Osteoporosis Recovery by Antrodia camphorata

Antrodia camphorata has previously demonstrated the efficacy in treating cancer and anti-inflammation. In this study, we are the first to evaluate Antrodia camphorata alcohol extract (ACAE) for osteoporosis recovery in vitro with preosteoblast cells (MC3T3-E1) and in vivo with an osteoporosis mouse model established in our previous studies, ovariectomized senescence accelerated mice (OVX-SAMP8). Our results demonstrated that ACAE treatment was slightly cytotoxic to preosteoblast at 25 μg/mL, by which the osteogenic gene expression (RUNX2, OPN, and OCN) was significantly upregulated with an increased ratio of OPG to RANKL, indicating maintenance of the bone matrix through inhibition of osteoclastic pathway. Additionally, evaluation by Alizarin Red S staining showed increased mineralization in ACAE-treated preosteoblasts. For in vivo study, our results indicated that ACAE inhibits bone loss and significantly increases percentage bone volume, trabecular bone number, and bone mineral density in OVX-SAMP8 mice treated with ACAE. Collectively, in vitro and in vivo results showed that ACAE could promote osteogenesis and prevent bone loss and should be considered an evidence-based complementary and alternative medicine for osteoporosis therapy through the maintenance of bone health

Unambiguous molecular detections with multiple genetic approach for the complicated chromosome 22q11 deletion syndrome

<p>Abstract</p> <p>Background</p> <p>Chromosome 22q11 deletion syndrome (22q11DS) causes a developmental disorder during the embryonic stage, usually because of hemizygous deletions. The clinical pictures of patients with 22q11DS vary because of polymorphisms: on average, approximately 93% of affected individuals have a de novo deletion of 22q11, and the rest have inherited the same deletion from a parent. Methods using multiple genetic markers are thus important for the accurate detection of these microdeletions.</p> <p>Methods</p> <p>We studied 12 babies suspected to carry 22q11DS and 18 age-matched healthy controls from unrelated Taiwanese families. We determined genomic variance using microarray-based comparative genomic hybridization (array-CGH), quantitative real-time polymerase chain reaction (qPCR) and multiplex ligation-dependent probe amplification (MLPA).</p> <p>Results</p> <p>Changes in genomic copy number were significantly associated with clinical manifestations for the classical criteria of 22q11DS using MPLA and qPCR (<it>p </it>< 0.01). An identical deletion was shown in three affected infants by MLPA. These reduced DNA dosages were also obtained partially using array-CGH and confirmed by qPCR but with some differences in deletion size.</p> <p>Conclusion</p> <p>Both MLPA and qPCR could produce a clearly defined range of deleted genomic DNA, whereas there must be a deleted genome that is not distinguishable using MLPA. These data demonstrate that such multiple genetic approaches are necessary for the unambiguous molecular detection of these types of complicated genomic syndromes.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Reciprocal regulation between GCM1 and human chorionic gonadotropin controls placental cell fusion

