Epigenome-wide association study of diabetic chronic kidney disease progression in the Korean population: the KNOW-CKD study.

Since the etiology of diabetic chronic kidney disease (CKD) is multifactorial, studies on DNA methylation for kidney function deterioration have rarely been performed despite the need for an epigenetic approach. Therefore, this study aimed to identify epigenetic markers associated with CKD progression based on the decline in the estimated glomerular filtration rate in diabetic CKD in Korea. An epigenome-wide association study was performed using whole blood samples from 180 CKD recruited from the KNOW-CKD cohort. Pyrosequencing was also performed on 133 CKD participants as an external replication analysis. Functional analyses, including the analysis of disease-gene networks, reactome pathways, and protein-protein interaction networks, were conducted to identify the biological mechanisms of CpG sites. A phenome-wide association study was performed to determine the associations between CpG sites and other phenotypes. Two epigenetic markers, cg10297223 on AGTR1 and cg02990553 on KRT28 indicated a potential association with diabetic CKD progression. Based on the functional analyses, other phenotypes (blood pressure and cardiac arrhythmia for AGTR1) and biological pathways (keratinization and cornified envelope for KRT28) related to CKD were also identified. This study suggests a potential association between the cg10297223 and cg02990553 and the progression of diabetic CKD in Koreans. Nevertheless, further validation is needed through additional studies.

Identification of a novel PARP4 gene promoter CpG locus associated with cisplatin chemoresistance.

The protein family of poly (ADP-ribose) polymerases (PARPs) is comprised of multifunctional nuclear enzymes. Several PARP inhibitors have been developed as new anticancer drugs to combat resistance to chemotherapy. Herein, we characterized PARP4 mRNA expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. PARP4 mRNA expression was significantly upregulated in cisplatin-resistant ovarian cancer cell lines, and this upregulation was associated with the hypomethylation of specific cytosine-phosphate-guanine (CpG) sites (cg18582260 and cg17117459) on its promoter. Reduced PARP4 expression was restored by treating cisplatin-sensitive cell lines with a demethylation agent, implicating the epigenetic regulation of PARP4 expression by promoter methylation. Depletion of PARP4 expression in cisplatin-resistant cell lines reduced cisplatin chemoresistance and promoted cisplatin-induced DNA fragmentation. The differential mRNA expression and DNA methylation status at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) according to cisplatin responses, was further validated in primary ovarian tumor tissues. The results showed significantly increased PARP4 mRNA expressions and decreased DNA methylation levels at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) in cisplatin-resistant patients. Additionally, the DNA methylation status at cg18582260 CpG sites in ovarian tumor tissues showed fairly clear discrimination between cisplatin-resistant patients and cisplatin-sensitive patients, with high accuracy (area under the curve = 0.86, P = 0.003845). Our findings suggest that the DNA methylation status of PARP4 at the specific promoter site (cg18582260) may be a useful diagnostic biomarker for predicting the response to cisplatin in ovarian cancer patients. [BMB Reports 2023; 56(6): 347-352].

Novel Genetic Variants Associated with Chronic Kidney Disease Progression.

