In the present study short, conserved sequence profiles were generated for all peptidase families with more than four members in the comprehensive Merops peptidase database. The sequence profiles were combined with the Homology to Peptide Pattern (Hotpep) method for automatic annotation of peptidases. This method is a stand-alone software that annotates protease sequences to Merops family and subgroup and is suitable for large-scale sequence analysis. Compared to the Mammalian Degradome Database Hotpep-protease had an accuracy of 92% and a sensitivity of 96% for annotation of the human degradome. Annotation by commonly used methods (Blast and conserved domains) had an accuracy of 69% and a sensitivity of 78%. For fungal genomes, there were large differences between annotation with Hotpep-protease, Blast- and Hidden Markov Model-based annotation and the Merops annotation, which confirms the difficulty of large-scale peptidase annotation. Manual annotation indicated that Hotpep-protease had a positive prediction rate of 0.90 compared to a positive prediction rate of 0.67 for Blast search. Hence, Hotpep-protease is highly accurate method for fast and accurate annotation of peptidases.

Identification of an l-serine/l-threonine dehydratase with glutamate racemase activity in mammals

Recent investigations have shown that multiple d-amino acids are present in mammals and these compounds have distinctive physiological functions. Free d-glutamate is present in various mammalian tissues and cells and in particular, it is presumably correlated with cardiac function, and much interest is growing in its unique metabolic pathways. Recently, we first identified d-glutamate cyclase as its degradative enzyme in mammals, whereas its biosynthetic pathway in mammals is unclear. Glutamate racemase is a most probable candidate, which catalyzes interconversion between d-glutamate and l-glutamate. Here, we identified the cDNA encoding l-serine dehydratase-like (SDHL) as the first mammalian clone with glutamate racemase activity. This rat SDHL had been deposited in mammalian databases as a protein of unknown function and its amino acid sequence shares ∼60% identity with that of l-serine dehydratase. Rat SDHL was expressed in Escherichia coli, and the enzymatic properties of the recombinant were characterized. The results indicated that rat SDHL is a multifunctional enzyme with glutamate racemase activity in addition to l-serine/l-threonine dehydratase activity. This clone is hence abbreviated as STDHgr. Further experiments using cultured mammalian cells confirmed that d-glutamate was synthesized and l-serine and l-threonine were decomposed. It was also found that SDHL (STDHgr) contributes to the homeostasis of several other amino acids.

First Genome Sequence of the Gunnison's Prairie Dog (Cynomys gunnisoni), a Keystone Species and Player in the Transmission of Sylvatic Plague.

Prairie dogs (genus Cynomys) are a charismatic symbol of the American West. Their large social aggregations and complex vocalizations have been the subject of scientific and popular interest for decades. A large body of literature has documented their role as keystone species of western North America’s grasslands: They generate habitat for other vertebrates, increase nutrient availability for plants, and act as a food source for mammalian, squamate, and avian predators. An additional keystone role lies in their extreme susceptibility to sylvatic plague (caused by Yersinia pestis), which results in periodic population extinctions, thereby generating spatiotemporal heterogeneity in both biotic communities and ecological processes. Here, we report the first Cynomys genome for a Gunnison’s prairie dog (C. gunnisoni gunnisoni) from Telluride, Colorado (USA). The genome was constructed using a hybrid assembly of PacBio and Illumina reads and assembled with MaSuRCA and PBJelly, which resulted in a scaffold N50 of 824 kb. Total genome size was 2.67 Gb, with 32.46% of the bases occurring in repeat regions. We recovered 94.9% (91% complete) of the single copy orthologs using the mammalian Benchmarking Universal Single-Copy Orthologs database and detected 49,377 gene models (332,141 coding regions). Pairwise Sequentially Markovian Coalescent showed support for long-term stable population size followed by a steady decline beginning near the end of the Pleistocene, as well as a recent population reduction. The genome will aid in studies of mammalian evolution, disease resistance, and the genomic basis of life history traits in ground squirrels.

