Month: March 2019

In the present study, we identified miR-346 as a positive regulator of the osteogenic differentiation of hBMSCs. We found that miR-346 was highly expressed during the course of osteogenic differentiation. Overexpression of miR-346 in hBMSCs enhanced osteogenic differentiation, whereas inhibition of miR-346 suppressed their osteogenic potential. miRNAs, as novel regulators of target gene expression, play important roles in regulation of stem cell differentiation into osteoblasts by controlling the levels of critical factors. For instance, miR-138, which is down-regulated during AbMole Capromorelin tartrate osteoblast differentiation of hBMSCs, functions as a negative regulator of osteogenic differentiation by targeting FAK and suppressing the FAK-ERK1/2 signaling pathway. miR-204 inhibits osteoblast differentiation of BMSCs, while adipocyte differentiation is promoted when miR-204 is overexpressed in these cells. miR20a AbMole Diniconazole promotes the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARc, Bambi and Crim1, the negative regulators of BMP signaling. Here, we report that GSK-3b is a direct target of miR-346 in hBMSCs. Our data indicate that miR-346 overexpression significantly down-regulates GSK-3b by directly targeting the 39UTR of GSK-3b mRNA confirmed using luciferase-reporter-gene assays, and this effect was largely eliminated when the sites in GSK-3b 39UTR targeted by miR-346 were mutated. Moreover, miR-346 overexpression in hBMSCs resulted in upregulation of GSK-3b protein level without changing its mRNA expression, confirming that miR-346 regulates GSK-3b expression at a post-transcription level. GSK-3b, an isoform of GSK-3, is implicated in various biological processes including cell growth, differentiation and apoptosis. Mounting evidence indicates that GSK-3b inhibition promotes bone formation in vivo. In recent years, GSK-3b has been reported to play important roles in regulating osteoblast differentiation. Gambardella et al. have shown that inhibition of GSK-3b promotes osteogenic differentiation of mesenchymal progenitors but not adipogenic differentiation. Jang et al. found that GSK-3b inactivation upon receptor activator of NF-kB ligand stimulation is crucial for osteoclast differentiation. In this study, we demonstrate that GSK-3b is a functional target of miR-346. Our data show that osteogenic differentiation of hBMSCs is suppressed by overexpression of GSK-3b inmiR-346-overexpressing cells, while GSK3b siRNA almost completely blockes the inhibitory role of antimiR-346 on osteogenic differentiation, suggesting that miR-346regulated osteogenic differentiation is GSK-3b-dependent. Our data further demonstrate that miR-346-mediated downregulation of GSK-3b leads to activation of Wnt/b-catenin signaling in hBMSCs. Firstly, miR-346 overexpression significantly increased total and activated b-catenin expression. Secondly, immunofluorescence staining showed increased b-catenin nuclear accumulation in miR-346�Coverexpressing cells compared to miRcontrol cells. Thirdly, miR-346 overexpression increased TCF/ LEF transcriptional activity, and this effect was blocked by antimiR-346. In addition, b-catenin knockdown in miR-346-overexpressing cells almost completely blocked the positive effect of miR346 on osteogenic differentiation. Consistent with these findings, miR-346 overexpression was found to enhance the expression of several b-catenin downstream genes in hBMSCs. In conclusion, this study identified miR-346 as a positive regulator of human osteogenesis, acting by targeting GSK-3b and activating Wnt/b-catenin signaling in hBMSCs. Our findings suggest that miR-346 may be a useful target in the treatment of pathological conditions of bone loss.