SIGNIFICANCE STATEMENT: eGFR slope has been used as a surrogate outcome for progression of CKD. However, genetic markers associated with eGFR slope among patients with CKD were unknown. We aimed to identify genetic susceptibility loci associated with eGFR slope. A two-phase genome-wide association study identified single nucleotide polymorphisms (SNPs) in TPPP and FAT1-LINC02374 , and 22 of them were used to derive polygenic risk scores that mark the decline of eGFR by disrupting binding of nearby transcription factors. This work is the first to identify the impact of TPPP and FAT1-LINC02374 on CKD progression, providing predictive markers for the decline of eGFR in patients with CKD. BACKGROUND: The incidence of CKD is associated with genetic factors. However, genetic markers associated with the progression of CKD have not been fully elucidated. METHODS: We conducted a genome-wide association study among 1738 patients with CKD, mainly from the KoreaN cohort study for Outcomes in patients With CKD. The outcome was eGFR slope. We performed a replication study for discovered single nucleotide polymorphisms (SNPs) with P <10 -6 in 2498 patients with CKD from the Chronic Renal Insufficiency Cohort study. Several expression quantitative trait loci (eQTL) studies, pathway enrichment analyses, exploration of epigenetic architecture, and predicting disruption of transcription factor (TF) binding sites explored potential biological implications of the loci. We developed and evaluated the effect of polygenic risk scores (PRS) on incident CKD outcomes. RESULTS: SNPs in two novel loci, TPPP and FAT1-LINC02374 , were replicated (rs59402340 in TPPP , Pdiscovery =7.11×10 -7 , PCRIC =8.13×10 -4 , Pmeta =7.23×10 -8 ; rs28629773 in FAT1-LINC02374 , Pdiscovery =6.08×10 -7 , PCRIC =4.33×10 -2 , Pmeta =1.87×10 -7 ). The eQTL studies revealed that the replicated SNPs regulated the expression level of nearby genes associated with kidney function. Furthermore, these SNPs were near gene enhancer regions and predicted to disrupt the binding of TFs. PRS based on the independently significant top 22 SNPs were significantly associated with CKD outcomes. CONCLUSIONS: This study demonstrates that SNP markers in the TPPP and FAT1-LINC02374 loci could be predictive markers for the decline of eGFR in patients with CKD.

Increased Caveolin-2 Expression in Brain Endothelial Cells Promotes Age-Related Neuroinflammation.

Aging is a major risk factor for common neurodegenerative diseases. Although multiple molecular, cellular, structural, and functional changes occur in the brain during aging, the involvement of caveolin-2 (Cav-2) in brain ageing remains unknown. We investigated Cav-2 expression in brains of aged mice and its effects on endothelial cells. The human umbilical vein endothelial cells (HUVECs) showed decreased THP-1 adhesion and infiltration when treated with Cav-2 siRNA compared to control siRNA. In contrast, Cav-2 overexpression increased THP-1 adhesion and infiltration in HUVECs. Increased expression of Cav-2 and iba-1 was observed in brains of old mice. Moreover, there were fewer iba-1-positive cells in the brains of aged Cav-2 knockout (KO) mice than of wild-type aged mice. The levels of several chemokines were higher in brains of aged wild-type mice than in young wild-type mice; moreover, chemokine levels were significantly lower in brains of young mice as well as aged Cav-2 KO mice than in their wild-type counterparts. Expression of PECAM1 and VE-cadherin proteins increased in brains of old wild-type mice but was barely detected in brains of young wild-type and Cav-2 KO mice. Collectively, our results suggest that Cav-2 expression increases in the endothelial cells of aged brain, and promotes leukocyte infiltration and age-associated neuroinflammation.

Identification of Serum Metabolites for Predicting Chronic Kidney Disease Progression according to Chronic Kidney Disease Cause.

Early detection and proper management of chronic kidney disease (CKD) can delay progression to end-stage kidney disease. We applied metabolomics to discover novel biomarkers to predict the risk of deterioration in patients with different causes of CKD. We enrolled non-dialytic diabetic nephropathy (DMN, n = 124), hypertensive nephropathy (HTN, n = 118), and polycystic kidney disease (PKD, n = 124) patients from the KNOW-CKD cohort. Within each disease subgroup, subjects were categorized as progressors (P) or non-progressors (NP) based on the median eGFR slope. P and NP pairs were randomly selected after matching for age, sex, and baseline eGFR. Targeted metabolomics was performed to quantify 188 metabolites in the baseline serum samples. We selected ten progression-related biomarkers for DMN and nine biomarkers each for HTN and PKD. Clinical parameters showed good ability to predict DMN (AUC 0.734); however, this tendency was not evident for HTN (AUC 0.659) or PKD (AUC 0.560). Models constructed with selected metabolites and clinical parameters had better ability to predict CKD progression than clinical parameters only. When selected metabolites were used in combination with clinical indicators, random forest prediction models for CKD progression were constructed with AUCs of 0.826, 0.872, and 0.834 for DMN, HTN, and PKD, respectively. Select novel metabolites identified in this study can help identify high-risk CKD patients who may benefit from more aggressive medical treatment.