122 Proteome of extracellular vesicles from follicular fluid of bovine 3- to 6-mm follicles: Similarity and specificity compared with granulosa cells

Extracellular vesicles (EVs) are released from cells and are present in most bodily fluids. Small EVs, 30-150nm in diameter, known as exosomes, transport regulatory noncoding RNA, lipids, and proteins, and thus participate in cell-to-cell communications and signalling. Exosomes from bovine ovarian follicular fluid (FF) can stimulate proliferation of granulosa cells (GCs), cellular uptake, and cumulus expansion, and therefore might be useful in reproductive technologies. The aim of our study was to characterise the protein content of bovine FF exosomes and compare it with the GC proteome in order to better understand the origin of these EVs and their possible functions. We extracted EVs from FF of bovine 3- to 6-mm ovarian follicles (enclosed oocytes routinely used for invitro embryo production (IVEP)) via a series of centrifugations at 2,000g to 100 000g. Transmission electron microscopy analysis revealed that 87.9% of extracted EVs had a larger size of exosomes, and 11.8% had smaller size (&amp;lt;30nm); only 0.3% of EVs ranged from 150 to 250nm. For proteome analysis, 80µg of total proteins extracted from EVs and GCs originating from the same follicles were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After trypsin proteolytic digestion, peptide mixtures were analysed via nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (MS/MS) using LTQ Orbitrap Velos (Thermo Fisher Scientific). We used Mascot engine (Matrix Science) against the NCBIprot mammalian database to perform MS/MS ion searches; proteins identified with at least two peptides were clustered with Scaffold 4.0 software. We identified 542 protein isoforms that represented 221 and 299 clusters of unique proteins in EVs and GCs, respectively. Of these, 158 are common among EVs and GCs and demonstrate significant enrichment (P&amp;lt;0.01) in Gene Ontology (GO) terms related to protein folding in endoplasmic reticulum, receptor-mediated endocytosis, cell-cell adhesion, translation processes, binding of proteins, RNA or cholesterol, and antioxidant activity. Of common proteins, 69.6% are mapped to the GO “extracellular exosome.” Of the 63 proteins over-represented in EVs, 37 are ribosomal proteins involved in translation, rRNA processing, and mRNA catabolic processes. Of the proteins over-represented in GCs, 141 are mainly involved in carbon, fatty acid, and tricarboxylic acid (TCA) metabolism, oxidation-reduction processes, NAD and poly(A) RNA binding, and cell cycle regulation. These proteins are enriched in GO terms related to membrane, mitochondrion, and extracellular exosomes. Our findings indicate that proliferating GCs of antral follicles actively synthesise and secrete exosomes, which contain significant quantity of different ribosomal proteins. These RNA-binding proteins may serve to compact the cargo of different types of RNAs-both regulatory and designated for degradation, which are transported by FF exosomes. Funds were provided by INRA PHASE France and by the Russian Science Foundation (project 19-16-00115).

A seven-DNA methylation signature as a novel prognostic biomarker in breast cancer.

Breast cancer (BC) is a common malignant tumor and its incidence and mortality rates are ranked first among female cancers. So far, there has been no effective biomarkers for BC prognosis. The DNA methylation data of BC was downloaded from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus, and Functional ANnoTation of The Mammalian Genome databases. The RNA-Seq data and clinical information of patients were downloaded from TCGA. R packages edgeR and minfi were used for differentially methylated genes (DMGs) screening. Then, the DMGs were collected for gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis by the online tool database for annotation, visualization and integrated discovery (DAVID) and Reactome. Cox regression analysis was used to screen candidate differentially methylated sites (DMSs) for BC prognosis. Logrank test was used to explore the correlation between DMSs and survival time. Correlation analysis was used to investigate the correlation between DNA methylation and gene expression. We identified 276 DMGs which contained 1454 DMSs in those three datasets. Also, six DMGs that contained seven DMSs were identified by Cox regression analysis. Interestingly, their expression levels were negatively correlated with the DNA methylation level and not affected by age, subtypes, or tumor stages. We proposed that these seven differentially DNA methylation sites can be used as a novel prognostic biomarker for BC area under curve (AUC) = 0.74), which may facilitate research and benefit the clinical treatment of BC.