Myostatin serves as a negative feedback molecule during the processes of muscular growth and regeneration in order to limit the final muscle mass. In this model, myostatin is switched on only during muscle growth or regeneration. In the Pompe disease patients included in this study, suggesting a low level of muscle growth or regeneration. After ERT completion, myostatin levels in the Pompe disease patients increased significantly. This elevation in myostatin may have occurred as a signals of muscle regeneration. ERT stops the process of muscular destruction in order that new muscle fibers can be generated. Since myostatin negatively regulates human myoblast proliferation, antagonism of myostatin may enhance the therapeutic effect of ERT in those patients who respond poorly. Follistatin was originally found in the ovarian follicular fluid, and broader expression was later shown in the reproductive, endocrine, digestive, and neurological systems of humans. Follistatin binds to members of the transforming growth factor-b superfamily, and exerts an inhibitory effect on these growth factors. The follistatin reference value for a term neonate is 0.4360.02 ng/ml, which is correlated with fat mass and gestational age. In our study, baseline serum levels of follistatin were higher than normal for both the Pompe disease group and the control group, and increased at follow-up. A possible explanation could be high inter-individual variation resulting from a small sample size. In addition, because follistatin augments adipogenesis, higher adipose tissue growth might correlate with higher follistatin serum levels. Follistatin is a cytokine that does not originate in muscle; we therefore regarded it as a control marker. This study has some limitations. Due to the rarity of the disease, the sample was small and heterogeneous and duration of follow-up was short. However, we have still obtained positive results from a small sample size. By recruiting more patients for a larger sample size, it should be possible to obtain more detailed information in the future through subgroup analyses, e.g. by looking at IOPD versus LOPD or good responders versus poor responders. In addition, due to ethical considerations, the present study does not include a Pompe disease cohort that did not receive ERT. Therefore, it is not known if the effects are due to the treatment or related to the disease’s natural course. Furthermore, assessments of muscle mass and strength may be needed to delineate the correlation between muscle regeneration and serum marker levels. A future longitudinal study is also needed to analyze dynamic changes in these markers over the disease course. In conclusion, serum IGF-1 levels were significantly lower in Pompe disease patients relative to normal control subjects, and serum myostatin also showed a trend for lower levels.

Addition of additives after complete binding of lysozyme to PS particles desorbed the bound lysozyme from the PS particles. This desorption by Arg and NaCl occurred even 14 h after lysozyme binding to the PS particles and the protein concentration was fivefold higher than in the absence of additives. Moreover, the recovery of enzyme activity was sixfold higher than without additives after 14 h of binding. However, the value of the enzyme activity was lower when lysozyme was first bound to the PS particles in the absence of additives than when lysozyme was bound in the presence of additives. These observations indicated that a portion of lysozyme adsorbed on PS particles is deactivated. Moreover, protein concentration and enzyme activity decreased in a similar time-dependent manner when Arg and NaCl were mixed after lysozyme was bound to PS particles. These observations suggest that the amount of reversibly bound inactive proteins increased and further became irreversible. These results may be explained by assuming three populations of bound lysozyme, i.e., irreversibly bound inactive protein, reversibly bound inactive protein, and reversibly bound active protein. The latter can be fully recovered by Arg and NaCl. Arg can also recover some portion of irreversibly bound protein. In summary, we have shown that the addition of Arg markedly suppresses protein adsorption on PS particles, which is a commonly-used material for vessels such as disposable test tubes and microtubes, and moreover can detach adsorbed proteins from PS particles. It has already been shown that Arg is one of the most versatile solution additives for proteins. Arg has a number of advantages in that it is a natural cell metabolite, biodegradable, nontoxic, and inexpensive. Arg has been used to enhance protein refolding, augment acid-induced viral inactivation, suppress protein aggregation during long-term storage, and enhance protein recovery during column chromatography. These effects of Arg may be at least in part due to its ability to suppress multiple interactions between proteins and chemical structures of solid surfaces as observed with the model PS particles. Prostatitis is a common yet uncertain condition without clear diagnostic criteria and treatment strategies. In 1995, The National Institutes of Health organized the International Prostatitis Collaborative Network workshop and proposed a new classification of prostatitis, and since then asymptomatic inammatory prostatitis has been regarded as a separate clinical entity among other prostatitis syndromes. This condition is characterized by the presence of inammatory cells in expressed prostatic secretion or in histologically prostate biopsy specimens in an otherwise asymptomatic patient and is therefore diagnosed solely in the laboratory. Unfortunately, while many investigators have studied symptomatic prostatitis, little research has been done with regard to asymptomatic prostatitis.

