Comprehensive probiogenomics analysis of the commensal Escherichia coli CEC15 as a potential probiotic strain.

BACKGROUND: Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis. RESULTS: CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis. CONCLUSIONS: These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

Environmental Biofilms from an Urban Community in Salvador, Brazil, Shelter Previously Uncharacterized Saprophytic Leptospira.

Biofilms are complex microecosystems with valuable ecological roles that can shelter a variety of microorganisms. Spirochetes from the genus Leptospira have been observed to form biofilms in vitro, in rural environments, and in the kidneys of reservoir rats. The genus Leptospira is composed of pathogenic and non-pathogenic species, and the description of new species is ongoing due to the advent of whole genome sequencing. Leptospires have increasingly been isolated from water and soil samples. To investigate the presence of Leptospira in environmental biofilms, we collected three distinct samples of biofilms formed in an urban setting with poor sanitation: Pau da Lima, in Salvador, Bahia, Brazil. All biofilm samples were negative for the presence of pathogenic leptospires via conventional PCR, but cultures containing saprophytic Leptospira were identified. Whole genomes were generated and analyzed for twenty isolates obtained from these biofilms. For species identification, we used digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis. The obtained isolates were classified into seven presumptive species from the saprophytic S1 clade. ANI and dDDH analysis suggest that three of those seven species were new. Classical phenotypic tests confirmed the novel isolated bacteria as saprophytic Leptospira. The isolates presented typical morphology and ultrastructure according to scanning electron microscopy and formed biofilms under in vitro conditions. Our data indicate that a diversity of saprophytic Leptospira species survive in the Brazilian poorly sanitized urban environment, in a biofilm lifestyle. We believe our results contribute to a better understanding of Leptospira biology and ecology, considering biofilms as natural environmental reservoirs for leptospires.

Draft Genome Sequence of Seven Pigmented Strains of Xanthomonas citri pv. anacardii, the Causal Agent of Cashew Angular Spot.

Cashew (Anacardium occidentale) angular leaf spot is caused by pigmented and non-pigmented strains of Xanthomonas citri pv. anacardii, which have been isolated from infected plants in Brazil. The disease symptoms can be observed in leaves, stems, and fruits. Given that infection in young fruits results in fruits unsuitable for commercialization, angular leaf spot represents a serious threat to the cashew crop in Brazil. Here, we report the genomic sequencing of seven pigmented strains of X. citri pv. anacardii, obtained from the leaves of cashew trees from São Paulo state, Brazil, in 2009. The construction of the libraries was carried out according to the manufacturer, and whole-genome sequencing was performed using the Illumina HiSeq 2500 platform. Genome size, number of coding sequences, largest contig length, and N50 ranged from 4,996,984 to 5,003,485 bp, 4,621 to 4,643 bp, 212,513 to 362,232 bp, and 113,582 to 141,003 bp, respectively. GC content and RNA numbers were 64.68% and 54, respectively, for all strains. ANIm and dDDH analyses showed values above 99.5 and 92.1% among these strains and the non-pigmented pathotype strain of X. citri pv. anacardii (IBSBF2579PT). A maximum likelihood tree built with 2,708 core genes grouped all X. citri pv. anacardii strains in the same clade, with a 100% bootstrap. These resources will contribute in a relevant way to help understand the ecological, taxonomic, evolutionary, pathogenicity, and virulence aspects of X. citri pv. anacardii, which will be useful for the study and development of techniques for managing cashew angular leaf spot.

Phylogenomics and gene selection in Aspergillus welwitschiae: Possible implications in the pathogenicity in Agave sisalana.

