Month: October 2020

The importance of LV fibrosis on cardiac function during hyperthyroidism is not well characterized. Using trichrome staining, we were able to confirm in our study that prolonged hyperthyroidism was associated with increased LV fibrosis when compared with age matched control hamsters. The majority of fibrotic deposits were found within the perivascular space and cardiac interstitium without evidence of myocyte necrosis and replacement fibrosis. Moreover, we observed severe relaxation impairment and ultimately systolic dysfunction in hyperthyroid hearts. Our findings further support the notion that LV fibrosis inversely affects LV function in the setting of hyperthyroidism. While increased collagen deposition certainly can impair global cardiac function, it may not similarly affect intrinsic cardiomyocyte mechanics. Accordingly, we also examined the influence of chronic hyperthyroidism on the mechanical function of individual myocytes. We hypothesized that mechanical impairment at the level of individual ventricular myocytes would strongly correlate with the decline observed in global cardiac function. Contrary to our hypothesis, we found that isolated ventricular myocytes from hyperthyroid hamsters had enhanced mechanical function when compared to age matched control hamsters, despite the aforementioned adverse chamber remodeling and diminished global cardiac function. Given the close proximity of fibrillar collagen to myocytes and the finding that fibrillar collagen is a relatively stiff material with a tensile strength greater than steel, it is likely that even a small increase in collagen can impair cardiomyocyte function. Indeed, this notion is supported by reports that small changes in collagen concentration can have a profound impact on passive mechanical properties of cardiac tissue. In agreement, our findings suggest that relatively modest but significant increases in myocardial collagen deposition can impede myocyte contractile ability even when individual myocyte function is enhanced. Consistent with our previous report, we did not observe noticeable areas of fibronecrosis. This suggests that the depressed global cardiac function observed during chronic hyperthyroidism is at least in part the product of inhibited myocyte function caused by increased perivascular and interstitial fibrotic deposition and does not appear to be a product of extensive myocyte loss. Our study has BAY-60-7550 several limitations. While a standard experimental protocol was closely followed to replicate the same experimental conditions for each animal, it is possible that myocytes selected for functional assessment do not represent the total myocyte population within the intact heart. It is also important to note that isolated myocytes in the current study were not tested under loading conditions. Loading conditions can influence muscle function and altered loading theoretically could impact isolated myocyte shortening and tension development. Unfortunately, the technical difficulty of myocyte loading experiments limits its utility and widespread implementation. Unloaded isolated myocyte assessment still is an important tool for evaluating shortening velocity/cross bridge turnover rate and comparisons between treatment groups can be made under similar experimental conditions without influence of potential confounders. Finally, we acknowledge that comprehensive isolated myocyte morphometric analysis would have complemented the echocardiographic and isolated myocyte functional data presente.

Smg also regulates mRNA translation through a separate mechanism involving an NVP-BKM120 interaction with the eIF4E-binding protein Cup. Cup binds to the mRNA cap binding protein eIF4E through a canonical eIF4E-binding motif. Cap-dependent translation initiation involves eIF4E recruiting eIF4G to an mRNA, which indirectly mediates recruitment of the 40S ribosome. eIF4G also interacts with eIF4E through an eIF4E-binding motif and thus recruitment of Cup to an mRNA inhibits translation by blocking the eIF4E/ eIF4G interaction. The role of Cup in Smg function led us to speculate that Vts1p might also regulate target mRNAs through an eIF4E-binding protein. While there is no Cup homolog in yeast, two eIF4Ebinding proteins, Caf20p and Eap1p, have been identified. In addition, global genetic analysis revealed synthetically lethal interactions between Eap1p and two deadenylase components, Ccr4p and Pop2p, suggesting a functional relationship, either direct or indirect, among the gene products. This genetic interaction combined with the role of the Ccr4pPop2p-Not deadenylase in Vts1p-mediated regulation prompted us to test if Eap1p might function with Vts1p to regulate target mRNAs. Using two different Vts1p target mRNAs we demonstrate that Eap1p is required for efficient Vts1p-mediated transcript degradation. Eap1p does not stimulate deadenylation but is instead required for efficient removal of the 59 cap. In addition, Eap1p-mediated stimulation of transcript decay requires binding to eIF4E. We also find that Eap1p biochemically interacts with Vts1p and is able to mediate an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby the Vts1p/Eap1p/eIF4E complex stimulates transcript decapping. Here we demonstrate a role for Eap1p in Vts1p-mediated mRNA decay. The ability of Eap1p to interact with the cap binding protein eIF4E is required for this function and, consistent with its proximity to the 59 cap, Eap1p stimulates mRNA decapping. While Eap1p is required for efficient decay of a reporter mRNA bearing wild-type SREs, it is not required for degradation of a reporter bearing mutated non-functional SREs. This suggests a specific role for Eap1p in the degradation of Vts1p target transcripts. In addition, we show that Vts1p interacts with Eap1p and that Eap1p is able to mediate an indirect interaction between Vts1p and eIF4E. Taken together these data suggest a model whereby recruitment of Eap1p to target transcripts, through its interaction with Vts1p, stimulates decapping via binding to eIF4E. Interestingly, another RNA binding protein, Puf5p, also functions with Eap1p to enchance the decapping of its target mRNAs. Alternatively, since the poly tail is known to stimulate translation, Vts1p-mediated deadenylation of target mRNAs could simultaneously repress translation and induce transcript decay; if so, the two processes would not be separable. A lack of a role for Eap1p in translational repression of Vts1p target mRNAs is consistent with the fact that a genome-wide survey of mRNAs that are translationally regulated by Eap1p show no statistically significant overlap with mRNAs that are bound by Vts1p. Thus the pool of Eap1p target mRNAs that are translationally regulated are distinct from the pool of Vts1p target mRNAs that are degraded. Since a poly tail is required for mRNA translation, perhaps Vts1p-mediated deadenylation represses mRNA translation to the point where Eap1p can no longer augment this effect.

