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MicroRNAs (miRNAs) and mRNAs establish a complex regulatory network influencing diverse biological pathways including muscle development and growth. Elucidating miRNA-dependent regulatory networks involved in muscle development could provide additional insights into muscle traits largely predefined during prenatal development. The present study aimed to determine differentially expressed transcripts and functional miRNA- mRNA relationships associated with different stages of skeletal muscle development in two pig breeds, German Landrace and Pietrain, distinct in muscle characteristics. A comparative transcriptional profiling of longissimus dorsi muscle tissues from fetuses at 35, 63 and 91 days post-conception as well as adult pigs (180 days postnatum) was performed using the Affymetrix GeneChip porcine genome microarray. Differential expression patterns were identified to be associated with muscularly developmental stages and breed types. The integration of miRNA expression data and ingenuity pathways analysis (ipa) pathway analysis revealed several miRNA-dependent regulatory networks related to muscle growth and development. The present results provide insights into muscle biology for further improvement of porcine meat quality. [ABSTRACT FROM AUTHOR]

Recently, the incidence of canine infection by the tick-borne parasites Babesia spp., Hepatozoon canis , Ehrlichia canis and Anaplasma platys has been increasing globally. We have developed a multiplex high-resolution melting analysis (mHRM) technique to reduce the time demands and costs associated with detecting haemoparasites in canine blood, while increasing the degree of reliability of this method of analysis. We have designed primers that are specific for protozoans (B. vogeli and H. canis) and Rickettsia -like bacteria (E. canis and A. platys) based on the 18S or 16S rDNA sequences, respectively. Two primer pairs (Protz18S-C and Bact16S-A) were found to be suitable for detecting these agents since their melting temperatures (T m) exhibited discernible differences among the four haemoparasites, A. platys , B. vogeli , E. canis and H. canis (83.10 °C, 82.41 °C, 80.37 °C and 78.56 °C, respectively). The sequences acquired from these PCR products were >94 % identical to those of A. platys , B. vogeli , E. canis and H. canis in GenBank. The limit of detection (LOD) for B. vogeli , E. canis and A. platys was 103 copies/μl, while the LOD for H. canis was 104 copies/μl. Of the 68 dogs tested, 28 (41 %) were infected with these agents. The most commonly occurring infection involved E. canis , followed by B. vogeli , A. platys and H. canis , with infection percentages of 26 %, 13 %, 7 % and 6 %, respectively. These results demonstrate that mHRM can serve as a rapid, economical and reliable tool for the detection of parasitic diseases in canine blood for diagnosis and epidemiology. [ABSTRACT FROM AUTHOR]

Numbers of wild Asian elephants (Elephas maximus) have been decreasing gradually throughout Asia due primarily to human activities, such as poaching, and habitat encroachment and destruction that lead to human–elephant conflict. Sustainability problems exist in captive populations as well, where morbidity and mortality rates are high and reproduction is low. Determining the genetic diversity of these populations is essential for conservation and sustainable utilization efforts. Intersimple sequence repeat markers were used to assess the genetic variation and differentiation in 97 captive Asian elephants from seven elephant camps in Chiang Mai, Thailand. The nine primers chosen for the analysis revealed 88 bands in male and 115 bands in female elephants, of which 37 (42.05%) and 83 (63.64%) were polymorphic, respectively. Shannon's index information (I = 2.415 ± 0.054) and expected heterozygosity (He = 0.892 ± 0.008) indicated high species-level genetic diversity. The fixation index (Fst) was −0.130 ± 0.016, demonstrating there was no genetic subdivision between populations. A cluster analysis was performed using Unweight Pair-Group Method with Arithmetic Mean and dendrograms, which illustrated genetic relationships among captive Asian elephants that included 2 main clusters across the seven camps and 27 clusters for the 97 individual elephants. This high variability may be due to the different origins of these individuals, including originating from other Asian countries. Thus, this study showed that intersimple sequence repeat marker analysis was effective in demonstrating high genetic diversity among captive Asian elephants in Chiang Mai province and found cluster differences that could be used to guide breeding management to decrease the risk of inbreeding among Asian elephant groups. [ABSTRACT FROM AUTHOR]

Abstract Background Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the MMP-3 gene in in vitro human chondrosarcoma using siRNA. Methods Cells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and MMP-3 siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR). Results In IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (p < 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (p < 0.05). MMP-3 gene expression was downregulated significantly (p < 0.05). Conclusion MMP-3 specific siRNA can inhibit the expression of MMP-3 in chondrosarcoma. This suggests that MMP-3 siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.

Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell's metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including KLF1, NFATC3, ZNF148, ASCL1, FOXI1 , and KLF5 . These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation.

Hepatozoonosis, caused by the protozoan Hepatozoon canis, is a prevalent blood disease affecting owned and stray dogs and cats. The prevalence of these parasites among companion animals in Thailand remains poorly understood. Diagnosing the old-world form of the disease is challenging due to the wide range of nonspecific clinical signs and the reliance on finding low levels of Hepatozoon gamonts in blood smears for conventional diagnosis. PCR demonstrates high specificity and sensitivity but it requires sophisticated instrumentation. Therefore, we established recombinase polymerase amplification (RPA) coupled with Cas12a for H. canis detection based on 18S rRNA. Our findings showed that RPA-Cas12a using gRNA&#95;H was highly specific to H. canis, without yielding positives for other pathogen species including Babesia species. Even in cases of co-infection, RPA-Cas12a only detected positives in samples containing H. canis. This approach detected minimal amounts of H. canis18S rRNA-harboring plasmid at 10 copies per reaction, whereas plasmid-spiked canine blood enabled detection at a minimal amount of 100 copies per reaction. The performance of RPA-Cas12a was validated by comparing it with quantitative PCR-high resolution melting analysis (qPCR-HRM) and sequencing based on 35 canine blood samples. RPA-Cas12a demonstrated precision and accuracy values of 94 % and 90 %, respectively comparable to qPCR-HRM. Overall, these results indicate that RPA-Cas12a serves as a promising tool for H. canis detection as indicated by comparable performance to qPCR-HRM and is suitable for implementation in small animal hospitals or clinics due to its minimal resource requirements, thereby contributing to effective diagnosis and treatment for infected dogs., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kittisak Buddhachat reports financial support was provided by National Research Council of Thailand (NRCT), Thailand. Korakot Nganvongpanit reports a relationship with Excellence Center in Veterinary Bioscience, Chiang Mai University, Chiang Mai, Thailand that includes: board membership and employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rapid climate change is associated with frequent extreme heat events and the resulting thermal stress has consequences for the health, welfare, and growth of farm animals. The aim of this study was to characterize the transcriptional changes and the effects on energy metabolism in proliferating porcine myoblasts derived from piglets of different ages, representing differences in thermoregulatory abilities, and cultivated below (35°C) and above (39°C, 41°C) the standard cultivation temperature (37°C). Satellite cells originating from Musculus rhomboideus of piglets isolated on days 5 (P5, thermolabile) and 20 (P20, thermostable) of age were used. Our expression analyses highlighted differentially expressed genes in porcine myoblasts cultures under heat or cold induced stress. These gene sets showed enrichment for biological processes and pathways related to organelle fission, cell cycle, chromosome organization, and DNA replication. Culture at 35°C resulted in increased metabolic flux as well as a greater abundance of transcripts of the cold shock protein-encoding gene RBM3 and those of genes related to biological processes and signaling pathways, especially those involving the immune system (cytokine-cytokine receptor interaction, TNF and IL-17 signaling pathways). For cultivation at 39°C, differences in the expression of genes related to DNA replication and cell growth were identified. The highest glutathione index ratio was also found under 39°C. Meanwhile, cultivation at 41°C induced a heat stress response, including the upregulation of HSP70 expression and the downregulation of many biological processes and signaling pathways related to proliferative ability. Our analysis also identified differentially expressed genes between cells of donors with a not yet (P5) and already fully developed (P20) capacity for thermoregulation at different cultivation temperatures. When comparing P5 and P20, most of the changes in gene expression were detected at 37°C. At this optimal temperature, muscle cells can develop to their full capacity. Therefore, the most diverse molecular signaling pathways, including PI3K-Akt signaling, Wnt signaling, and EGFR tyrosine kinase inhibitor, were found and are more pronounced in muscle cells from 20-day-old piglets. These results contribute to a better understanding of the mechanisms underlying the adaptation of skeletal muscle cells to temperature stress in terms of their thermoregulatory ability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Metzger, Kalbe, Siengdee and Ponsuksili.)