We estimated that 70% of all individuals diagnosed with HIV in Denmark were virally suppressed. The Center for Disease Control and Prevention has estimated that the corresponding proportion in the United States is only 35%. This difference is mainly due to lower rates of linkage to and retention in care. Each of the steps of the continuum of HIV care in the United States as well as possible effects of addressing gaps in care have been reviewed by Gardner et al. They found that incompletely engaged individuals account for the largest proportion of HIV infected individuals with detectable viremia and that multiple sequential barriers have to be overcome, since improvements in any single component of the continuum of care will have minimal impact on the proportion of HIV infected individuals with vireamia. In Denmark 95% were linked to care and 93% of those linked were retained in care, whereas the corresponding numbers for the United States were 77% and 66%. In the United States socially disadvantaged groups are disproportionally affected by HIV, which likely partly explains the difference. But dissimilarities in the organization of the health care systems may also be important. User’s fees and lack of health insurance may be barriers to engagement in care. However, differential methods for data collection and analyses may affect estimates. If nationwide data are not available or if it is not possible to link data from different registers at the individual level, the proportion of patients not retained in care may be overestimated due to migration. In spite of the marked increase in the proportion of HIV patients with viral suppression during the study period, no decrease in incidence of HIV diagnoses has been observed. On the contrary there has been a slight increase in the annual number of newly diagnosed MSM. This is discouraging given the hopes to the strategy of ��treatment as prevention�� fuelled by trials showing high efficacy of HAART in reduction of mother to child transmission and prevention of transmission in serodiscordant couples. It has been speculated that increases in unprotected sex counterbalance the effect of reductions in community viral load. An additional explanation might be that primary HIV infection drives a considerable part of HIV transmission or that a large fraction of individuals living with HIV are undiagnosed. There are no well documented estimates of numbers of individuals living with undiagnosed HIV in Denmark and whether this number has changed during the study period is unknown. Generally there has been a slight increase in the median CD4 count at time of HIV diagnosis and a reduction in the proportion of patients with advanced disease at HIV diagnosis. Reviews of the evidence on strategies to curtail the HIV epidemic conclude that treatment as prevention is unlikely to eliminate HIV on its own and that a combination of interventions are needed to control the epidemic.

In these sections, the glycogen load, indicated by the percentage of AbMole Oleandrin muscle fibers containing intracytoplasmic vacuoles under high power field, was calculated by an experienced pathologist using the formula: glycogen load number of total muscle fibers. The other half of the specimen was fixed in 3% glutaraldehyde, processed into epoxy resin blocks, sectioned into one-micron sections and stained with a combination of periodic acid-Schiff stain and Richardson’s stain for high resolution light microscopy, as previously described. Glycogen load in these sections was measured by computer morphometry with MetaMorph software and expressed as the percentage of tissue area occupied by glycogen, as previously described. In patients with Pompe disease, we demonstrated lower baseline levels of serum IGF-1 compared with control subjects and elevations in serum IGF-1 and myostatin levels following ERT. The significant increases in serum levels of myostatin and IGF-1 might be related to muscle regeneration, and could also correspond with muscle recovery as shown in pathological examinations. IGF-1 is an age-related serum protein that has insulin-like metabolic activities and growth-controlling properties. IGF-1 expression counteracts muscle function decline in mouse models of muscular dystrophy and amyotrophic lateral sclerosis, activates satellite cells, and improves the survival of motor neurons. Higher IGF-1 levels at baseline in control subjects might reflect the stage of life that is consistent with rapid muscle growth, while subsequent decreases at follow-up could be caused by slowdown in muscle growth with older age. In the present study, the baseline IGF-1 level of Pompe disease patients was lower than for control subjects. Lack of regeneration signals that activate satellite cells �C due to muscular destruction caused by glycogen accumulation – could be one explanation for this finding. After completion of ERT, we found that glycogen accumulation had decreased, and that IGF-1 expression had increased to approach normal levels. IGF-I levels are highly age-dependent in children. Immediately following birth, infants have a prominent postnatal surge in circulating IGF-1 levels. The level then declines to reach a nadir before one year of age, but increases slowly thereafter, surging again in adolescence. In our study, the serum level of IGF-1 in the control group declined during follow-up. This is to be expected as most of the IOPD patients in our study started ERT before one month of age, representing a time-window when IGF-1 levels in normal controls would be at their highest. In addition, the four LOPD patients who started ERT during adolescence had higher reference IGF-1 values. By including agematched controls in our study, we were able to minimize sample heterogeneity caused by age-associated differences in marker levels.