Although a bit surprising, a negative effect of axial mechanical load on trabecular bone of WT mice is consistent with previous studies from our group in aged mice, showing that trabecular bone volume actually decreases in young adult mice upon axial compression using the waveform used here. This is in contrast to findings from other groups who have used tibial compression under different loading regimes. Preliminary results from our laboratory suggest this discrepancy may depend on the waveform and cycle number. On the other hand, BV/TV significantly increased in cKO mice with 1200 me load, further suggesting that Cx43 is involved in response to load, though the trabecular compartment in the cKO may experience greater compressive strains than WT due to the altered architecture. Interestingly, axial load increased periosteal formation rate in cKO loaded at 1200 me as well as in WT loaded at 1900 me; however, periosteal mineral apposition rate did not significantly change in either group. Such a discrepancy implies a higher number of VE-822 osteoblasts depositing new bone, rather than increased activity of existing osteoblasts. Thus, these results are consistent with the notion that axial compression loading promotes osteoblast recruitment and differentiation on the periosteal surface, an effect enhanced by Cx43 deficiency. It is possible that signaling from osteocytes to the periosteal surface is enhanced in the cKO. However, since periosteal cells in cKO mice are exposed to a lower degree of strain for a given force, the fact that periosteal bone formation is increased in Cx43 deficient bones relative to WT bone even in animals living under normal loading conditions strongly suggests that periosteal cells from cKO are more sensitive to mechanical strain than are periosteal WT cells. that 1200 me was sufficient in cKO to elicit the same bone formation response as did 1900 me in WT animals. Notably, a periosteal increase in bone diameter contributes more to load bearing strength than do increases in cortical thickness associated with endocortical apposition. Thus, the increased periosteal bone formation observed in the cKO may be related to the need for generating bone at the diaphysis to withstand loads that are sensed as abnormally higher than they actually are. Increased periosteal bone apposition but decreased endosteal formation after mechanical loading had been previously reported in 9 week old C3H/HeJ mice. Such a paradoxical effect on the endocortical surface may reflect the young age of the mice used in these studies, and the decrease in bone formation rate may represent a slowing of the rapid bone formation rate occurring in growing mice. Since such an effect is more pronounced in mice with an osteoblast/osteocyte specific deletion of Gja1, it can be concluded that Cx43 deficiency also increases the sensitivity of the endocortical cells to mechanical loading. Decreased endocortical bone formation by axial load is in apparent contrast with our previous study showing increased endosteal bone formation after application of a 3-point bending load. Aside the different loading approaches, other differences in the experimental settings may contribute to the discrepancy, including a substantially larger strain and older mice used in the 3- point bending experiment relative to the present study, as well as different promoters used to drive Gja1 ablation. In both cases, however, Cx43 deficiency reduces bone formation at the endocortex upon mechanical loading.