人類絨毛膜性腺激素(hCG)在懷孕之初，由胎盤滋養葉融合層細胞所分泌。hCG對胎盤發育時期所須的黃體素､血管生成､免疫系統調節及滋養葉細胞分化扮演重要角色。母體hCG不足跟流產､胎兒發育不良及子癲前症之發生有關。hCG包括alpha及beta單元，後者是胎盤獨有並俱有代表性。哺乳動物GCM1是胎盤獨有的轉錄因子，調控胎盤細胞融合､入侵及血管生成。剔除GCM1基因之小鼠在懷孕10天即因胎盤發育不全而死亡。曾有報告指出hCGb受TFAP2及Sp1等轉錄因子所調控，但對GCM1而言是否可調控hCGb未曾被報導。 在本實驗我們透過ChIP-chip發現hCGb是GCM1的目標基因。TFAP2C､GCM1及hCGb三者在胎盤滋養葉融合層細胞有共同表現。hCGb也隨著GCM1的表現高低而同步增減。相反地，我們也證實hCG可以透過LH/CG接受器及cAMP訊息傳遞調控GCM1的轉錄､轉繹後修飾（磷酸化及乙醯化）､生物活性及其目標基因表現，包括syncytin-1及hCGb。證實GCM1對hCGb的自我調控是非常重要的。剔除LH/CG接受器或hCGb基因也會降低GCM1之表現。我們利用多種方法驗證並找出GCM1在hCGb promoter上的直接結合位置。雖然這位置與TFAP2C的結合位置相同，不過我們發現TFAP2C對hCGb的影響須經由GCM1作用。另外，LH/CG接受器-cAMP-GCM1-hCGb的訊息傳遞受到PKA抑制劑H89的阻撓，這表示cAMP-PKA為GCM1-hCGb互相調節迴路的主要路徑。最後，我們發現hCG促進BeWo細胞的融合也必須依賴GCM1才能達成。本實驗發現在hCGb調節及胎盤細胞分化上的一項新機制。Human chorionic gonadotropin (hCG) is a hormone secreted mainly by syncytiotrophoblasts governing many crucial functions of placental development, such as progesterone production from corpus lutein of ovaries in the first trimester of pregnancy, angiogenesis of uterine vasculature, immune modulation and adaptation, and cytotrophoblast differentiation. hCG contains two components, alpha and beta subunits, and the latter is placenta-specific. A low maternal serum level of hCG in early pregnancy could indicate miscarriage, fetal growth retardation, and subsequent pre-eclampsia. Maternal serum hCG levels increase dramatically from the implantation of the embryo, and reach a peak at approximately 10 weeks of gestation. Mechanisms contributing to this phenomenon are not yet known. Mammalian glial cells missing 1 (GCM1) is a placenta-specific transcription factor involved in placental cell invasion, cell-cell fusion, and angiogenesis. A knockout of mouse GCM1 induces malformation of placenta and early embryonic lethality approximately 10 dpc. It is reported that hCGb is regulated by two transcriptional factors, Sp1 and TFAP2. Whether GCM1 regulates hCGb remains elusive. In this study, we have identified that hCGb is among the GCM1 target genes through ChIP-chip analysis. We further discovered that TFAP2C, GCM1, and hCGb are co-localized at the syncytiotrophoblasts through immunohistochemical study of the placental tissue. The protein expression and secretion levels of hCGb are up- and down-regulated by GCM1 overexpression and knockdown in choriocarcinoma cell lines, respectively. hCG treatment reciprocally enhances GCM1 transcription, post-translational modification (Serine269/275 phosphorylation and acetylation), and contributes to GCM1 stability through the LH/CG receptor (LH/CGR), cAMP signaling pathway and CBP transactivation, and further enhances the target genes of GCM1, including syncytin-1 and hCGb. These data indicate that GCM1 plays an important and positive role in hCGb production and auto-regulation; either knockdown of LH/CGR or hCGb suppress GCM1 and its target gene expressions. We have identified the precise binding region of GCM1 on the hCGb promoter using a luciferase reporter assay, ChIP, and electrophoretic mobility shift assay, which was previously found to be the same binding region of TFAP2, suggesting that GCM1 and TFAP2C simultaneously regulate hCGb. Indeed, the regulation of hCGb by TFAP2C is through GCM1. The LH/CGR-cAMP-GCM1-hCGb pathway is inhibited by the PKA inhibitor, H89, indicating that the cAMP-PKA pathway is the dominant pathway in the GCM1-hCGb positive loop. Finally, by use of a functional assay, the fusion of BeWo cells was enhanced by hCG treatment, which is GCM1-dependent. Our study reveals a novel mechanism for regulating hCGb and placental cell differentiation

Connective tissue growth factor mediates transforming growth factor β-induced collagen expression in human endometrial stromal cells.

BACKGROUND:Adenomyosis is a medical condition defined by the abnormal presence of endometrial tissue within the myometrium, in which fibrosis occurs with new collagen deposition and myofibroblast differentiation. In this study, the effect of several mediators and growth factors on collagen expression was investigated on human endometrial stromal cells (fibroblasts) derived from adenomyotic endometrium. EXPERIMENTAL APPROACH:RT-PCR, Western blot analysis, pharmacological interventions and siRNA interference were applied to primary cultured human endometrial stromal cells (fibroblasts). Immunohistochemistry was used to analyze protein expression in adenomyotic endometrium tissue specimens. RESULTS:Of the tested mediators, transforming growth factor β1 (TGFβ1) and its isoforms were effective to induce collagen and connective tissue growth factor (CTGF) expression. Collagen and CTGF induction by TGFβ1 could be reduced by the inhibitors targeting DNA transcription, protein translation, and Smad2/3 signaling. Interestingly, TGFβ1 induced Smad2/3 phosphorylation and CTGF mRNA expression, but not collagen mRNA expression, suggesting that TGFβ1 mediates collagen expression through CTGF induction and Smad2/3 activation. In parallel, TGFβ1 and CTGF also induced expression of heat shock protein (HSP) 47, a protein required for the synthesis of several types of collagens. However, only CTGF siRNA knockdown, could compromise TGFβ1-induced collagen expression. Finally, the immunohistochemistry revealed vimentin- and α-SMA-positive staining for (myo)fibroblasts, TGFβ1, collagen, and CTGF in the subepithelial stroma region of human adenomyotic endometria. CONCLUSION AND IMPLICATIONS:We reveal here that TGFβ1, collagen, and CTGF are expressed in the stroma of adenomyotic endometria and demonstrate that TGFβ1 can induce collagen production in endometrium-derived fibroblasts through cellular Smad2/3-dependent signaling pathway and CTGF expression, suggesting that endometrial TGFβ may take part in the pathogenesis of adenomyosis and ectopic endometrium may participate in uterine adenomyosis