Salivary proteomics of canine oral tumors using MALDI-TOF mass spectrometry and LC-tandem mass spectrometry.

Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein–chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Mammalian diversity in West Java, Indonesia

Abstract. Husodo T, Shanida SS, Febrianto P, Pujianto MP, Megantara EN. 2019. Mammalian diversity in West Java, Indonesia. Biodiversitas 20: 1846-1858. Protected forests in West Java are wider than conservation forests, whereas mammalian diversity in protected forests is as high as mammalian diversity in conservation forests. Mammals in protected forests are not protected by regional protection regulations, while anthropogenic factors in Java are quite high. This is possible that mammals who have high conservation status will experience local extinction. This study aims to determine (i) the composition of mammalian species and (ii) the species that are always found in studies of mammalian diversity in West Java. The study was conducted through a qualitative approach by combining several methods such as interview, camera trapping, sign survey, observation and transect, and collapsible traps. Mammalia in West Java found 54 species, 21 families, and nine orders with details of three species of Artiodactyla, 12 species of Carnivores, seven species of Chiroptera, one species of Dermoptera, one species of Euphotyphla, one species of Pholidota, five species of Primates, 21 species of Rodentia, and three species of Scandentia. The species that are always found in all study locations are nine species, including wild boar, Javan leopard, leopard cat, Javan mongoose, oriental small-clawed otter, Asian palm civet, grizzled leaf monkey, Javan langur, and Javan tree shrew. The results presented here may facilitate improvements in the mammalian diversity database, especially in the non-conservation area.

Prioritizing candidate genes for fertility in dairy cows using gene-based analysis, functional annotation and differential gene expression

BackgroundAn unfavorable genetic correlation between milk production and fertility makes simultaneous improvement of milk production and fertility difficult in cattle breeding. Rapid genetic improvement in milk production traits in dairy cattle has been accompanied by decline in cow fertility. The genetic basis of this correlation remains poorly understood. Expanded reference populations and large sets of sequenced animals make genome-wide association studies (GWAS) with imputed markers possible for large populations and thereby studying genetic architecture of complex traits.ResultsIn this study, we associated 15,551,021 SNPs with female fertility index in 5038 Nordic Holstein cattle. We have identified seven quantitative trait loci (QTL) on six chromosomes in cattle. Along with nearest genes to GWAS hits, we used gene-based analysis and spread of linkage disequilibrium (LD) information to generate a list of potential candidate genes affecting fertility in cattle. Subsequently, we used prior knowledge on gene related to fertility from Gene Ontology terms, Kyoto Encyclopedia of Genes and Genomes pathway analysis, mammalian phenotype database, and public available RNA-seq data to refine the list of candidate genes for fertility. We used variant annotations to investigate candidate mutations within the prioritized candidate genes. Using multiple source of information, we proposed candidate genes with biological relevance underlying each of these seven QTL. On chromosome 1, we have identified ten candidate genes for two QTL. For the rest of chromosomes, we proposed one candidate gene for each QTL. In the candidate genes list, differentially expressed genes from different studies support FRAS1, ITGB5, ADCY5, and SEMA5B as candidate genes for cow fertility.ConclusionThe GWAS result not only confirmed previously mapped QTL, but also made new findings. Our findings contributes towards dissecting the genetics for female fertility in cattle. Moreover, this study shows the usefulness of adding independent information to pick candidate genes during post-GWAS analysis.

