Klotho Overexpression Is Frequently Associated With Upstream Rearrangements in Fusion-Negative Phosphaturic Mesenchymal Tumors of Bone and Sinonasal Tract.

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.

CLIN_SKAT: an R package to conduct association analysis using functionally relevant variants.

BACKGROUND: Availability of next generation sequencing data, allows low-frequency and rare variants to be studied through strategies other than the commonly used genome-wide association studies (GWAS). Rare variants are important keys towards explaining the heritability for complex diseases that remains to be explained by common variants due to their low effect sizes. However, analysis strategies struggle to keep up with the huge amount of data at disposal therefore creating a bottleneck. This study describes CLIN_SKAT, an R package, that provides users with an easily implemented analysis pipeline with the goal of (i) extracting clinically relevant variants (both rare and common), followed by (ii) gene-based association analysis by grouping the selected variants. RESULTS: CLIN_SKAT offers four simple functions that can be used to obtain clinically relevant variants, map them to genes or gene sets, calculate weights from global healthy populations and conduct weighted case-control analysis. CLIN_SKAT introduces improvements by adding certain pre-analysis steps and customizable features to make the SKAT results clinically more meaningful. Moreover, it offers several plot functions that can be availed towards obtaining visualizations for interpretation of the analyses results. CLIN_SKAT is available on Windows/Linux/MacOS and is operative for R version 4.0.4 or later. It can be freely downloaded from https://github.com/ShihChingYu/CLIN_SKAT , installed through devtools::install_github("ShihChingYu/CLIN_SKAT", force=T) and executed by loading the package into R using library(CLIN_SKAT). All outputs (tabular and graphical) can be downloaded in simple, publishable formats. CONCLUSIONS: Statistical association analysis is often underpowered due to low sample sizes and high numbers of variants to be tested, limiting detection of causal ones. Therefore, retaining a subset of variants that are biologically meaningful seems to be a more effective strategy for identifying explainable associations while reducing the degrees of freedom. CLIN_SKAT offers users a one-stop R package that identifies disease risk variants with improved power via a series of tailor-made procedures that allows dimension reduction, by retaining functionally relevant variants, and incorporating ethnicity based priors. Furthermore, it also eliminates the requirement for high computational resources and bioinformatics expertise.

Comparison of DNA Methylation Changes Between the Gestation Period and the After-Delivery State: A Pilot Study of 10 Women.

Background: Gestational adaptation occurs soon after fertilization and continues throughout pregnancy, whereas women return to a pre-pregnancy state after delivery and lactation. However, little is known about the role of DNA methylation in fine-tuning maternal physiology. Understanding the changes in DNA methylation during pregnancy is the first step in clarifying the association of diet, nutrition, and thromboembolism with the changes in DNA methylation. In this study, we investigated whether and how the DNA methylation pattern changes in the three trimesters and after delivery in ten uncomplicated pregnancies. Results: DNA methylation was measured using a Human MethylationEPIC BeadChip. There were 14,018 cytosine-guanine dinucleotide (CpG) sites with statistically significant changes in DNA methylation over the four time periods (p < 0.001). Overall, DNA methylation after delivery was higher than that of the three trimesters (p < 0.001), with the protein ubiquitination pathway being the top canonical pathway involved. We classified the CpG sites into nine groups according to the changes in the three trimesters and found that 38.37% of CpG sites had DNA methylation changes during pregnancy, especially between the first and second trimesters. Conclusion: DNA methylation pattern changes between trimesters, indicating possible involvement in maternal adaptation to pregnancy. Meanwhile, DNA methylation patterns during pregnancy and in the postpartum period were different, implying that puerperium repair may also function through DNA methylation mechanisms.

The Gut Microbiome, Seleno-Compounds, and Acute Myocardial Infarction.

Background: Gut microbiome alterations might be considered a metabolic disorder. However, the relationship between the microbiome and acute myocardial infarction (AMI) has not been properly validated. Methods: The feces of 44 subjects (AMI: 19; control: 25) were collected for fecal genomic DNA extraction. The variable region V3−V4 of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. The metabolite amounts were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Results: The bacteria were more enriched in the AMI group both in the observed operational taxonomic units (OTUs) and faith phylogenetic diversity (PD) (p-value = 0.01 and <0.001 with 95% CI, individually). The Selenomonadales were less enriched in the AMI group at the family, genus, and species levels (all linear discriminant analysis (LDA) scores > 2). Seleno-compounds were more abundant in the AMI group at the family, genus, and species levels (all LDA scores > 2). Conclusions: This is the first study to demonstrate the association of Selenomonadales and seleno-compounds with the occurrence of AMI. Our findings provide an opportunity to identify a novel approach to prevent and treat AMI.

Transcript annotation tool (TransAT): an R package for retrieving annotations for transcript-specific genetic variants.