Aspergillus welwitschiae causes bole rot disease in sisal (Agave sisalana and related species) which affects the production of natural fibers in Brazil, the main worldwide producer of sisal fibers. This fungus is a saprotroph with a broad host range. Previous research established A. welwitschiae as the only causative agent of bole rot in the field, but little is known about the evolution of this species and its strains. In this work, we performed a comparative genomics analysis of 40 Aspergillus strains. We show the conflicting molecular identity of this species, with one sisal-infecting strain sharing its last common ancestor with Aspergillus niger, having diverged only 833 thousand years ago. Furthermore, our analysis of positive selection reveals sites under selection in genes coding for siderophore transporters, Sodium­calcium exchangers, and Phosphatidylethanolamine-binding proteins (PEBPs). Herein, we discuss the possible impacts of these gene functions on the pathogenicity in sisal.

Protein speciation is likely to increase the chance of proteins to be determined in 2-DE/MS.

Multiple spotting due to protein speciation might increase a protein's chance of being captured in a random selection of 2-DE spots. We tested this expectation in new (PXD015649) and previously published 2-DE/MS data of porcine and human tissues. For comparison, we included bottom-up proteomics studies (BU-LC/MS) of corresponding biological materials. Analyses of altogether ten datasets proposed that amino acid modification fosters multispotting in 2-DE. Thus, the number of 2-DE spots containing a particular protein more tightly associated with a peptide diversity measure accounting for amino acid modification than with an alternative one disregarding it. Furthermore, every 11th amino acid was a post-translational modification candidate site in 2-DE/MS proteins, whereas in BU-LC/MS proteins this was merely the case in every 21st amino acid. Alternative splicing might contribute to multispotting, since genes encoding 2-DE/MS proteins were found to have on average about 0.3 more transcript variants than their counterparts from BU-LC/MS studies. Correspondingly, resolution completeness as estimated from the representation of transcript variant-rich genes was higher in 2-DE/MS than BU-LC/MS datasets. These findings suggest that the ability to resolve proteomes down to protein species can lead to enrichment of multispotting proteins in 2-DE/MS. Low sensitivity of stains and MS instruments appears to enhance this effect.

RNA-seq of nine canine prostate cancer cell lines reveals diverse therapeutic target signatures.

BACKGROUND: Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. METHODS: This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. RESULTS: Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. CONCLUSION: Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

α/ß-Hydrolase D16B Truncation Results in Premature Sperm Capacitation in Cattle.

Recently it was shown that a specific form of male infertility in Holstein cattle was caused by a nonsense variant in the α/ß-hydrolase domain-containing 16B (ABHD16B) gene resulting in a protein truncation at amino acid position 218 (p.218Q*) and loss of function. Lipidomics showed that the absence of ABHD16B influenced the content of phosphatidylcholine (PC), ceramide (Cer), diacylglycerol (DAG), and sphingomyelin (SM) in variant carrier sperm membranes. However, the exact cause of infertility in affected sires has remained unclear until now. To elucidate the cause of infertility, we analyzed (i) standard sperm parameters (i.e., total sperm number, morphological intact sperm, total sperm motility), (ii) in vitro fertilizability and effects on early embryonic development, and (iii) sperm survival rates (i.e., capacitation time). The affected spermatozoa showed no changes in the usual sperm parameters and were also capable of fertilization in vitro. Furthermore, the absence of ABHD16B did not affect early embryonic development. Based on these results, it was concluded that the affected spermatozoa appeared to be fertilizable per se. Consequently, the actual cause of the inability to fertilize could only be due to a time- and/or place-dependent process after artificial insemination and before fertilization. A process fundamental to the ability to fertilize after insemination is capacitation. Capacitation is a biochemical maturation process that spermatozoa undergo in the female genital tract and is inevitable for the successful fertilization of the oocyte. It is known that the presence and concentration of certain sperm membrane lipids are essential for the correct course of capacitation. However, precisely these lipids are absent in the membrane of spermatozoa affected by the ABHD16B truncation. Since all other causes of fertilization inability were excluded in the previous experiments, consequently, the only remaining hypothesis was that the loss of function of ABHD16B leads to a capacitation disruption. We were able to show that heterozygous and homozygous affected spermatozoa exhibit premature capacitation and therefore decay before fertilization. This effect of the loss of function of ABHD16B has not been described before and our studies now revealed why sires harboring the variant in the ABHD16B gene are infertile.

