Identification and bioinformatics analysis of differentially expressed milk exosomal microRNAs in milk exosomes of heat-stressed Holstein cows.

In summer, heat stress is one of the primary reasons for the compromised health and low milk productivity of dairy cows. Hyperthermia affects milk synthesis and secretion in the mammary glands of dairy cows. As molecules for intercellular communication, milk-derived exosomes carry genetic material, proteins, and lipids, playing a crucial role in mammary tissue growth and milk synthesis in dairy cows. The aim of this study was to explore the milk exosomal miRNA profile of heat-stressed and normal Holstein cows. We isolated and identified milk exosomes to screening for differentially expressed miRNAs using small RNA sequencing. Then, TargetScan and miRanda algorithms were used to predict the putative targets of the differentially expressed miRNAs, whereas GO and KEGG pathway enrichment analyses were performed for the differentially expressed miRNA-target genes. Our results showed that 215 miRNAs were significantly differentially expressed in heat-stressed milk exosomes, of which one was upregulated and 214 were significantly downregulated. GO and KEGG enrichment analyses indicated that differentially expressed miRNAs might play a role in apoptosis, autophagy, and the p38 MAPK pathway. qRT-PCR assay verified that the expression of miRNAs was consistent with the sequencing results, warranting further verification of their specific targets of action. In conclusion, changes in the miRNA expression profile of milk exosomes indicated the role of exosomal miRNAs in regulating heat stress resistance and apoptosis in dairy cows. Our results suggested that milk-derived exosomal miRNAs could increase mammary gland resistance to heat stress, thereby enhancing milk synthesis in dairy cows.

Depletion of serum-derived exosomes aggravates heat stress-induced damage of bovine mammary epithelial cells.

BACKGROUND: Exosomes are involved in intercellular communication, affecting many physiological and pathological process. The present study evaluated the effects of serum exosomes on the function of bovine mammary epithelial cells (BMECs) and milk synthesis under heat stress. METHODS AND RESULTS: We cultured the BMECs in fetal bovine serum (FBS) or exosome-free FBS medium and examined, their viability using CCK-8 kit. The results showed that culturing the cells in an exosome-free medium decreased viability and increased the levels of reactive oxygen species. The BMECs cultured in the exosome-free medium had reduced mitochondrial membrane potential, decreased manganese superoxide dismutase activity, and disrupted mitochondrial dynamics. They exhibited apoptosis due to upregulated Drp1, Fis1, Bax and HSP70. Lastly, we observed downregulation of milk fat and lactoprotein-related genes: mTOR, PPARγ, p-mTOR and ADD1 and SREBP1, ELF5, and CSN2, respectively, after culturing the cells in an exosome-free medium. These negative effects of the exosome-free medium on the BMECs could be further reinforced under heat stress. CONCLUSION: Our results demonstrated that exosomes from serum are critical for maintaining the normal function of BMECs.

SIRT4 Expression Ameliorates the Detrimental Effect of Heat Stress via AMPK/mTOR Signaling Pathway in BMECs.

Sirtuin 4 (SIRT4), a member of the SIRT family, has been reported to be a key factor involved in antioxidant defense in mitochondria. This study aimed to explore the potential molecular mechanism via which SIRT4 regulates heat stress-induced oxidative stress and lactoprotein synthesis in bovine mammary epithelial cells (BMECs). Our results showed that SIRT4 was significantly decreased in heat stressed mammary tissue. Depletion of SIRT4 in BMECs induced the generation of ROS, which, as exhibited by the decreased activity of antioxidant enzymes, changed mitochondrial morphology through mediating protein and mRNA levels related to mitochondrial fission and fusion. Moreover, we found that depletion of SIRT4 or stress conditions inhibited the expression of milk proteins, as well as lipid and glucose synthesis-related genes, and activated the AMPK/mTOR signaling pathway. Increased SIRT4 expression was found to have the opposite effect. However, blocking the AMPK/mTOR signaling pathway could inhibit the regulatory function of SIRT4 in milk synthesis-related gene expression. In summary, our results suggest that SIRT4 may play critical roles in maintaining mammary gland function by regulating the AMPK/mTOR signaling pathway in dairy cows, indicating that SIRT4 may be a potential molecular target for curing heat stress-induced BMEC injury and low milk production in dairy cows.