Surface Stabilization of Ni-Rich Layered Cathode Materials via Surface Engineering with LiTaO3 for Lithium-Ion Batteries.

Recently, Ni-rich layered cathode materials have become the most common material used for lithium-ion batteries. From a structural viewpoint, it is crucial to stabilize the surface structures of such materials, as they are prone to undesirable side reactions and particle cracking in which intergranular microcracks form at the particle surfaces and then propagate inside. As a simplified engineering technique for obtaining Ni-rich cathode materials with high reversibility and long-term cycling stability, we propose a facile surface coating of piezoelectric LiTaO3 onto a Ni-rich cathode material to enhance the charge transfer reaction and surface structural integrity. Based on theoretical and experimental investigation, we demonstrate that this surface protection approach is effective at enhancing the reversibility and mechanical strength of Ni-rich cathode materials, leading to a stable cycle performance at up to 150 cycles, even at 60 °C. Furthermore, the piezoelectric characteristics of the surface LiTaO3 can enhance the rate capability of Ni-rich cathode materials at current densities of up to 2.0C. The results of this study provide a practical insight on the development of Ni-rich cathode materials for practical use in electric vehicle applications.

Truncated Milk Fat Globule-EGF-like Factor 8 Ameliorates Liver Fibrosis via Inhibition of Integrin-TGFß Receptor Interaction.

Milk fat globule-EGF factor 8 (MFG-E8) protein is known as an immunomodulator in various diseases, and we previously demonstrated the anti-fibrotic role of MFG-E8 in liver disease. Here, we present a truncated form of MFG-E8 that provides an advanced therapeutic benefit in treating liver fibrosis. The enhanced therapeutic potential of the modified MFG-E8 was demonstrated in various liver fibrosis animal models, and the efficacy was further confirmed in human hepatic stellate cells and a liver spheroid model. In the subsequent analysis, we found that the modified MFG-E8 more efficiently suppressed transforming growth factor ß (TGF-ß) signaling than the original form of MFG-E8, and it deactivated the proliferation of hepatic stellate cells in the liver disease environment through interfering with the interactions between integrins (αvß3 & αvß5) and TGF-ßRI. Furthermore, the protein preferentially delivered in the liver after administration, and the safety profiles of the protein were demonstrated in male and female rat models. Therefore, in conclusion, this modified MFG-E8 provides a promising new therapeutic strategy for treating fibrotic diseases.

Protective role of ABCA1 in ischemic preconditioning is mediated by downregulation of miR-33-5p and miR-135-5p.

Ischemic preconditioning (IPC) significantly reduces ischemia-reperfusion injury in the brain by inducing ischemic tolerance. Although emerging evidence suggests that microRNAs (miRNAs) contribute to the pathogenesis of brain ischemia and IPC-induced neuroprotection, the role of miRNAs and their underlying mechanisms are still unclear. IPC was induced in male C57BL/6 mice by brief bilateral common carotid artery occlusion. After 24 h, mice underwent transient middle cerebral artery occlusion followed by 3 h of reperfusion. Expression levels of messenger RNAs (mRNAs) and proteins were examined in the ipsilateral cortex, and mimics and inhibitors of selective miRNAs were transfected into Neuro-2a cells before oxygen-glucose deprivation (OGD). Post-IPC miRNA expression profiling identified neuroprotection-associated changes in miRNA expression in the ipsilateral cortex after ischemic stroke. Among them, miR-33-5p and miR-135b-5p were significantly downregulated by IPC. Inhibition of miR-33-5p and miR-135b-5p expression protected Neuro-2a cells from OGD-induced apoptosis. Inhibition of these two miRNAs significantly increased mRNA and protein levels of ATP-binding cassette subfamily A member 1 (ABCA1), and a binding assay showed that these two miRNAs showed specificity for Abca1 mRNA. Overexpression of ABCA1 decreased the Bax/Bcl2 mRNA ratio and activation of caspase-9 and caspase-3, whereas knockdown of ABCA1 expression increased the Bax/Bcl2 mRNA ratio and the percentage of Neuro-2a cells with a loss of mitochondrial membrane potential after OGD-treatment. In conclusion, ABCA1 expression is regulated by miR-33-5p and miR-135b-5p. Increased ABCA1 expression following IPC exerts a protective influence against cerebral ischemia via suppression of a mitochondria-dependent apoptosis pathway.