Shotgun metagenomic analysis of kombucha mutualistic community exposed to Mars-like environment outside the International Space Station.

Kombucha is a multispecies microbial ecosystem mainly composed of acetic acid bacteria and osmophilic acid-tolerant yeasts, which is used to produce a probiotic drink. Furthermore, Kombucha Mutualistic Community (KMC) has been recently proposed to be used during long space missions as both a living functional fermented product to improve astronauts' health and an efficient source of bacterial nanocellulose. In this study, we compared KMC structure and functions before and after samples were exposed to the space/Mars-like environment outside the International Space Station in order to investigate the changes related to their re-adaptation to Earth-like conditions by shotgun metagenomics, using both diversity and functional analyses of Community Ecology and Complex Networks approach. Our study revealed that the long-term exposure to space/Mars-like conditions on low Earth orbit may disorganize the KMC to such extent that it will not restore the initial community structure; however, KMC core microorganisms of the community were maintained. Nonetheless, there were no significant differences in the community functions, meaning that the KMC communities are ecologically resilient. Therefore, despite the extremely harsh conditions, key KMC species revived and provided the community with the genetic background needed to survive long periods of time under extraterrestrial conditions.

Sperm Lipid Markers of Male Fertility in Mammals.

Sperm plasma membrane lipids are essential for the function and integrity of mammalian spermatozoa. Various lipid types are involved in each key step within the fertilization process in their own yet coordinated way. The balance between lipid metabolism is tightly regulated to ensure physiological cellular processes, especially referring to crucial steps such as sperm motility, capacitation, acrosome reaction or fusion. At the same time, it has been shown that male reproductive function depends on the homeostasis of sperm lipids. Here, we review the effects of phospholipid, neutral lipid and glycolipid homeostasis on sperm fertilization function and male fertility in mammals.

An RNA-Seq-Based Framework for Characterizing Canine Prostate Cancer and Prioritizing Clinically Relevant Biomarker Candidate Genes.

Prostate cancer (PCa) in dogs is a highly malignant disease akin to its human counterpart. In contrast to the situation in humans, multi-gene approaches facilitating risk stratification of canine PCa are barely established. The aims of this study were the characterization of the transcriptional landscape of canine PCa and the identification of diagnostic, prognostic and/or therapeutic biomarkers through a multi-step screening approach. RNA-Sequencing of ten malignant tissues and fine-needle aspirations (FNA), and 14 nonmalignant tissues and FNAs was performed to find differentially expressed genes (DEGs) and deregulated pathways. The 4098 observed DEGs were involved in 49 pathways. These 49 pathways could be grouped into five superpathways summarizing the hallmarks of canine PCa: (i) inflammatory response and cytokines; (ii) regulation of the immune system and cell death; (iii) cell surface and PI3K signaling; (iv) cell cycle; and (v) phagosome and autophagy. Among the highly deregulated, moderately to strongly expressed DEGs that were members of one or more superpathways, 169 DEGs were listed in relevant databases and/or the literature and included members of the PCa pathway, oncogenes, prostate-specific genes, and druggable genes. These genes are novel and promising candidate diagnostic, prognostic and/or therapeutic canine PCa biomarkers.

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells.

Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3ß, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

Intra-Protein Coevolution Is Increasingly Functional with Greater Proximity to Fertilization.

