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Simple Summary: This study aimed to identify stable reference genes in early-passage and late-passage cultured canine skin fibroblasts. The early-passage fibroblasts retained their spindle-shaped morphology, exhibited a short doubling time, and had low β-galactosidase activity. In contrast, the late-passage fibroblasts displayed an elongated morphology, a prolonged doubling time, and elevated β-galactosidase activity. To assess the stability of the reference genes, the Ct values obtained using qRT-PCR were analyzed using three algorithms: geNorm, NormFinder, and BestKeeper. As a result, HPRT1, YWHAZ, and GUSB were identified as the most stable reference genes across all three algorithms in canine skin fibroblasts. When comparing early-passage to late-passage fibroblasts, the normalization of Vimentin expression using both stable and unstable reference genes showed a decrease in late-passage cells. Although the use of less stable reference genes did not result in a significant difference in Vimentin expression, the use of stable reference genes revealed a significant difference. This study provides a foundation for the further application of RT-qPCR in the gene expression analysis of long-term expanded canine skin fibroblasts. Fibroblasts are cells that reside within the fibrous or loose connective tissues of most mammalian organs. For research purposes, fibroblasts are often subjected to long-term culture under defined conditions, during which their properties can significantly change. It is essential to understand and document these changes to obtain reliable outcomes. For the quantification of specific gene expressions, the most reliable and widely used technique is quantitative real-time polymerase chain reaction (qRT-PCR). Here, we assessed the impact of a reference gene's stability on a qRT-PCR analysis of long-term cultured canine skin fibroblasts. After successfully isolating the fibroblasts from canine skin tissues, they were cultured and evaluated for proliferation and β-galactosidase activity at different passage numbers. With extended culture, the fibroblasts showed a long doubling time and elevated β-galactosidase activity. Using three widely used algorithms, geNorm, Normfinder, and Bestkeeper, we identified HPRT1, YWHAZ, and GUSB as the most stable reference genes for both early- and late-passage fibroblasts. Conventional reference genes such as GAPDH were found to be less stable than those genes. The normalization of Vimentin by the stable genes showed statistical differences, whereas normalization by an unstable gene did not. Collectively, this study indicates that using stable reference genes is essential for accurately and reliably measuring gene expression in both early- and late-passage fibroblasts. These findings provide valuable insights into internal controls for gene expression studies and are expected to be utilized for analyzing gene expression patterns in molecular biology research. [ABSTRACT FROM AUTHOR]

Simple Summary: This study focused on understanding how radiation therapy, a common treatment for cancer, affects the skin of dogs. While radiation is effective at destroying cancer cells, it can also cause damage to healthy skin, leading to various side effects like redness, peeling, changes in skin color, and sores. Over nine weeks, we monitored these skin changes in dogs, observing that radiation led to increased inflammation and stress in the skin cells, as well as significant disruptions in how skin cells grow and heal. We also noticed changes in specific proteins and genes related to skin inflammation, healing, and cell death. These findings help us better understand how radiation therapy impacts the skin and provide valuable information for managing these side effects in dogs. Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX–2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX–2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy. [ABSTRACT FROM AUTHOR]

Studies on somatic mutations in cloned animals have revealed slight genetic variances between clones and their originals, but have yet to identify the precise effects of these differences within the organism. Somatic mutations contribute to aging and are implicated in tumor development and other age-related diseases. Thus, we compared whole genome sequencing data from an original dog with that of cloned dogs, identifying candidate somatic mutations that were disproportionately located within genes previously implicated in aging. The substitutional signature of cloning-specific somatic mutations mirrored the uniform distribution characteristic of the signature associated with human aging. Further analysis of genes revealed significant enrichment of traits associated with body size as well as the molecular mechanisms underlying neuronal function and tumorigenesis. Overall, the somatic mutations found in cloned dogs may indicate a conserved mechanism driving aging across species and a broad spectrum of pathway alterations. [ABSTRACT FROM AUTHOR]