Daily and seasonal temperature fluctuations are inevitable due to climate change, which highlights the importance of studying the detrimental effects of temperature fluctuations on the health, productivity, and product quality of farm animals. Muscle membrane composition and the molecular signals are vital for muscle cell differentiation and muscle growth, but their response to temperature stress is not well characterized. Temperature changes can lead to modification of membrane components of the cell, which may affect its surroundings and intracellular signaling pathways. Using C2C12 myoblast cells as a model of skeletal muscle development, this study was designed to investigate the effects of high temperature (39 °C and 41 °C) and low temperature (35 °C) on molecular pathways in the cells as well as the cell membrane fatty acid composition. Our results show that several genes were differentially expressed in C2C12 cells cultured under heat or cold stress, and these genes were enriched important KEGG pathways including PI3K-Akt signaling pathway, lysosome and HIF- signaling pathway, Wnt signaling pathway and AMPK signaling pathway. Our analysis further reveals that several membrane transporters and genes involved in lipid metabolism and fatty acid elongation were also differentially expressed in C2C12 cells cultured under high or low temperature. Additionally, temperature stress shifts the fatty acid composition in the cell membranes, including the proportion of saturated, monounsaturated and polyunsaturated fatty acids. This study revealed an interference between fatty acid composition in the membranes and changing molecular pathways including lipid metabolism and fatty acids elongation mediated under thermal stress. These findings will reinforce a better understanding of the adaptive mechanisms in skeletal muscle under temperature stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Temperature stress is one of the main environmental stressors affecting the welfare, health and productivity of livestock. Temperature changes can modify cell membrane components, disrupting the crosstalk between the cell and its surroundings by affecting signaling pathways including Wnt signaling pathway, which subsequently disrupts cell energy metabolism. The present study aims to understand the effect of temperature stress on the expression of genes involved in Wnt signaling pathways, and their interaction with energy metabolism in C2C12 myoblasts cells. The C2C12 cells were exposed to cold stress (35 °C), mild heat stress (39 °C) and severe heat stress (41 °C), whereas 37 °C was used as control temperature. Transcript levels of important genes involved in Wnt signaling including Axin2, Tnks2, Sfrp1, Dkk1, Dact1, Cby1, Wnt5a, Wnt7a, Wnt11, Porcn, Ror2, Daam1 , and Ppp3ca were significantly altered under severe heat stress (41 °C), whereas eight Wnt signaling-related transcripts ( Daam1, Ppp3ca, Fzd7, Wnt5a, Porcn, Tnks2, Lrp6, and Aes ) were significantly altered under cold stress (35 °C) compared to control. Under heat stress transcripts of the Wnt/β-catenin inhibitors ( Sfrp1, Dkk1 , and Cby1 ) and negative regulators ( Dact1 and Axin2 ) are activated. A positive correlation between oxidative phosphorylation and Wnt-related transcripts was found under high temperatures. Transcripts of the cell membrane receptors, including Lrp6 and Fzd7 , and the members of Wnt/Ca +2 signaling pathway, including Ppp3ca and Porcn were downregulated under cold stress. Many Wnt signaling-related transcripts were positively correlated with glycolysis under cold stress. These findings indicate a cross-talk between Wnt signaling and energy metabolism under thermal stress., Competing Interests: The authors declare there are no competing interests., (©2021 Risha et al.)