S-allyl cysteine ameliorates heat stress-induced oxidative stress by activating Nrf2/HO-1 signaling pathway in BMECs.

Heat stress-induced oxidative stress in bovine mammary epithelial cells (BMECs) threatens the normal growth and development of bovine mammary tissue, resulting in lower milk production of dairy cows. The aim of the present study is to investigate the protective effects of S-allyl cysteine (SAC), an organosulfur component extracted from aged garlic, on heat stress-induced oxidative stress and apoptosis in BMECs and to explore its underlying mechanisms. Our results showed that heat stress treatment considerably decreased cell viability, whereas SAC treatment dose-dependently restored cell viability of BMECs under heat-stress conditions. In addition, SAC protected BMECs from heat stress-induced oxidative damage by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited heat stress-induced apoptosis by reducing the ratio of Bax/Bcl-2 and blocking proteolytic the cleavage of caspase-3 in BMECs. Interestingly, we found that the protective effect of SAC on heat stress-induced oxidative stress and apoptosis was dependent on the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. SAC promoted the Nrf2 nuclear translocation in heat stress-induced BMECs. The results were also validated by Nrf2 and Keap1 knockdown experiments further demonstrating that Nrf-2 was indeed involved in the protective effect of SAC on heat stress-induced oxidative damage and apoptosis. In summary, our results showed that SAC could protect BMECs from heat stress-induced injury by mediating the Nrf2/HO-1 signaling pathway, suggesting that SAC could be considered as a therapeutic drug for attenuating heat stress-induced mammary gland diseases.

Dihydromyricetin attenuates heat stress-induced apoptosis in dairy cow mammary epithelial cells through suppressing mitochondrial dysfunction.

It is well known that the dairy cow production is very sensitive to environmental factors, including high temperature, high humidity and radiant heat sources. High temperature-induced heat stress is the main environmental factor that causes oxidative stress and apoptosis, which affects the development of mammary glands in dairy cows. Dihydromyricetin (DMY) is a nature flavonoid compound extracted from Ampelopsis grossedentata; it has been shown to have various pharmacological functions, such as anti-inflammation, antitumor and liver protection. The present study aims to evaluate the protective effect of DMY on heat stress-induced dairy cow mammary epithelial cells (DCMECs) apoptosis and explore the potential mechanisms. The results show that heat stress triggers heat shock response and reduces cell viability in DCMECs; pretreatment of DCMECs with DMY (25 µM) for 12 h significantly alleviates the negative effects of heat stress on cells. DMY can provide cytoprotective effects by suppressing heat stress-caused mitochondrial membrane depolarization and mitochondrial dysfunction, Bax and Caspase 3 activity, and modulation of oxidative enzymes, thereby preventing ROS production and apoptosis in DCMECs. Importantly, DMY treatment could attenuate heat stress-induced mitochondrial fragmentation through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (Mfn1, 2). Above all, our findings demonstrate that DMY could protect DCMECs against heat stress-induced injury through preventing oxidative stress, the imbalance of mitochondrial fission and fusion, which provides useful evidence that DMY can be a promising therapeutic drug for protecting heat stress-induced mammary glands injury and mastitis.

Structural Characterization of the CD44 Stem Region for Standard and Cancer-Associated Isoforms.