BACKGROUND: An individual's genetics play a role in how RNA transcripts are generated from DNA and consequently in their translation into protein. Transcriptional and translational profiling of patients furnishes the information that a specific marker is present; however, it fails to provide evidence whether the marker correlates with response to a therapeutic agent. A comparative analysis of the frequency of genetic variants, such as single nucleotide polymorphisms (SNPs), in diseased and general populations can identify pathogenic variants in individual patients. This is in part because SNPs have considerable effects on protein function and gene expression when they occur in coding regions and regulatory sequences, respectively. Therefore, a tool that can help users to obtain the allele frequency for a corresponding transcript is the need of the day. Several annotation tools such as SNPnexus and VariED are publicly available; however, none of them can use transcript IDs as input and provide the corresponding genomic positions of variants. RESULTS: In this study, we developed an R package, called transcript annotation tool (TransAT), that provides (i) SNP ID and genomic position for a user-provided transcript ID from patients, and (ii) allele frequencies for the SNPs from publicly available global populations. All data elements are extracted, collected, and displayed in an easily downloadable format in two simple command lines. TransAT is available on Windows/Linux/MacOS and is operative for R version 4.0.4 or later. It is available at https://github.com/ShihChingYu/TransAT and can be downloaded and installed using devtools::install_github("ShihChingYu/TransAT", force=T) on the R execution page. Thereafter, all functions can be executed by loading the package into R with library(TransAT). CONCLUSIONS: TransAT is a novel tool that seamlessly provides genetic annotations for queried transcripts. Such easily obtainable information would be greatly advantageous for physicians, assisting them to make individualized decisions about specific drug treatments. Moreover, allele frequencies from user-chosen global ethnic populations will highlight the importance of ethnicity and its effect on patient pathogenicity.

Long-term outcomes and left ventricular diastolic function of sarcomere mutation-positive and mutation-negative patients with hypertrophic cardiomyopathy: a prospective cohort study.

AIMS: Hypertrophic cardiomyopathy (HCM) is an inheritable disease that leads to sudden cardiac death and heart failure (HF). Sarcomere mutations (SMs) have been associated with HF. However, the differences in ventricular function between SM-positive and SM-negative HCM patients are poorly characterized. METHODS AND RESULTS: Of the prospectively enrolled 374 unrelated HCM patients in Taiwan, 115 patients underwent both 91 cardiomyopathy-related gene screening and cardiovascular magnetic resonance (45.6 ± 10.6 years old, 76.5% were male). Forty pathogenic/likely pathogenic mutations were identified in 52 patients by next-generation sequencing. The SM-positive group were younger at first cardiovascular event (P = 0.04) and progression to diastolic HF (P = 0.02) with higher N-terminal pro-brain natriuretic peptide (NT-proBNP) [New York Heart Association (NYHA) Class III/IV symptoms with left ventricular ejection fraction > 55%] than the SM-negative group (P < 0.001). SM-positive patients had a greater extent of late gadolinium enhancement (P = 0.01), larger left atrial diameter (P = 0.03), higher normalized peak filling rate (PFR) and PFR ratio, and a greater reduction in global longitudinal strain than SM-negative patients (all P ≤ 0.01). During mean lifelong follow-up time (49.2 ± 15.6 years), SM-positive was a predictor of earlier HF (NYHA Class III/IV symptoms) after multivariate adjustment (hazard ratio 3.5; 95% confidence interval 1.3-9.7; P = 0.015). CONCLUSION: SM-positive HCM patients had a higher extent of myocardial fibrosis and more severe ventricular diastolic dysfunction than those without, which may contribute to earlier onset of advanced HF, suggesting the importance of close surveillance and early treatment throughout life.

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-ß/-γ (PPAR-ß/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-ß/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-ß/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-ß/-γ antagonists. Collectively, these results demonstrate that PPAR-ß/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

Antiplatelet activity of nifedipine is mediated by inhibition of NF-κB activation caused by enhancement of PPAR-ß/-γ activity.

BACKGROUND AND PURPOSE: The transcription factor NF-κB, stimulates platelet aggregation through a non-genomic mechanism. Nifedipine, a voltage-gated L-type calcium channel blocker, is widely used to treat hypertension. Nifedipine also displays antiplatelet activity, but the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiplatelet effects of nifedipine are mediated by regulating NF-κB-dependent responses. EXPERIMENTAL APPROACH: Platelet aggregation was measured turbidimetrically using an aggregometer. NF-κB and PPAR activation, intracellular Ca2+ mobilization, PKCα activity, surface glycoprotein IIb/IIIa (GPIIb/IIIa) expression and platelet activation-related signalling pathways were determined in control and nifedipine-treated platelets in the presence or absence of PPAR antagonists or betulinic acid, a NF-κB activator. KEY RESULTS: Exposure of platelets to nifedipine significantly increased the PPAR-ß/-γ activity in activated human platelets. Treatment with nifedipine reduced collagen-induced NF-κB events, including the phosphorylation of IκB kinase-ß, IκBα and p65NF-κB, which were markedly attenuated by GSK0660, a PPAR-ß antagonist, or GW9662, a PPAR-γ antagonist. Furthermore, the interaction of PPAR-ß/-γ with NF-κB and the PPAR-ß/-γ-up-regulated NO/cGMP/PKG1 cascade may contribute to inhibition of NF-κB activation by nifedipine. Suppressing PPAR-ß/-γ activity or increasing NF-κB activation greatly reversed the inhibitory effect of nifedipine on collagen-induced platelet aggregation, intracellular Ca2+ mobilization, PKCα activity and surface GPIIb/IIIa expression.CONCLUSIONS AND IMPLICATIONSPPAR-ß/-γ-dependent inhibition of NF-κB activation contributes to the antiplatelet activity of nifedipine. These findings provide a novel mechanism underlying the beneficial effects of nifedipine on platelet hyperactivity-related vascular and inflammatory diseases.