Dissecting closely linked association signals in combination with the mammalian phenotype database can identify candidate genes in dairy cattle

BackgroundGenome-wide association studies (GWAS) have been successfully implemented in cattle research and breeding. However, moving from the associations to identify the causal variants and reveal underlying mechanisms have proven complicated. In dairy cattle populations, we face a challenge due to long-range linkage disequilibrium (LD) arising from close familial relationships in the studied individuals. Long range LD makes it difficult to distinguish if one or multiple quantitative trait loci (QTL) are segregating in a genomic region showing association with a phenotype. We had two objectives in this study: 1) to distinguish between multiple QTL segregating in a genomic region, and 2) use of external information to prioritize candidate genes for a QTL along with the candidate variants.ResultsWe observed fixing the lead SNP as a covariate can help to distinguish additional close association signal(s). Thereafter, using the mammalian phenotype database, we successfully found candidate genes, in concordance with previous studies, demonstrating the power of this strategy. Secondly, we used variant annotation information to search for causative variants in our candidate genes. The variant information successfully identified known causal mutations and showed the potential to pinpoint the causative mutation(s) which are located in coding regions.ConclusionsOur approach can distinguish multiple QTL segregating on the same chromosome in a single analysis without manual input. Moreover, utilizing information from the mammalian phenotype database and variant effect predictor as post-GWAS analysis could benefit in candidate genes and causative mutations finding in cattle. Our study not only identified additional candidate genes for milk traits, but also can serve as a routine method for GWAS in dairy cattle.

Mammalian Annotation Database for improved annotation and functional classification of Omics datasets from less well-annotated organisms

Next-generation sequencing technologies and the availability of an increasing number of mammalian and other genomes allow gene expression studies, particularly RNA sequencing, in many non-model organisms. However, incomplete genome annotation and assignments of genes to functional annotation databases can lead to a substantial loss of information in downstream data analysis. To overcome this, we developed Mammalian Annotation Database tool (MAdb, https://madb.ethz.ch) to conveniently provide homologous gene information for selected mammalian species. The assignment between species is performed in three steps: (i) matching official gene symbols, (ii) using ortholog information contained in Ensembl Compara and (iii) pairwise BLAST comparisons of all transcripts. In addition, we developed a new tool (AnnOverlappeR) for the reliable assignment of the National Center for Biotechnology Information (NCBI) and Ensembl gene IDs. The gene lists translated to gene IDs of well-annotated species such as a human can be used for improved functional annotation with relevant tools based on Gene Ontology and molecular pathway information. We tested the MAdb on a published RNA-seq data set for the pig and showed clearly improved overrepresentation analysis results based on the assigned human homologous gene identifiers. Using the MAdb revealed a similar list of human homologous genes and functional annotation results regardless of whether starting with gene IDs from NCBI or Ensembl. The MAdb database is accessible via a web interface and a Galaxy application.

Integrative analysis of shared genetic pathogenesis by obsessive‑compulsive and eating disorders.

A number of common pathological features have been observed in obsessive‑compulsive disorder (OCD) and eating disorders (EDs). The present study examined the association between OCD and EDs at the genetic level in order to gain an improved understanding of the shared genetic basis of the diseases and identify novel potential risk genes for the two diseases. An integrated analysis using large‑scale disease‑gene association data and gene expression data was conducted. Disease‑gene association data were acquired from the Pathway Studio Mammalian database. Gene expression data were acquired from samples of 133 subjects, including 15 ED cases, 16 OCD cases and 102 normal controls. Genes associated with OCD and ED presented significant overlap (21genes, P=6.70x10‑34), serving roles within multiple common genetic pathways (top 10 pathway enrichment P<4.30x10‑7) that were implicated in the two diseases. A genetic network of 17genes was constructed, through which OCD and ED were observed to influence each other. Expression analysis revealed four novel common significant genes for OCD and ED (oxytocin receptor, glutamate decarboxylase 2, neuropeptide Y and glutamate ionotropic receptor kainate type subunit 3). These genes demonstrated a strong functional association with the two diseases. The results of the present study supported the presence of complex genetic associations between OCD and ED. Genes associated with one disease are worthy of further investigation as potential risk factors for the other. The findings of the present study may provide novel insights into the understanding of the pathogenesis of OCD and ED.