CD44 is widely expressed in most vertebrate cells, whereas the expression of CD44v6 is restricted to only a few tissues and has been considered to be associated with tumor progression and metastasis. Thus, CD44v6 has been recognized as a promising prognostic biomarker and therapeutic target for various cancers for more than a decade. However, despite many experimental studies, the structural dynamics and differences between CD44s and CD44v6, particularly in their stem region, still remain elusive. Here, a computational study was conducted to address these problems. We found that the stem of CD44s adopted predominantly two conformations, one featuring antiparallel ß-sheets and the other featuring parallel ß-sheets, whereas the stem of CD44v6 adopted mainly one conformation with relatively highly suppressed ß-sheet contents. Moreover, Phe215 was found to be essential in the ß-sheets of both CD44s and CD44v6. We finally found intramolecular Phe215-Trp224 hydrogen-bonding interactions and hydrophobic interactions with Phe215 that cooperatively drove conformational differences upon the addition of the v6 region to CD44. Our study elucidated the structural differences between the stem regions of CD44s and CD44v6 and thus can offer useful structural information for drug design to specifically target CD44v6 in promising clinical applications.

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway.

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.

Chemerin induces lipolysis through ERK1/2 pathway in intramuscular mature adipocytes of dairy bull calves.

The adipokine Chemerin has been reported to regulate differentiation and metabolism of adipocytes, but the mechanism underlying lipolysis is still largely unknown. The purpose of this study was to explore whether ERK1/2 pathway is involved in regulating Chemerin during bovine intramuscular mature adipocyte lipolysis. Intramuscular mature adipocytes of dairy bull calves were cultured in vitro and were treated with Chemerin or U0126, which is an inhibitor of ERK1/2 pathway. The results showed that TG content in cells was significantly decreased, glycerol and free fatty acid were significantly increased in cell culture media, and the expression of phosphorylated ERK1/2 in cells was increased in Chemerin-treated group, suggested that ERK1/2 pathway was involved in regulation of lipolysis by Chemerin. In addition, the expression of lipolytic-related critical factors ATGL, HSL, LPL, PPARα, UCP3, and CPT1 were upregulated, but the expression of adipogenic key factors, including PPARγ and C/EBPα were downregulated by Chemerin. Interestingly, all the effects of Chemerin on genes expression in intramuscular mature adipocytes or fat tissue were inhibited by U0126, showed that the function of Chemerin to promote adipose decomposition will be significantly weakened if the ERK1/2 pathway is suppressed, and confirmed that ERK1/2 pathway is involved in mediate Chemerin-enhanced lipolysis. In conclusion, the study demonstrated that Chemerin induce intramuscular mature adipocytes lipolysis through activation of the ERK1/2 pathway. Our research at least provide partial mechanisms of Chemerin on lipolysis and deposition of intramuscular fat tissue of dairy bull calves.

SIRT7 depletion inhibits cell proliferation, migration, and increases drug sensitivity by activating p38MAPK in breast cancer cells.

SIRT7 is a member of the sirtuin family of proteins that are known to be associated with tumor development. However, the functional roles and molecular mechanisms underlying the function of SIRT7 in breast cancer cell survival and tumor development remain unclear. Recent studies demonstrated that SIRT7 is upregulated in breast cancer cells and tissues. In the present study, we systematically explored the roles of SIRT7 in the growth of breast cancer cells and tumors both in vitro and in vivo. Our results showed that SIRT7 plays a major role in facilitating cell survival by promoting cell proliferation and inhibiting apoptosis. SIRT7 depletion significantly inhibited cell invasion and wound healing by blocking cell cycle progression and inducing cell apoptosis. Meanwhile, SIRT7 depletion can increase the sensitivity of breast cancer cells to doxorubicin (DOX). Xenograft model studies showed that stable silencing of SIRT7 inhibited tumor growth and enhanced tumor sensitivity to DOX. Further research revealed that p38MAPK is involved in SIRT7-mediated regulation of breast cancer cell proliferation and tumor growth. Taken together, our results showed that SIRT7 plays a critical role in breast cancer cell survival, migration, and tumor growth, and increased the efficiency of DOX treatment both in vitro and in vivo. Therefore, SIRT7 is a promising therapeutic target in breast cancer treatment.

The small GTPase RhoA regulates the LIMK1/2-cofilin pathway to modulate cytoskeletal dynamics in oocyte meiosis.