Knowledge of gene expression profiles reflecting functional features and specific responsiveness of parathyroid glands (PTGs) contributes to understanding mineral homeostasis and parathyroid function in healthy and diseased conditions. The study aims to reveal effector molecules driving the maintenance of phosphorus (P) homeostasis and parathyroid hormone (PTH) responsiveness to variable P supply throughout fetal and postnatal life. In this study, a long-term dietary intervention was performed by keeping pig offspring on distinct mineral P levels throughout fetal and postnatal life. Respective adaptation processes of P homeostasis were assessed in mRNA profiles of PTGs and serum minerals. RNA sequencing data and resulting molecular pathways of PTGs showed that the PTH abundance is very strictly controlled via e.g., PIN1 , CaSR , MAfB , PLC and PKA signaling to regulate PTH expression, stability, and secretion. Additionally, the observed dietary effects on collagen expression indicate shifts in the ratio between connective tissue and parenchyma, thereby affecting cell-cell contacts as another line of PTH regulation. Taken together, the mRNA profiles of porcine PTGs reflect physiological responses in-vivo following variable dietary P supplies during fetal and postnatal life. The results serve to evaluate a long-term nutrition strategy with implications for improving the mineral balance in individuals with pathological disorders.

Polyunsaturated fatty acids (PUFAs) are the main components of cell membrane affecting its fluidity, signaling processes and play a vital role in muscle cell development. The effects of docosahexaenoic acid (DHA) on myogenesis are well known, while the effects of arachidonic acid (AA) are largely unclear. The purpose of this study is to evaluate the effect of two PUFAs (DHA and AA) on cell fate during myogenic processes, Wnt signaling and energy metabolism by using the C2C12 cells. The cells were treated with different concentrations of AA or DHA for 48 h during the differentiation period. PUFA treatment increased mRNA level of myogenic factor 5 ( Myf5 ), which is involved in early stage of myoblast proliferation. Additionally, PUFA treatment prevented myoblast differentiation, indicated by decreased myotube fusion index and differentiation index in parallel with reduced mRNA levels of myogenin ( MyoG ). After PUFA withdrawal, some changes in cell morphology and myosin heavy chain mRNA levels were still observed. Expression of genes associated with Wnt signaling pathway, and energy metabolism changed in PUFA treatment in a dose and time dependent manner. Our data suggests that PUFAs affect the transition of C2C12 cells from proliferation to differentiation phase by prolonging proliferation and preventing differentiation.

Synovial mesenchymal stem cells (SMSCs) have become a great cell source for musculoskeletal stem cell research, especially related to cartilage and bone tissue regeneration, due to their superior cell proliferation properties and multidifferentiation potential into various cell lineages. This study revealed isolation methods, culture conditions, and morphological and molecular characterization of SMSCs derived fibrous synovium (FS) and adipose synovium (FP) of two pig breeds differing in growth performance [German Landrace (DL), and fat deposition (Angeln Saddleback (AS)]. Herein, FS possessed nucleated cell numbers nearly twice as high as those of FP at Passage 0. SMSCs derived from different types of synovial membrane and genetic background show similar cell morphologies and immunophenotypes, which were assessed by cell surface epitopes and multilineage differentiation potential, but differ significantly in their molecular characteristics. In addition, transcripts of SMSCs from AS were more enriched in IGF-1 signaling and VEGF ligand receptor, while SMSCs from DL were more enriched in growth hormone signaling and bone metabolism. The results indicate that genetics and tissues play significant roles for SMSC characteristics so that SMSCs can be traced back to the original cell donor and be used for fine turning in applications of medical research and therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Siengdee, Oster, Reyer, Viergutz, Wimmers and Ponsuksili.)

