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Abstract Background Unraveling the intricate and tightly regulated process of adipogenesis, involving coordinated activation of transcription factors and signaling pathways, is essential for addressing obesity and related metabolic disorders. The molecular pathways recruited by mesenchymal stem cells (MSCs) during adipogenesis are also dependent on the different sources of the cells and genetic backgrounds of donors, which contribute to the functional heterogeneity of the stem cells and consequently affect the developmental features and fate of the cells. Methods In this study, the alteration of transcripts during differentiation of synovial mesenchymal stem cells (SMSCs) derived from fibrous synovium (FS) and adipose synovial tissue (FP) of two pig breeds differing in growth performance (German Landrace (DL)) and fat deposition (Angeln Saddleback (AS)) was investigated. SMSCs from both tissues and breeds were stimulated to differentiate into adipocytes in vitro and sampled at four time points (day 1, day 4, day 7 and day 14) to obtain transcriptomic data. Results We observed numerous signaling pathways related to the cell cycle, cell division, cell migration, or cell proliferation during early stages of adipogenesis. As the differentiation process progresses, cells begin to accumulate intracellular lipid droplets and changes in gene expression patterns in particular of adipocyte-specific markers occur. PI3K-Akt signaling and metabolic pathways changed most during adipogenesis, while p53 signaling and ferroptosis were affected late in adipogenesis. When comparing MSCs from FS and FP, only a limited number of differentially expressed genes (DEGs) and enriched signaling pathways were identified. Metabolic pathways, including fat, energy or amino acid metabolism, were highly enriched in the AS breed SMSCs compared to those of the DL breed, especially at day 7 of adipogenesis, suggesting retention of the characteristic metabolic features of their original source, demonstrating donor memory in culture. In contrast, the DL SMSCs were more enriched in immune signaling pathways. Conclusions Our study has provided important insights into the dynamics of adipogenesis and revealed metabolic shifts in SMSCs associated with different cell sources and genetic backgrounds of donors. This emphasises the critical role of metabolic and genetic factors as important indications and criteria for donor stem cell selection.

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.

Abstract Synovial membrane mesenchymal stem cells (SMSCs) often serve as in vitro model for bone disease, but the molecular mechanisms driving osteogenesis in SMSCs from different donor cells of various sources and breeds remain unclear. In this study, porcine SMSCs isolated from adipose synovium (FP) and fibrous synovium (FS) of Angeln Saddleback (AS) and German Landrace (DL) were used to discover the signaling network change after osteogenic induction. During osteogenic differentiation, mineral deposition was first observed at day 14 and further increased until day 21. Transcriptional changes between day 1 and day 21 were enriched in several signaling pathways, including Wnt, PI3K-Akt, and TGF-beta pathway. Certain pathways related to osteogenesis, including osteoblast differentiation, regulation of bone mineralization, and BMP signaling pathway, were enriched at late time points, as confirmed by the osteogenic markers ALPL, COL1A1, and NANOG. A fraction of differentially expressed genes (DEGs) were found between FP and FS, while DEGs between AS and DL increased during the differentiation phase until day 7 and then decreased from day 14 to day 21. These genes are involved in several important signaling pathways, including TGF-beta, Wnt, and lipid-related signaling pathways, suggesting that SMSCs from these two breeds have different osteogenic capabilities.

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.

Abstract Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/μl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

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.

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.

Non-thermal plasma (NTP) is being developed for a wide-range of medical applications such as improvement of wound healing, elimination of infective microorganisms, and treatment of cancer. This study investigated the effect of culture medium exposed to NTP on the proliferation in-vitro of skin fibroblasts from Asian elephants. Dulbecco"s Modified Eagle"s Medium (DMEM) was used as culture medium and was exposed to NTP with three different intensities. The NTP reactive species Nitrite (NO2-) was measured in the treated medium before addition to cells. Fibroblasts were incubated for 24 h with NTP-treated complete medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic/antimycotic. Cell proliferation, the number of cells and viability rate were analysed using flow cytometry 24, 48 and 72 h after the start of the incubation. The proliferation rate of fibroblasts incubated with NTP treated medium was significantly higher (P

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.