LIM kinases (LIMK1/2) are LIM domain-containing serine/threonine/tyrosine kinases that mediate multiple cellular processes in mitosis. In the present study, we explored the functional roles and potential signaling pathway of LIMK1/2 during mouse oocyte meiosis. Disruption of LIMK1/2 activity and expression significantly decreased oocyte polar body extrusion. Live-cell imaging revealed that spindle migration was disturbed after both LIMK1 and LIMK2 knock down, and this might be due to aberrant distribution of actin filaments in the oocyte cytoplasm and cortex. Meanwhile, our results demonstrated that the function of LIMK1 and LIMK2 in actin assembly was related to cofilin phosphorylation levels. In addition, disruption of LIMK1/2 activity significantly increased the percentage of oocytes with abnormal spindle morphologies, which was confirmed by the abnormal p-MAPK localization. We further, explored the upstream molecules of LIMK1/2, and we found that after depletion of ROCK, phosphorylation of LIMK1/2 and cofilin were significantly decreased. Moreover, RhoA inhibition caused the decreased expression of ROCK, p-LIMK1/2, and cofilin. In summary, our results indicated that the small GTPase RhoA regulated LIMK1/2-cofilin to modulate cytoskeletal dynamics during mouse oocyte meiosis.

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes.

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells' lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

ROCK inhibition prevents early mouse embryo development.

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.

The protective effect of rosmarinic acid on hyperthermia-induced C2C12 muscle cells damage.

High temperature will cause animal tissues or cells damage. Rosmarinic acid (RA) is a good antioxidant and health care product, but the roles of RA in muscle cells damage and the mechanisms which caused by high temperature is still unknown. In this study, the roles of RA on hyperthermia-induced apoptosis and damage of C2C12 muscle cells were investigated. C2C12 cells were cultured in medium with different concentration (0, 25, 50, 100 µM) RA and treated in 42 °C high temperature to induce cellular apoptosis and damage. Then, these cells were analyzed effect of different dose of RA on cells apoptosis and damage. The results indicated that RA has protective effect on heat-stress induced cellular damage, and the cells have the higher cell viability at the dose of 50 µM RA by MTT assay. Hochest33342/PI double staining showed that the cellular apoptosis of C2C12 cells were decreased in the presence of selected 50 µM RA. Malondialdehyde formation and reactive oxygen species levels were also decreased significantly, but cellular superoxide dismutase activity was increased significantly in the presence of RA even in the condition of 42 °C. Meanwhile, Caspase-3 mRNA expression, Caspase-3 activity, and Bax/Bcl-2 ratio were reduced significantly, but the mRNA expression of Hsp72 was increased significantly in those hyperthermia-induced C2C12 cells in the presence of 50 µM RA. Taken together, the results at least discovered that RA has protective effects on hyperthermia-induced cellular apoptosis and damage of muscle cells by change the expression of stress-genes and increasing intracellular antioxidant capability.

miR-27a-3p relieves heat stress-induced mitochondrial damage and aberrant milk protein synthesis through MEK/ERK pathway in BMECs.

With global warming, heat stress has become a primary factor that compromises the health and milk quality of dairy cows. Here, we investigated the function and underlying regulatory mechanism of miR-27a-3p in bovine mammary epithelial cells (BMECs) under heat-stress conditions. The current study showed that miR-27a-3p could prevent heat stress-induced BMEC oxidative stress and mitochondrial damage by regulating the balance between mitochondrial fission and fusion processes. Importantly, we found that miR-27a-3p could increase cell proliferation under heat stress conditions by regulating the MEK/ERK pathway and cyclin D1/E1. Interestingly, miR-27a-3p is also involved in the regulation of milk protein synthesis-related protein expression, such as CSN2 and ELF5. Inhibition of the MEK/ERK signaling pathway by AZD6244 blocked the regulatory function of miR-27a-3p in cell proliferation and milk protein synthesis in BMECs under heat stress conditions. Our findings demonstrated that miR-27a-3p protects BMECs from heat stress-induced oxidative stress and mitochondrial damage through the MEK/ERK pathway, thereby promoting BMECs proliferation and lactation in dairy cows. The potential regulatory mechanism of miR-27a-3p in attenuating heat stress-induced apoptosis and lactation defect in BMECs.