Myxomatous mitral valve disease (MMVD) is the most common cardiovascular disorder in dogs with a high prevalence, accounting for approximately 75% of all canine heart disease cases. MMVD is a complex disease and shows variable progression from mild valve leakage to severe regurgitation, potentially leading to heart failure. However, the molecular mechanisms and age-related changes that govern disease progression, especially at the early stage (B1) before the development of discernable clinical signs, remain poorly understood. In this prospective study, we aimed to compare gene expression differences between blood samples of aged beagle dogs with stage B1 MMVD and those of healthy controls using RNA sequencing. Clinical evaluation was also conducted, which revealed minimal differences in radiographic and echocardiographic measurements despite distinct biomarker variations between the two groups. Comparative transcriptomics revealed differentially expressed genes associated with extracellular matrix remodeling, prostaglandin metabolism, immune modulation, and interferon-related pathways, which bear functional relevance for MMVD. In particular, the top 10 over- and under-expressed genes represent promising candidates for influencing pathogenic changes in MMVD stage B1. Our research findings, which include identified variations in clinical markers and gene expression, enhance our understanding of MMVD. Furthermore, they underscore the need for further research into early diagnosis and treatment strategies, as, to the best of our knowledge, no prior studies have explored the precise molecular mechanisms of stage B1 in MMVD through total RNA sequencing. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1β) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1β but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton plays a crucial role not only in maintaining cell shape and viability but also in homing/engraftment properties of mesenchymal stem cells (MSCs), a valuable source of cell therapy. Therefore, during the cryopreservation process of MSCs, protecting the actin cytoskeleton from the freezing/thawing stress is critical in maintaining their functionality and therapeutic potential. In this study, the safety and cryoprotective potential of sphingosine-1-phosphate (S1P), which has a stabilizing effect on actin cytoskeleton, on dental pulp-derived MSCs (DP-MSCs) was investigated. Our results demonstrated that S1P treatment did not adversely affect viability and stemness of DP-MSCs. Furthermore, S1P pretreatment enhanced cell viability and proliferation properties of post-freeze/thaw DP-MSCs, protecting them against damage to the actin cytoskeleton and adhesion ability as well. These findings suggest that a new cryopreservation method using S1P pretreatment can enhance the overall quality of cryopreserved MSCs by stabilizing the actin cytoskeleton and making them more suitable for various applications in regenerative medicine and cell therapy. [ABSTRACT FROM AUTHOR]

Reference genes are crucial in molecular biological studies as an internal control for gene re-search as they exhibit consistent expression patterns across many tissue types. In canines, radiation therapy is the most important therapeutic tool to cure various diseases like cancer. However, when using radiation for therapeutic strategy, radiation exposure to healthy tissues leads to some possible side effects such as acute radiation-induced skin injury and alters gene expression. Therefore, the analysis of a change in reference gene expression during the skin recovery process after radiation therapy is essential in healthy canine tissue. In the present study, we analyzed eight reference genes (ACTB, GAPDH, YWHAZ, GUSB, HPRT1, RPL4, RPS5, and TBP) in canine dermal tissues at 0, 1, 2, 3, 4, 5, 7, and 9 weeks of radiation exposure that affected the skin condition of canines. The stability of reference genes is determined by evaluating radiation therapy's effect on healthy canine dermal tissue. Epidermal marker, Keratin 10 expression varies each week after irradiation, and HPRT1 is found to be the most suitable for normalization of mRNA expression in radiation-exposed canine dermal tissues. Changes in the gene expression level were evaluated by using a reliable tool such as quantitative real-time polymerase chain reaction (qRT-PCR). In order to achieve a valid qRT-PCR result, the most stable reference genes used for normalization after the radiation exposure process are important. Therefore, the current study was designed to evaluate the most stable reference gene for the post-irradiation canine tissues. After radiation exposure, the alternation of reference gene expression was estimated by three algorithms (geNorm, Normfinder, and Bestkeeper). The RG validation programs (GeNorm and NormFinder) suggested that HPRT1, RPL4, and TBP were suitable for normalization in qRT-PCR. Furthermore, three algorithms suggested that HPRT1 was the most stable reference gene for normalization with qRT-PCR results, regardless of before and after radiation exposure. Whereas GAPDH was found to be the most unstable reference gene. In addition, the use of stable or unstable reference genes for the normalization of Keratin 10 expression showed statistical differences. Therefore, we observed that, to obtain accurate and suitable PCR results of the canine tissues with and without radiation exposure, the HPRT1 reference gene is recommended for normalization with its high stability. Additionally, the use of RGs such as HPRT1, RPL4, and TBP for normalization in qRT-PCR experiments is recommended for post-radiation canine tissues to generate more accurate and reliable data. These results will provide fundamental information regarding internal controls for gene expression studies and can be used for the analysis of gene patterns in regenerative medicine. [ABSTRACT FROM AUTHOR]