The identification of differing physical characteristics of dogs is an uncomplicated and straightforward way to categorize dog breeds. However, many dog owners and veterinarians still struggle to distinguish between pure breed and mixed variations in certain breeds of dogs. Presently, the absence of the tools and methods needed to confirm a pure breed dog is a significant problem since the only method available to validate pure or mongrel breeds is the official pedigree system. Inter-simple sequence repeat markers have been successfully used to assess genetic variations and differentiations. Notably, inter-simple sequence repeat markers coupled with high resolution melting analysis were effectively used for the breed identification of 43 breeds of dogs (total 463 dogs). The 10 primers chosen for analysis resulted in a range of 31-78.6% of breed discrimination when using one primer, while a combination of two primers was able to successfully discriminate between all of the 43 dog breeds (100%). Shannon's index information ( I  = 2.586 ± 0.034) and expected heterozygosity ( H e  = 0.908 ± 0.003) indicated a high level of genetic diversity among breeds. The fixation index ( F st ) revealed a value of 10.4%, demonstrating that there was a high level of genetic subdivision between populations. This study showed that inter-simple sequence repeat marker analysis was effective in demonstrating high genetic diversity among varying breeds of dogs, while a combination of Inter-simple sequence repeat marker analysis and high resolution melting analysis could provide an optional technique for researchers to effectively identify breeds through genetic variations., Competing Interests: Korakot Nganvongpanit is an Academic Editor for PeerJ., (©2020 Kriangwanich et al.)

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1 , SMAD3 , IGSF10 , Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

A major concern associated with the use of drugs is their adverse side effects. Specific examples of the drugs of concern include antibiotic agents and non-steroidal anti-inflammatory drugs. Despite the presence of a high degree of efficacy for specific conditions, these drugs may deteriorate the surrounding tissues that are exposed to them. Often, carprofen is used for joint inflammation; however, it may stimulate cartilage degradation which can then lead to osteoarthritis progression. In this study, hyaluronan was combined with carprofen treatment in three different applications (pre-treatment, co-treatment and post-treatment) on normal canine chondrocytes to determine whether Hyaluronan (HA) is capable of mitigating the degree of chondrotoxicity of carprofen. Our findings revealed that carprofen at IC 20 (0.16 mg/mL) decreased viability and increased nitric oxide (NO) production. Importantly, carprofen induced the apoptosis of canine chondrocytes via the up-regulation of Bax , Casp3 , Casp8 , Casp9 and NOS2 as compared to the control group. Although the co-treatment of HA and carprofen appeared not to further alleviate the chondrotoxicity of carprofen due to the presence of a high number of apoptotic chondrocytes, post-treatment with HA (carprofen treatment for 24 h and then changed to HA for 24 h) resulted in a decrease in chondrocyte apoptosis by the down-regulation of Bax , Casp3 , Casp8 , Casp9 , NOS2 , along with NO production when compared with the treatment of carprofen for 48 h ( P < 0.05). These results suggest that HA can be used as a therapeutic agent to mitigate the degree of chondrotoxicity of carprofen., Competing Interests: The authors declare that they have no competing interests., (© 2020 Nganvongpanit et al.)

Background: Previous studies reported the effect of enrofloxacin (Enro) and marbofloxacin (Mar) on cell death and alteration of the key genes involved in catabolic and anabolic processes and demonstrated the beneficial effects of hyaluronan (HA) combined with fluoroquinolones (FQs) on primary canine chondrocytes. This study further determines the effects of these treatments on canine cartilage explants in both normal and interleukin-1 beta (IL-1β)-stimulated conditions., Methods: We examined sulfate glycosaminoglycan (s-GAG) release, uronic acid (UA) content, and safranin-O staining, as well as the expression patterns of inflammatory, extracellular matrix (ECM) component and enzymes., Results: Enro treatment alone effectively stimulated proteoglycan anabolism by increasing UA content and glycosaminoglycans (GAGs) in normal and pre-IL-1β-stimulated explant, whereas Mar showed opposite results. The combination of HA and FQs increased s-GAG release and UA content in normal explants in addition to effective down-regulated expression of MMP3 . HA reduced the adverse effects of Mar by enhancing UA and GAG contents in both normal and pre-IL-1β-explants. Moreover, HA effectively induced HAS1 and ACAN up-regulation and reduced MMP9, TNF, PTGS2, and NFKB1 expression for a long term., Discussion: Our results suggest the direct effects of Enro and Mar may selectively stimulate the conditioned explants to express MMP-codinggenes and promote gene expression involved in matrix production, pro-inflammatory cytokines, and cell degradation in different directions. HA successfully reduced the adverse effects of FQs by enhancing s-GAG and UA contents and down-regulated expression of MMPs., Competing Interests: The authors declare there are no competing interests.