SIRT3 protects bovine mammary epithelial cells from heat stress damage by activating the AMPK signaling pathway.

With global warming, heat stress has become an important challenge for the global dairy industry. Sirtuin 3 (SIRT3), an important mitochondrial NAD+dependent decarboxylase and a major regulator of cellular energy metabolism and antioxidant defense, is integral to maintaining normal mitochondrial function. The aim of this study was to assess the protective effect of SIRT3 on damage to bovine mammary epithelial cells (BMECs) induced by heat stress and to explore its potential mechanism. Our results indicate that SIRT3 is significantly downregulated in heat-stressed mammary tissue and high-temperature-treated BMECs. SIRT3 knockdown significantly increased the expression of HSP70, Bax, and cleaved-caspase 3 and inhibited the production of antioxidases, thus promoting ROS production and cell apoptosis in BMECs. In addition, SIRT3 knockdown can aggravate mitochondrial damage by mediating the expression of genes related to mitochondrial fission and fusion, including dynamin-related protein 1, mitochondrial fission 1 protein, and mitochondrial fusion proteins 1and 2. In addition, SIRT3 knockdown substantially decreased AMPK phosphorylation in BMECs. In contrast, SIRT3 overexpression in high-temperature treatment had the opposite effect to SIRT3 knockdown in BMECs. SIRT3 overexpression reduced mitochondrial damage and weakened the oxidative stress response of BMECs induced by heat stress and promoted the phosphorylation of AMPK. Taken together, our results indicate that SIRT3 can protect BMECs from heat stress damage through the AMPK signaling pathway. Therefore, the reduction of oxidative stress by SIRT3 may be the primary molecular mechanism underlying resistance to heat stress in summer cows.

Hexestrol Deteriorates Oocyte Quality via Perturbation of Mitochondrial Dynamics and Function.

Hexestrol (HES) is a synthetic non-steroidal estrogen that was widely used illegally to boost the growth rate in livestock production and aquaculture. HES can also be transferred to humans from treated animals and the environment. HES has been shown to have an adverse effect on ovarian function and oogenesis, but the potential mechanism has not been clearly defined. To understand the potential mechanisms regarding how HES affect female ovarian function, we assessed oocyte quality by examining the critical events during oocyte maturation. We found that HES has an adverse effect on oocyte quality, indicated by the decreased capacity of oocyte maturation and early embryo development competency. Specifically, HES-exposed oocytes exhibited aberrant microtubule nucleation and spindle assembly, resulting in meiotic arrest. In addition, HES exposure disrupted mitochondrial distribution and the balance of mitochondrial fission and fusion, leading to aberrant mitochondrial membrane potential and accumulation of reactive oxygen species. Lastly, we found that HES exposure can increase cytosolic Ca2+ levels and induce DNA damage and early apoptosis. In summary, these results demonstrate that mitochondrial dysfunction and perturbation of normal mitochondrial fission and fusion dynamics could be major causes of reduced oocyte quality after HES exposure.

Amyloid-ß peptide alteration of tau exon-10 splicing via the GSK3ß-SC35 pathway.

Amyloid-beta peptide (Aß) and Tau protein are the lead constituents in the pathogenesis of Alzheimer's disease (AD). However, their inter-relationship in the disease process remains to be established. Tauopathy refers to a characteristic neurodegenerative process in AD. In tauopathy, Tau accumulates as a consequence of altered pre-mRNA splicing of tau exon 10, resulting in 3R (without exon 10)/4R (with exon 10) imbalance. We studied Aß effects on tau exon 10 pre-mRNA splicing and relevant signaling events. This is the first demonstration of Aß alteration of tau exon 10 splicing with an increase in 3R/4R ratio caused by reduced 4R expression. This Aß ï€ action is causally related to its activation of GSK-3ß which in turn phosphorylates SC35, an enhancer in tau exon 10 splicing. The establishment of the Aß-GSK-3ß-SC35 cascade broadens insight into development of novel strategies to modulate Aß action on tau exon 10 splicing for possible prevention of tauopathy.