Deficiency of the intestinal enzyme acyl CoA:monoacylglycerol acyltransferase-2 protects mice from metabolic disorders induced by high-fat feeding.

Animals are remarkably efficient in absorbing dietary fat and assimilating this energy-dense nutrient into the white adipose tissue (WAT) for storage. Although this metabolic efficiency may confer an advantage in times of calorie deprivation, it contributes to obesity and associated metabolic disorders when dietary fat is abundant. Here we show that the intestinal lipid synthesis enzyme acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has a crucial role in the assimilation of dietary fat and the accretion of body fat in mice. Mice lacking MGAT2 have a normal phenotype on a low-fat diet. However, on a high-fat diet, MGAT2-deficient mice are protected against developing obesity, glucose intolerance, hypercholesterolemia and fatty livers. Caloric intake is normal in MGAT2-deficient mice, and dietary fat is absorbed fully. However, entry of dietary fat into the circulation occurs at a reduced rate. This altered kinetics of fat absorption apparently results in more partitioning of dietary fat toward energy dissipation rather than toward storage in the WAT. Thus, our studies identify MGAT2 as a key determinant of energy metabolism in response to dietary fat and suggest that the inhibition of this enzyme may prove to be a useful strategy for treating obesity and other metabolic diseases associated with excessive fat intake

Association between antenatal corticosteroid treatment and severe adverse events in pregnant women

Abstract Background Antenatal corticosteroids are considered the standard of care for pregnant women at risk for preterm birth, but studies examining their potential risks are scarce. We aimed to estimate the associations of antenatal corticosteroids with three severe adverse events: sepsis, heart failure, and gastrointestinal bleeding, in pregnant women. Methods Of 2,157,321 pregnant women, 52,119 at 24 weeks 0/7 days to 36 weeks 6/7 days of gestation were included in this self-controlled case series study during the study period of 2009–2018. We estimated incidence rates of three severe adverse events: sepsis, heart failure, and gastrointestinal bleeding. Conditional Poisson regression was used to calculate incidence rate ratios (IRRs) for comparing incidence rates of the adverse events in each post-treatment period compared to those during the baseline period among pregnant women exposed to a single course of antenatal corticosteroid treatment. Results Among 52,119 eligible participants who received antenatal corticosteroid treatment, the estimated incidence rates per 1000 person-years were 0.76 (95% confidence interval (CI): 0.69–0.83) for sepsis, 0.31 (95% CI: 0.27–0.36) for heart failure, and 11.57 (95% CI: 11.27–11.87) for gastrointestinal bleeding. The IRRs at 5 ~ 60 days after administration of antenatal corticosteroids were 5.91 (95% CI: 3.10–11.30) for sepsis and 4.45 (95% CI: 2.63–7.55) for heart failure, and 1.26 (95% CI: 1.02–1.55) for gastrointestinal bleeding; and the IRRs for days 61 ~ 180 were 2.00 (95% CI: 1.01–3.96) for sepsis, 3.65 (95% CI: 2.14–6.22) for heart failure, and 1.81 (95% CI: 1.56–2.10) for gastrointestinal bleeding. Conclusions This nationwide population-based study suggests that a single course of antenatal corticosteroids is significantly associated with a 1.3- to 5.9-fold increased risk of sepsis, heart failure, and gastrointestinal bleeding in pregnant women. Maternal health considerations, including recommendations for adverse event monitoring, should be included in future guidelines for antenatal corticosteroid treatment