Intramolecular coevolution of amino acid sites has repeatedly been studied to improve predictions on protein structure and function. Thereby, the focus was on bacterial proteins with available crystallographic data. However, intramolecular coevolution has not yet been compared between protein sets along a gradient of functional proximity to fertilization. This is especially true for the potential effect of external selective forces on intraprotein coevolution. In this study, we investigated both aspects in equally sized sets of mammalian proteins representing spermatozoa, testis, entire body, and liver. For coevolutionary analyses, we derived the proportion of covarying sites per protein from amino acid alignments of 10 mammalian orthologues each. In confirmation of the validity of our coevolution proxy, we found positive associations with the nonsynonymous or amino acid substitution rate in all protein sets. However, our coevolution proxy negatively correlated with the number of protein interactants (node degree) in male reproductive protein sets alone. In addition, a negative association of our coevolution proxy with protein hydrophobicity was significant in sperm proteins only. Accordingly, the restrictive effect of protein interactants was most pronounced in male reproductive proteins, and the tendency of sperm proteins to form internal structures decreased the more coevolutionary sites they had. Both aspects illustrate that the share of outward and thus functional coevolution increases with greater proximity to fertilization. We found this conclusion confirmed by additional comparisons within sperm proteins. Thus, sperm proteins with high hydrophobicity had the lowest proportions of covarying sites and, according to gene annotations, localized more frequently to internal cellular structures. They should therefore be less exposed to postcopulatory forms of sexual selection. Their counterparts with low hydrophobicity had larger proportions of covarying sites and more often resided at the cell membrane or were secreted. At the cellular level, they are thus closer to externally induced forces of postcopulatory selection which are known for their potential to increase substitution rates. In addition, we show that the intermediary status of the testicular protein set in correlation analyses is probably due to a special combination of reproductive and somatic involvements.

Using ITS2 metabarcoding and microscopy to analyse shifts in pollen diets of honey bees and bumble bees along a mass-flowering crop gradient.

Worldwide pollinator declines lead to pollination deficits in crops and wild plants, and managed bees are frequently used to meet the increasing demand for pollination. However, their foraging can be affected by flower availability and colony size. We investigated how mass-flowering oilseed rape (OSR) can influence the pollen resource use of small and large honey bee (Apis mellifera L.) and bumble bee (Bombus terrestris L.) colonies. Colonies were placed adjacent to strawberry fields along a gradient of OSR availability in the landscapes. We used ITS2 metabarcoding to identify the pollen richness based on ITS2 amplicon sequencing and microscopy for quantification of target pollen. Bumble bees collected pollen from more different plant genera than honey bees. In both species, strawberry pollen collection decreased with high OSR availability but was facilitated by increasing strawberry flower cover. Colony size had no effect. The relationship between next-generation sequencing-generated ITS2 amplicon reads and microscopic pollen counts was positive but pollen type-specific. Bumble bees and, to a lesser degree, honey bees collected pollen from a wide variety of plants. Therefore, in order to support pollinators and associated pollination services, future conservation schemes should sustain and promote pollen plant richness in agricultural landscapes. Both bee species responded to the availability of flower resources in the landscape. Although honey bees collected slightly more strawberry pollen than bumble bees, both can be considered as crop pollinators. Metabarcoding could provide similar quantitative information to microscopy, taking into account the pollen types, but there remains high potential to improve the methodological weaknesses.

Establishment and characterization of stable red, far-red (fR) and near infra-red (NIR) transfected canine prostate cancer cell lines.