The protective effect of rosmarinic acid on myotube formation during myoblast differentiation under heat stress.

High ambient temperature is one of the most important environmental factors that caused the reduction of livestock productivity and the increase of mortality. It has been shown that heat stress could affect the meat quality characteristics by physiological and metabolic perturbations in live livestock. Rosmarinic acid (RA) is a natural polyphenolic phytochemical compound that has many important biological activities, such as antioxidant, antimutagenic, and antitumor. The purpose of this study was to investigate the possible function and mechanism of RA on myoblast proliferation and differentiation under heat stress condition. The results showed that heat stress reduced the viability of myoblast and increased the percentage of apoptotic cells, and it also disrupted myotube formation by altering the expression of myogenic regulatory factors MyoD, myogenin, and MyHC. However, pretreatment of RA can protect C2C12 cells from heat stress-induced apoptosis, and it also increased the expression level of MyoD, myogenin, and MyHC under heat stress, which indicated that RA have protective effect on heat stress-caused failure of myotube formation during myoblast differentiation. Above all, our finding demonstrated that RA can promote the differentiation of C2C12 myoblast and maintain the formation of myotubes even under heat stress condition.

Heat stress induces apoptosis through disruption of dynamic mitochondrial networks in dairy cow mammary epithelial cells.

Heat stress-induced reductions in milk yield and the dysfunction of mammary glands are economically important challenges that face the dairy industry, especially during summer. The aim of the present study is to investigate the effects of heat stress on mitochondrial function by using dairy cow mammary epithelial cells (DCMECs) as an in vitro model. Live cell imaging shows that the mitochondria continually change shape through fission and fusion. However, heat stress induces the fragmentation of mitochondria, as well as the decreased of ATP level, membrane potential, and anti-oxidant enzyme activity and the increased of respiratory chain complex I activity. In addition, the cytosolic Ca2+ concentration and cytochrome c expression (Cyto-c) were increased after heat stress treatment. Both qRT-PCR and western blot analysis indicate that mitofusin1/2 (Mfn1/2) and optic atrophy protein-1 (Opa-1) are downregulated after heat stress, whereas dynamin-related protein 1 (Drp1) and fission 1 (Fis-1) are upregulated, which explains the observed defect of mitochondrial network dynamics. Accordingly, the present study indicated that heat stress induced the dysfunction of DCMEC through disruption of the normal balance of mitochondrial fission and fusion.

The epigenetic effects of amyloid-beta(1-40) on global DNA and neprilysin genes in murine cerebral endothelial cells.

Amyloid-beta (Abeta) is the core component of senile plaques, which are the pathological markers for Alzheimer's disease and cerebral amyloid angiopathy. DNA methylation/demethylation plays a crucial role in gene regulation and could also be responsible for presentation of senescence. Oxidative stress, which may be induced by Abeta, is thought to be an important contributor of DNA hyper-methylation; however, contradicting this is the fact that global DNA hypo-methylation has been found in aging brains. It therefore remains largely unknown as to whether Abeta does in fact cause DNA methylation/demethylation. Neprilysin (NEP) is one of the enzymes responsible for Abeta degradation, with its expression decreasing in both Alzheimer and aging brains. Using high-performance liquid chromatography (HPLC), we explore whether Abeta is responsible for alteration of the global DNA methylation status on a murine cerebral endothelial cells model, and also use methylation-specific PCR (MSPCR) to examine whether DNA methylation status is altered on the NEP promoter region. We find that Abeta reduces global DNA methylation whilst increasing NEP DNA methylation and further suppressing the NEP expression in mRNA and protein levels. Our results support that Abeta induces epigenetic effects, implying that DNA methylation may be part of a vicious cycle involving the reduction in NEP expression along with a resultant increase in Abeta accumulation, and that Abeta may induce global DNA hypo-methylation.