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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.

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-1beta- 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-1beta 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.