In the veterinary field, their ability to regulate gene expression and their stability make miRNAs useful tools to guide breeding programs for disease resistance in many types of livestock, including pigs. Although there are currently 1043 human miRNAs in the miRBase database, only about 250 pig miRNAs have been identified so far. Initially, miRNAs in livestock such as cattle and pigs were discovered using homology searches. However, after the high-throughput detection platform was developed, high-density small RNA expression profiles could be rapidly identified using gene chips, although these were limited to the detection of known miRNAs. In recent years, the emergence of Solexa and 454 highthroughput sequencing technologies have enabled the direct sequencing of miRNAs and thus the discovery new miRNAs. Xie et al. constructed a small RNA cDNA library for 16 tissues of Silmitasertib 1009820-21-6 different pig breeds, conducted deep sequencing on the miRNA transcriptome, and obtained 437 conserved and 86 predicted porcine miRNA sequences. Following this, Xie et al. customized gene chips and identified 58 miRNAs differentially expressed in native Chinese Tongcheng pigs and the Large White exotic breed that contain different lean meat percentages. Likewise, Li et al. constructed small RNA cDNA libraries of pig tissues at different growth stages from the fetal period to adulthood, and sequenced these using the Genome Analyzer GA-I. Our main research focus is diarrhea and edema disease of weaned pigs caused by E. coli F18. Previous studies have shown that E. coli F18 cells combine with the intestinal epithelial cell receptor of piglets through surface fimbriae, and then multiply and produce toxins, leading to piglet disease. Vogeli et al. found that FUT1 gene and the epithelial cell receptor gene were closely linked, and that a G to A mutation at locus M307 of the FUT1 gene that alters the structure of the receptor could be used as a genetic marker for screening. However, no AA genotype has been detected following screening of dozens of local breeds in China. Thus, the FUT1 gene is not a suitable genetic marker for the selection of E. coli F18-resistant local Chinese pig breeds. It therefore seems that exotic breeds and Chinese local pig breeds have different resistance mechanisms to E. coli F18 infection. We previously used gene chips to screen for differential gene expression in eight full-sib pair groups of E. coli F18-sensitive and – resistant Sutai pigs bred under the same conditions. We have now constructed small RNA duodenal libraries of individual E. coli F18-sensitive and -resistant weaned piglets in full-sib pair groups and analyzed these by Illumina Solexa high-throughput sequencing to identify differentially expressed miRNAs. This provides improved database information on pig miRNAs, better understanding of the genetic basis of E. coli F18 resistance in local Chinese and exotic pig breeds, and lays new foundation for identifing new markers in E. coli F18 resistance. In this study, sequencing of small RNAs from the duodenum of E. coli F18- sensitive and -resistant individuals was done using Illumina Solexa technology, and differentially expressed miRNAs were identified. We found that more than 90% of known swine miRNAs are expressed in duodenal tissues of all weaned piglets. In addition, a large number of small RNAs had sequences consistent with miRNA structure, indicating that physiological functions of the pig intestinal tract are regulated by miRNAs. As the swine and human miRNA sequences show a high degree of homology.

Interestingly, one of the miR-21 target genes is the PTEN tumor suppressor. In addition, both miR-192 and miR-215 are tumor suppressors and upregulation of miR-215 inhibits the differentiation of colon cancer stem cells. Therefore, the BAY-60-7550 miRNAs identified in this study have important functions in intestinal disease and contribute to variations in immune function between individuals. Our further studies will aim to investigate the function of candidate miRNAs in intestine epithelial cells by altering their expression and investigating the effect on the immune response to E. coli F18 infection by measuring levels of cytokine secretion and cell surface antigen expression. In addition, an ectopic target reporter gene could be developed to identify E. coli F18-resistant pigs for an improved pig-breeding program. Compared with mature milk, the initial milk is often referred to as colostrum, and is higher in immunological agents and other compounds that act against viruses, bacteria, and parasites. This helps to protect the newborn until its own immune system can function properly. MicroRNAs, an abundant class of evolutionarily conserved small non-coding RNAs of,22 nucleotides that are derived from 70 nt long stem-loop precursors, are post-transcriptional regulators that bind to complementary sequences on target mRNAs, usually resulting in translational repression in mammals. Emerging evidence suggests that miRNAs have important roles in regulating the development of immune cells and in modulating innate and adaptive immune responses. Extracellular miRNAs in various body fluids have recently been shown to be associated with various pathological conditions. These circulating miRNAs are mainly delivered by exosomes, which are membranous vesicles of endocytic origin that are released by a variety of cell types into the extracellular space. Exosomes appear to play a significant role in cellular communication in the immune system and elsewhere by transferring miRNAs, mRNAs, and proteins to neighboring cells. Exosomes are present in human breast milk and are packaged with abundant immune-related proteins, as well as miRNAs, have the potential to influence the immune system of the infant. Here, we present the miRNA expression profiles in porcine milk exosomes across six lactigenous stages. We particularly emphasize the differences between the colostrum and mature milk. We found that , and may have significant effects on the development of the immune system in infants. This study also highlights the use of pigs as a model organism for human breastfeeding medicine and immune diseases research. This study reports the comprehensive lactation-related miRNA expression profiles of porcine breast milk exosomes. We found that immune-related miRNAs are present and enriched in breast milk exosomes and are generally resistant to relatively harsh conditions. These immunerelated miRNAs are present in higher numbers in the colostrum compared with the mature milk or the blood of colostrum-only fed piglets compared with the mature milk-only fed piglets. The presence and enrichment of immune-related miRNAs in breast milk exosomes, especially in the colostrum, was demonstrated. These miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations call for further detailed investigations to obtain a thorough understanding of the essential effects of breast milk in the development of the infant’s immune system.