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 (He = 0.908 ± 0.003) indicated a high level of genetic diversity among breeds. The fixation index (Fst) 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.

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 IC20 (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.

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 (χ2) 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.

This study conducted an investigation on three exons of the endothelin receptor type B (EDNRB) gene of Thai odd-eyed cats to find out the association between the variations in the gene and heterochromia iridis. DNA sequencing analysis was performed on 11 odd-eyed cats compared to 11 normal-eyed cats. Seven variations were found across the studied region (XM_003980457.2: c.610A>G, c.820+40C>T, c.821-14C>T, c.916A>G, c.1025+36G>T, c.1025+69A>T, and c.1025+138C>T) with two of them (c.610A>G and c.916A>G) causing amino acid changes (P.Asn128Ser and P.Val230Ala). There was no statistical association between the variations near the three exons of EDNRB and feline heterochromia iridis (chi-square test, P-value >0.05).

The oral cavity of dogs and cats is colonized by hundreds of bacterial species. Here, we describe the bacterial composition in the dental calculus of dogs and cats. Dental calculus samples from 43 dogs and 4 cats were pooled into four different groups. Dogs were categorized into three groups: non-small breed dogs (NSB), non-brachycephalic small breed dogs (SB) and brachycephalic small breed dogs (SBb). The fourth group included cats. Bacterial communities were identified based on 16S rRNA sequencing (V3 and V4 hypervariable regions) with the Illumina platform. The numbers of operational taxonomic units (OTUs) identified in the three groups of dogs were 180, 190 and 150 and in NSB, SBb and SB, respectively, while in cats there were 111 OTUs. In dental calculus from both dogs and cats, the phylum Firmicutes had the highest proportion of read number, especially the class Clostridia. PCoA and UPGMA analysis revealed differences in the microbiomes of canine and feline calculus. Our findings demonstrated that the bacterial communities in calculus seemed to differ from those in other sites of the oral cavity. Calculus may serve as a potential habitßat for the growth of bacteria linked to canine and feline periodontal disease.

The aims of this study were to investigate the coding sequence and the deduced amino acid sequence of Asian elephant"s biglycan gene as well as its expression in different tissues and conditions using wound healing as a model. The results showed that Asian elephant biglycan coding sequence was 1,110 base pair (bp) long (accession number: JQ753329), encoding 369 amino acids. The coding and amino acid sequences between Asian and African elephants revealed 99% and 98% similarity, respectively. The conserved domains of biglycan protein were also observed. In addition, its expression was found in 15 tissues with a predominant expression in cartilage and spleen. For expression analysis in the wound healing process, it was found that the level of biglycan mRNA was influenced by many factors, including age, type of wound and stage of wound healing.

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 HAS1and ACANup-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.

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.

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 cm2 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% CO2. 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% CO2. 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.

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.

The purpose of this study was to determine the effect of bromelain treatment on canine articular chondrocytes in vitro. This research evaluated cell viability, levels of apoptotis and mitotis, proteoglycan concentrations and the expression of certain genes. Chondrocytes were exposed to 50 μg/ml bromelain for 4, 16 and 32 h. The rate of apoptotis in the treatment groups was significantly lower than in the control groups that were incubated with media only (P < 0.05); and the mitotic rate in treatment groups was significantly higher than in the control groups (P < 0.05), at all durations of exposure. The effect of bromelain on gene expression was measured by the real-time PCR technique. It was found that bromelain significantly decreased (P < 0.05) TIMP-1 and MMP-3 expression. These experimental bromelain treatments have shown positive results, and have increased the basic knowledge in regard to the healing and modulation of osteoarthritis, prior to the general use of bromelain in clinical practice.

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.

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.

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]

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.

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.)

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.

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.)