Objective: Environmental stress induces disturbances in cell energy metabolism and may cause epigenetic modifications. This study aimed to understand the possible impact of temperature stress (35 °C, 39 °C and 41 °C, compared to control 37 °C) on energy metabolism and epigenetic modifications, such as DNA methylation and histone H4 acetylation, as well as its effects on the expression of genes responsible for epigenetic changes, in mouse skeletal myoblasts (C2C12 cells). Methods: The results showed significantly reduced maximal respiration and spare respiratory capacity under heat stress (39 °C and 41 °C), suggesting that mitochondrial functions were compromised under these conditions. The glycolytic capacity and glycolysis markedly increased following low-temperature stress (35 °C). The results suggested that, under cold stress, cells prefer glycolysis as a rapid compensatory mechanism to meet energy requirements for adaptive thermogenic response. Results: Epigenetic changes (histone H4 acetylation and global DNA methylation) were observed under both heat and cold stress. Among the genes coding for DNA methyltransferases, the Dnmt3a was significantly increased under high-temperature conditions (39 °C and 41 °C), while Dnmt1 expression was significantly increased at low temperature (35 °C), indicating that under these conditions the cells preferred maintenance of methylation to de novo methylation activity. An expression pattern similar to Dnmt3a was observed for Gcn5 , encoding for a histone acetyltransferase. The study revealed that temperature stress induced changes in the metabolic profiles, as well as epigenetic modifications, including the dynamics of the key enzymes. Conclusion: The results indicated the existence of crosstalk mechanisms between energy metabolism and epigenetics during cell stress response.

Aim: The objective of this study was to uncover new candidate genes related to patellar luxation (PL) in dogs to select for those with low susceptibility for breeding purposes., Materials and Methods: The inter simple sequence repeat (ISSR) technique was performed to construct DNA fingerprints of 61 Chihuahua dogs with PL and 30 healthy Chihuahua dogs. DNA polymorphisms were detected by comparing the sequences between the affected and unaffected dogs, using the pairwise alignments in MultAlin. Genotyping was performed using allele-specific polymerase chain reaction (AS-PCR). The association analysis of ISSR DNA fingerprints and genotypes or phenotypes was performed using the Chi-square ( χ 2 ) model and generalized linear model (GLM), respectively., Results: Two single nucleotide polymorphisms (SNPs), namely SNP1UBC811 (g.91175C>G) and SNP2UBC811 (g.92259T>C), were found in the intron of the Dystroglycan 1 ( DAG1 ) gene, which was obtained using the PL-related marker UBC811 primer (p=0.02), and genotyped by AS-PCR. When investigated using the GLM, g.91175C>G had a significant association with PL (p=0.0424), whereas g.92259T>C did not have such an association (p=0.0959)., Conclusion: DAG1 might be one of the genes related to PL in Chihuahuas and could aid the process of marker-assisted selection in genetic breeding for Chihuahua dogs without PL.