Asthma is one of the most common inflammatory diseases of the lung worldwide. There has been considerable progress in recent studies to treat and prevent allergic asthma, however, various side effects are still observed in clinical practice. Six-week-old male BALB/c mice were orally administered with either sword bean pod extracts (SBP; 100 or 300 mg/kg) or dexamethasone (DEX; 5 mg/kg) once daily over 3 weeks, followed by ovalbumin sensitization (OVA/Alum.; intraperitoneal administration, 50 μg/2 mg/per mouse). Scoring of lung inflammation was performed to observe pathological changes in response to SBP treatment compared to OVA/Alum.-induced lung injury. Additionally, inflammatory cytokines were quantified in serum, bronchoalveolar lavage fluid (BALF), and lung tissue using ELISA and Western blot analyses. SBP treatment significantly reduced the infiltration of inflammatory cells, and release of histamine, immunoglobulin E, and leukotriene in serum and BALF. Moreover, the therapeutic effect of SBP was also assessed to analyze the inflammatory changes in the lung tissues. SBP markedly suppressed the activation of the MAPK signaling pathway and the expression of key inflammatory proteins (e.g., TNF-α) and Th2 type cytokines (IL-5 and IL-13). SBP was effective in ameliorating the allergic inflammation against OVA/Alum.-induced asthma by suppressing pulmonary inflammation. [ABSTRACT FROM AUTHOR]

Previous studies have shown that mesenchymal stem cells (MSCs) derived from various tissue sources can be differentiated into smooth muscle-like cells (SMLCs) in vitro. In this paper, dental pulp-derived mesenchymal stem cells (DPSCs) were evaluated for their differentiation ability towards smooth muscle-like cells (SMLCs) under the effect of widely used cytokines (TGF-β1 and PDGF-BB) with special focus on different culturing environments. For this purpose, both the commercially used culturing plates (Norm-c) and 0.1% gelatin-precoated (Gel-c) plates were used. Isolated cells displayed plastic adherence, pluripotency and cell surface marker profiling, and adipogenic and osteogenic differentiation potential with lineage specific marker expression. Differentiated cells induced under different culturing plates showed successful differentiation into SMLCs by positively expressing smooth muscle cell (SMC) specific markers both at the mRNA and protein levels. Gelatin coating could substantially enhance DPSC differentiation potential than Norm-c-induced cells. However, the absence of mature marker MHY-11 by immunostaining results from all treatment groups further indicated the development of immature and synthetic SMLCs. Finally, it was concluded that DPSC differentiation ability into SMLCs can be enhanced under cytokine treatment as well as by altering the cellular niche by precoating the culturing plates with suitable substrates. However, to get fully functional, contractile, and mature SMLCs, still many different cytokine cocktail combinations and more suitable coating substrates will be needed. [ABSTRACT FROM AUTHOR]

The present study investigated the terminal differentiation capacity into adipocytes and subsequent growth inhibition in A549 cancer cells treated with pioglitazone (PGZ), a PPARγ activator. The rate of cell growth in A549 cells was significantly (P <.05) inhibited in concentrations above 10 μM PGZ while maintaining less cytotoxic effects in MRC-5 fibroblasts. Following 50 μM PGZ treatment, population doubling time (PDT) was significantly (P <.05) increased by inhibition of cell growth, as per increasing PGZ exposure time by up to 4 weeks. The adiposome-like vesicles were commonly observed in the PGZ-treated A549 cells, and the vesicles were highly stained with Oil-Red O solution. In addition, the cell size and expression of GLUT4 and PPARγ were significantly (P <.05) increased, as per increasing PGZ exposure time by up to 4 weeks. The significant (P <.05) down-regulation of telomerase activity and up-regulation of senescence-associated β-galactosidase (SA β-GAL) activity was displayed in the PGZ-treated A549 cells, as per increasing PGZ exposure time by up to 4 weeks. The G1 phase of the cell cycle was also significantly (P <.05) increased in the PGZ-treated A549 cells compared with untreated A549 cells. The present results have demonstrated that activation of PPARγ using PGZ induces cellular differentiation into adipocytes and inhibits cell growth in the A549 cancer cells. The terminal differentiation into adipocytes could offer potent chemotherapy in the cancer cells showing high glucose metabolism. [ABSTRACT FROM AUTHOR]