BACKGROUND: Canine prostate cancer represents a unique model for human prostate cancer. In vitro systems offer various possibilities but Xenograft in vivo imaging allows studying complex tasks as tumor progression and drug intervention longitudinal. Herein, we established three canine prostate carcinoma cell lines stably expressing fluorescent proteins allowing deep tissue in vivo imaging. METHODS: Three canine prostate carcinoma (cPC) cell lines were stably transfected with fluorescent proteins in red, far-red and near infra-red spectrum, followed by G418 selection. Fluorescent protein expression was demonstrated by microscopy, flow cytometry and a NightOWL LB 983 in vivo imaging system. Cellular and molecular characteristics of the generated cell lines were compared to the parental cell line CT1258. Cell proliferation, metabolic activity and sphere formation capacity were analyzed. Stem cell marker expression was examined by qPCR and genomic copy number variation by genomic DNA whole genome sequencing. RESULTS: Three stably fluorescent protein transfected cPC cell lines were established and characterized. Compared to the parental cell line, no significant difference in cell proliferation and metabolic activity were detected. Genomic copy number variation analyses and stem cell marker gene expression revealed in general no significant changes. However, the generated cell line CT1258-mKate2C showed uniquely no distal CFA16 deletion and an elevated metabolic activity. The introduced fluorescencent proteins allowed highly sensitive detection in an in vivo imaging system starting at cell numbers of 0.156 × 106. Furthermore, we demonstrated a similar sphere formation capacity in the fluorescent cell lines. Interestingly, the clone selected CT1258-mKate2C, showed increased sphere formation ability. DISCUSSION: Starting from a well characterized cPC cell line three novel fluorescent cell lines were established showing high cellular and molecular similarity to the parental cell line. The introduction of the fluorescent proteins did not alter the established cell lines significantly. The red fluorescence allows deep tissue imaging, which conventional GFP labeling is not able to realize. CONCLUSION: As no significant differences were detected between the established cell lines and the very well characterized parental CT1258 the new fluorescent cell lines allow deep tissue in vivo imaging for perspective in vivo evaluation of novel therapeutic regimens.

Correction to: Unexpected high frequency of neurofibroma in the celiac ganglion of German cattle.

An amendment to this paper has been published and can be accessed via the original article.

Association of α/ß-Hydrolase D16B with Bovine Conception Rate and Sperm Plasma Membrane Lipid Composition.

We have identified a Holstein sire named Tarantino who had been approved for artificial insemination that is based on normal semen characteristics (i.e., morphology, thermoresistance, motility, sperm concentration), but had no progeny after 412 first inseminations, resulting in a non-return rate (NRdev) of -29. Using whole genome association analysis and next generation sequencing, an associated nonsense variant in the α/ß-hydrolase domain-containing 16B gene (ABHD16B) on bovine chromosome 13 was identified. The frequency of the mutant allele in the German Holstein population was determined to be 0.0018 in 222,645 investigated cattle specimens. The mutant allele was traced back to Whirlhill Kingpin (bornFeb. 13th, 1959) as potential founder. The expression of ABHD16B was detected by Western blotting and immunohistochemistry in testis and epididymis of control bulls. A lipidome comparison of the plasma membrane of fresh semen from carriers and controls showed significant differences in the concentration of phosphatidylcholine (PC), diacylglycerol (DAG), ceramide (Cer), sphingomyelin (SM), and phosphatidylcholine (-ether) (PC O-), indicating that ABHD16B plays a role in lipid biosynthesis. The altered lipid contents may explain the reduced fertilization ability of mutated sperms.

Genetic mechanism underlying sexual plasticity and its association with colour patterning in zebrafish (Danio rerio).

BACKGROUND: Elevated water temperature, as is expected through climate change, leads to masculinization in fish species with sexual plasticity, resulting in changes in population dynamics. These changes are one important ecological consequence, contributing to the risk of extinction in small and inbred fish populations under natural conditions, due to male-biased sex ratio. Here we investigated the effect of elevated water temperature during embryogenesis on sex ratio and sex-biased gene expression profiles between two different tissues, namely gonad and caudal fin of adult zebrafish males and females, to gain new insights into the molecular mechanisms underlying sex determination (SD) and colour patterning related to sexual attractiveness. RESULTS: Our study demonstrated sex ratio imbalances with 25.5% more males under high-temperature condition, resulting from gonadal masculinization. The result of transcriptome analysis showed a significantly upregulated expression of male SD genes (e.g. dmrt1, amh, cyp11c1 and sept8b) and downregulation of female SD genes (e.g. zp2.1, vtg1, cyp19a1a and bmp15) in male gonads compared to female gonads. Contrary to expectations, we found highly differential expression of colour pattern (CP) genes in the gonads, suggesting the 'neofunctionalisation' of those genes in the zebrafish reproduction system. However, in the caudal fin, no differential expression of CP genes was identified, suggesting the observed differences in colouration between males and females in adult fish may be due to post-transcriptional regulation of key enzymes involved in pigment synthesis and distribution. CONCLUSIONS: Our study demonstrates male-biased sex ratio under high temperature condition and support a polygenic SD (PSD) system in laboratory zebrafish. We identify a subset of pathways (tight junction, gap junction and apoptosis), enriched for SD and CP genes, which appear to be co-regulated in the same pathway, providing evidence for involvement of those genes in the regulation of phenotypic sexual dimorphism in zebrafish.

Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

Phenotypic plasticity induced using high ambient temperature during embryogenesis in domesticated zebrafish, Danio rerio.

Ambient temperature during early stages of life has a substantial effect on physiological processes, eliciting phenotypic plasticity during zebrafish developmental stages. Zebrafish are known to possess a noteworthy ability to modify their phenotype in dependence of environmental factors. However, there is a poor understanding of the effects of temperature during embryogenesis, which influences the biological functions such as survival ability and masculinization in later developmental stages. Since the middle embryonic phase (pharyngula period) is genetically the most conserved stage in embryogenesis, it is very susceptible to embryonic lethality in developmental processes of vertebrates. Here, we tested the effect of transient perturbations (heat shock) during early development (5-24 hr post-fertilization; hpf) at 35°C compared to control group at 28°C, on survival ability of zebrafish to study the embryonic and post-embryonic mortality. We studied the variation of heat-induced masculinization among and across the families in response to high temperature. Furthermore, morphometric traits of adult zebrafish at different developmental time points were measured in order to estimate the temperature × sex interaction effect. We found the highest embryonic mortality around the gastrula and segmentation periods in both experimental groups, with significantly lower survival ability in the temperature-treated group (73.30% ± 0.58% vs. 70.19% ± 0.57%, respectively). A higher hatching success was observed in the control group (71.08% ± 0.61%) compared to the heat-induced group (67.95% ± 0.60%). A distinct reduction in survival ability was also observed in both experimental groups during the first two weeks after hatching, followed by a reduced level of changes thereafter. We found sex ratio imbalances across all families, with 25.2% more males under temperature treatment. Our study on growth performance has shown a positive effect of increased temperature on growth plasticity, with a greater impact on female fish in response to high ambient temperature.

Genetic diversity analysis of Thai indigenous pig population using microsatellite markers

Objective: European pigs have been imported to improve the economically important traits of Thai pigs by crossbreeding and was finally completely replaced. Currently Thai indigenous pigs are particularly kept in a small population. Therefore, indigenous pigs risk losing their genetic diversity and identity. Thus, this study was conducted to perform large-scale genetic diversity and phylogenetic analyses on the many pig breeds available in Thailand. Methods: Genetic diversity and phylogenetics analyses of 222 pigs belonging to Thai native pigs (TNP), Thai wild boars (TWB), European commercial pigs, commercial crossbred pigs, and Chinese indigenous pigs were investigated by genotyping using 26 microsatellite markers. Results: The results showed that Thai pig populations had a high genetic diversity with mean total (TNA) and effective (Ne) number of alleles of 14.59 and 3.71, respectively, and expected heterozygosity (He) across loci (0.710). The polymorphic information content (PIC) per locus ranged between 0.651 and 0.914 leading to an average value above all loci of 0.789, and private alleles were found in six populations. The higher He compared to Ho in TNP, TWB, and the commercial pigs indicated some inbreeding within a population. The Nei's genetic distance, mean FST estimates, neighbour-joining tree of populations and individual, as well as multidimensional analysis indicated close genetic relationship between Thai indigenous pigs and some Chinese pigs, and they are distinctly different from European pigs. Conclusion: Our study reveals a close genetic relationship between Thai native pigs and Chinese pigs. The genetic introgression from European breeds is found in some Thai native pig populations, and signs of genetic erosion are shown. Private alleles found in this study should be taken into consideration for the breeding program. The genetic information from this study will be a benefit for both conservation and utilization of Thai pig genetic resources.