Background: Primary cultures from Asian elephants ( Elephas maximus ) allow scientists to obtain representative cells that have conserved most of their original characteristics, function, physiology and biochemistry. This technique has thus gained significant importance as a foundation for further cellular, cell biology and molecular research. Therefore, the aim of this study was to describe conditions for the successful establishment of primary adult fibroblasts from Asian elephant carcasses., Methods: Ear tissue sample collection from Asian elephant carcasses and our recommendations are given. We describe here a simple modified protocol for successful isolation and maintenance of primary adult fibroblasts from elephant ear skin. Ear samples from each individual (five 3 × 3 cm 2 pieces) were brought to the laboratory within 3 h after collection, kept in transportation medium at 0-4 °C. The ear tissues were prepared by a combination of 10% collagenase type II digestion procedure together with a simple explant procedure. Primary fibroblasts were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal calf serum (FCS) in a humidified atmosphere containing 5% CO 2 . After the third passage, fibroblasts were routinely trypsinized with 0.25% trypsin/EDTA and cultured in DMEM with 10% FCS at 37 °C and 5% CO 2 . Traditional cell counting method was used to measure cell viability and growth curve. Long-term storage of cells used freezing medium consisting of 40% FCS (v/v)., Results: We explored the most suitable conditions during sample collection (post-mortem storage time and sample storage temperature), which is the most important step in determining primary outgrowth. Our study successfully established and cultured primary adult skin fibroblasts obtained from post-mortem E. maximus ear skin tissues from six carcasses, with a success rate of around 83.3%. Outgrowth could be seen 4-12 days after explantation, and epithelial-like cells were found after 4-7 days of culture, while fibroblasts appeared at around day 7-10. The fibroblasts had viability and post-freezing recovery rates of around 97.3 ± 4.3% and 95.5 ± 7.3%, respectively, and doubling time was about 25 h (passage 6)., Discussion: To our knowledge, this report is the first to describe primary cell cultures derived from adult Asian elephant skin. Future studies should benefit from the information and useful suggestions herein, which may be used as a standard method for establishing primary skin fibroblast cultures in future experiments., Competing Interests: The authors declare there are no competing interests.

Mineralized tissues accumulate elements that play crucial roles in animal health. Although elemental content of bone, blood and teeth of human and some animal species have been characterized, data for many others are lacking, as well as species comparisons. Here we describe the distribution of elements in horn (Bovidae), antler (Cervidae), teeth and bone (humerus) across a number of species determined by handheld X-ray fluorescence (XRF) to better understand differences and potential biological relevance. A difference in elemental profiles between horns and antlers was observed, possibly due to the outer layer of horns being comprised of keratin, whereas antlers are true bone. Species differences in tissue elemental content may be intrinsic, but also related to feeding habits that contribute to mineral accumulation, particularly for toxic heavy metals. One significant finding was a higher level of iron (Fe) in the humerus bone of elephants compared to other species. This may be an adaptation of the hematopoietic system by distributing Fe throughout the bone rather than the marrow, as elephant humerus lacks a marrow cavity. We also conducted discriminant analysis and found XRF was capable of distinguishing samples from different species, with humerus bone being the best source for species discrimination. For example, we found a 79.2% correct prediction and success rate of 80% for classification between human and non-human humerus bone. These findings show that handheld XRF can serve as an effective tool for the biological study of elemental composition in mineralized tissue samples and may have a forensic application.

Background: Intra-articular injection of corticosteroids is used to treat the inflammatory pain of arthritis and osteoarthritis (OA), but our previous study found a deleterious effect of these steroids on chondrocyte cells. Hyaluronic acid (HA) injection has been suggested as a means to counteract negative side effects through replenishment of synovial fluid that can decrease pain in affected joints. To better understand the effects of corticosteroids on these processes, dexamethasone (Dex) and prednisolone (Pred) were administered to porcine cartilage explants at several concentrations with and without HA. We examined corticoid effects by determining sulfate-glycosaminoglycan (s-GAG) and uronic acid (UA) content of the explant media, and safranin-O staining of the cells. Analysis of lactate dehydrogenase (LDH) activity was conducted to assess cell cytotoxicity., Results: Dex treatment significantly reduced cellular cytotoxicity compared to the other treatment groups, especially with regards to the release of s-GAG, and protects against superficial proteoglycan damage. However, there was no difference between Pred and Dex, with and without HA, in the UA content remaining in porcine cartilage explants., Conclusions: The data suggest that combinations of Dex and Pred with HA did not have a significant effect on protection or enhancement of the articular cartilage matrix under the current conditions.