Mechanisms of antiplatelet activity of nifedipine: role of peroxisome proliferator-activated receptor-ß-γ-dependent processes.

OBJECTIVE: Nifedipine, an L-type calcium channel blocker, is widely used in the treatment of hypertension and coronary heart diseases, and also exhibits an antiplatelet activity. Activation of peroxisome proliferator-activated receptors (PPARs; α, ß/δ, and γ) inhibits the platelet aggregation. Therefore, the purpose of this study was to evaluate the contribution of PPAR-mediated processes to the antiplatelet activity of nifedipine. METHODS AND RESULTS: We assessed human platelet aggregation by using an aggregometer and measured several platelet activating markers and related signaling pathways in platelets treated with nifedipine in the presence or absence of PPAR agonists. Nifedipine treatment (1, 5  µmol/l) dose-dependently increased the activity and intracellular expression of PPAR-ß/-γ by inhibiting the release of PPAR-ß/-γ from activated platelets. Nifedipine treatment also upregulated cyclic 3',5'-cyclic monophosphate (GMP)/protein kinase G (PKG) expression, and increased PI(3)K/Akt pathway, endothelial nitric oxide synthase, and soluble guanylyl cyclase activities. In the presence of a selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibitory effects of nifedipine on collagen-induced platelet aggregation, intracellular Ca mobilization, and protein kinase C (PKC-α) activation were abrogated. Similarly, PPAR-ß-γ antagonists markedly attenuated nifedipine-mediated upregulation of nitric oxide/cyclic GMP/PKG cascade. In a mouse model of thrombosis, the administration of nifedipine substantially inhibited fluorescein sodium-induced vessel thrombus formation; however, the antithrombotic effect was considerably reduced in the presence of PPAR-ß/-γ antagonists. CONCLUSION: This study is the first to show that the PPAR-ß/-γ-dependent upregulation of PI(3)K/Akt/nitric oxide/cyclic GMP/PKG pathway and the inhibition of PKC-α activity and intracellular Ca(+) mobilization in platelets may be the mechanisms underlying the antiplatelet and antithrombotic activities of nifedipine.

The antiplatelet activity of magnolol is mediated by PPAR-ß/γ.

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, ß/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 µM) dose-dependently enhanced the activity and intracellular level of PPAR-ß/γ in platelets. In the presence of selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-ß/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-ß/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-ß/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-ß/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-ß/γ-dependent pathways.

Detailed balance model for intermediate band solar cells with photon conservation.

We developed a comprehensive detailed balance model of intermediate band solar cell (IBSC). The key feature of our model is based on the conservation of photons in solar spectrum. Together with parametric analysis of carrier partition, we calculated the power conversion efficiency and found an enhancement of 1.5 times in wide band gap material IBSC (such as GaN). On the other hand, this model can also explain the inferior performance of GaAs-based IBSC through the degradation of open-circuit voltages, which can be attributed to the strong non-radiative recombination and the increased photo-generated carriers. The resulting maximum efficiency is complied with the classical Shockley-Queisser limit, and should be considered for the future IBSC design.

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs.

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, ß/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.

Antiplatelet activity of alpha-lipoic acid.

Alpha-lipoic acid (ALA) is often used as a dietary supplement to prevent and treat chronic diseases associated with excessive oxidative stress. The aim of this study was to investigate the mechanisms of the antiplatelet activity of ALA. ALA significantly inhibited collagen-induced platelet aggregation, thromboxane B(2) (TXB(2)) formation, Ca(2+) mobilization, and protein kinase Calpha (PKCalpha) activation, but ALA itself increased cyclic AMP formation in rabbit washed platelets. However, the effects of ALA on the above platelet responses were markedly reversed by the addition of 2'5'-ddAdo, an adenylate cyclase inhibitor. Additionally, increased reactive oxygen species (ROS) formation and cyclooxygenase-1 activity stimulated by arachidonic acid were inhibited by ALA. In conclusion, we demonstrated that ALA possesses an antiplatelet activity, which may be associated with an elevation of cyclic AMP formation, involving subsequent inhibition of TXA(2), Ca(2+) mobilization, and PKCalpha-mediated pathways. Moreover, inhibition of ROS formation and increase of platelet membrane fluidity may also involve its actions.