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.

miRNAs regulate the expression of target genes in diverse cellular processes and hence play important roles in physiological processes including developmental timing, patterning, embryogenesis, organogenesis, cell lineage, myogenesis and growth control. A comparative expression analysis of miRNAs expressed in the longissimus dorsi muscle at two prenatal stages (63 and 91 days post-conception (dpc)), and one adult stage (180 days post-natum) in both German Landrace (DL) and Pietrain (Pi) pig breeds was performed using a custom-designed array. During the prenatal stages, miR-199 and the miR-17 families were significantly up-regulated at 63 dpc, whereas miR-1 and miR-133a were overexpressed at 91 dpc. The abundance of several miRNAs was increased in the adult stage compared to 91 dpc including miR-1, miR-133, miR-22(a/b) and miR-29a. Some miRNAs were breed-specific, such as miR-199 and the miR-17 families which were all up-regulated in Pi pigs, while miR-133, miR-181 and miR-214 were up-regulated in DL pigs. Several pathways related to muscle development were enriched with predicted targets for the differentially expressed miRNAs. The dynamic expression and breed-associated regulation of porcine muscle miRNAs suggests a functional role for miRNA-mediated gene regulation during muscle development and phenotypic variations of muscle traits., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Understanding the genetic contributions behind skeletal muscle composition and metabolism is of great interest in medicine and agriculture. Attempts to dissect these complex traits combine genome-wide genotyping, expression data analyses and network analyses. Weighted gene co-expression network analysis (WGCNA) groups genes into modules based on patterns of co-expression, which can be linked to phenotypes by correlation analysis of trait values and the module eigengenes, i.e. the first principal component of a given module. Network hub genes and regulators of the genes in the modules are likely to play an important role in the emergence of respective traits. In order to detect common regulators of genes in modules showing association with meat quality traits, we identified eQTL for each of these genes, including the highly connected hub genes. Additionally, the module eigengene values were used for association analyses in order to derive a joint eQTL for the respective module. Thereby major sites of orchestrated regulation of genes within trait-associated modules were detected as hotspots of eQTL of many genes of a module and of its eigengene. These sites harbor likely common regulators of genes in the modules. We exemplarily showed the consistent impact of candidate common regulators on the expression of members of respective modules by RNAi knockdown experiments. In fact, Cxcr7 was identified and validated as a regulator of genes in a module, which is involved in the function of defense response in muscle cells. Zfp36l2 was confirmed as a regulator of genes of a module related to cell death or apoptosis pathways. The integration of eQTL in module networks enabled to interpret the differentially-regulated genes from a systems perspective. By integrating genome-wide genomic and transcriptomic data, employing co-expression and eQTL analyses, the study revealed likely regulators that are involved in the fine-tuning and synchronization of genes with trait-associated expression.

Background: Physiological processes aiding the conversion of muscle to meat involve many genes associated with muscle structure and metabolic processes. MicroRNAs regulate networks of genes to orchestrate cellular functions, in turn regulating phenotypes., Results: We applied weighted gene co-expression network analysis to identify co-expression modules that correlated to meat quality phenotypes and were highly enriched for genes involved in glucose metabolism, response to wounding, mitochondrial ribosome, mitochondrion, and extracellular matrix. Negative correlation of miRNA with mRNA and target prediction were used to select transcripts out of the modules of trait-associated mRNAs to further identify those genes that are correlated with post mortem traits., Conclusions: Porcine muscle co-expression transcript networks that correlated to post mortem traits were identified. The integration of miRNA and mRNA expression analyses, as well as network analysis, enabled us to interpret the differentially-regulated genes from a systems perspective. Linking co-expression networks of transcripts and hierarchically organized pairs of miRNAs and mRNAs to meat properties yields new insight into several biological pathways underlying phenotype differences. These pathways may also be diagnostic for many myopathies, which are accompanied by deficient nutrient and oxygen supply of muscle fibers.