Dose Optimization of Vancomycin Using a Mechanism-based Exposure-Response Model in Pediatric Infectious Disease Patients.

PURPOSE: This study aimed to determine the appropriate vancomycin dosage, considering patient size and organ maturation, by simulating the bacterial count and biomarker level for drug administration in pediatric patients with gram-positive bacterial (GPB) infections. METHODS: Natural language processing for n-gram analysis was used to detect appropriate pharmacodynamic (PD) markers in infectious disease patients. In addition, a mechanism-based model was established to describe the systemic exposure and evaluate the PD marker simultaneously in pediatric patients. A simulation study was then conducted by using a mechanism-based model to evaluate the optimal dose of vancomycin in pediatric patients. FINDINGS: C-reactive protein (CRP) was selected as a PD marker from an analysis of ~270,000 abstracts in PubMed. In addition, clinical results, including the vancomycin plasma concentrations and CRP levels of pediatric patients (n = 93), were collected from electronic medical records. The vancomycin pharmacokinetic model with allometric scaling and a maturation function was built as a one-compartment model, with an additional compartment for bacteria. Both the effects of vancomycin plasma concentrations on the destruction of bacteria and those of bacteria on CRP production rates were represented by using a maximum achievable effect model (Emax model). Simulation for dose optimization was conducted not only by using the final model but also by exploring the possibility of therapeutic failure based on the MICs of vancomycin for GPB. Clinical cure was defined as when the CRP level fell below the upper limit of the normal range. Our dose optimization simulations suggested a vancomycin dosage of 10 mg/kg every 8 h as the optimal maintenance dose for pediatric patients with a postconceptual age <30 weeks and 10 mg/kg every 6 h for older children, aged up to 12 years. In addition, the MIC of 3 µg/mL was assessed as the upper concentration limit associated with successful vancomycin treatment of GPB infections. IMPLICATIONS: This study confirmed that the changes in bacterial counts and CRP levels were well described with mechanistic exposure-response modeling of vancomycin. This model can be used to determine optimal empiric doses of vancomycin and to improve therapeutic outcomes in pediatric patients with GPB.

Enhanced repair processes and iron uptake by ischemic preconditioning in the brain during the recovery phase after ischemic stroke.

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood-brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.

The effects of extracting procedures on occurrence of polycyclic aromatic hydrocarbons in edible oils.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most hazardous naturally occurring chemical contaminants of food. Edible oils are easily contaminated by PAHs generated during high-temperature processing steps such as oil extraction and refining. In this study, the effects of different extraction methods on the levels of PAHs in sesame oils and red pepper seed oils were assessed. GC-MS was used to determine the levels of PAHs in edible oils. Sesame oils extracted from seeds by plate-pressing extraction method had lower levels of PAHs than those extracted by screw-expeller extraction method from sesame flour. Furthermore, the levels of PAHs increased by 62.2% when the extraction time was longer. Notably, the PAHs already present in oils could be effectively reduced by refining procedures such as sinking, centrifugation, filtration, and neutralization with alkali.

Ahnak scaffolds p11/Anxa2 complex and L-type voltage-gated calcium channel and modulates depressive behavior.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the ß subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

Correction to: The small molecule WNT/ß-catenin inhibitor CWP232291 blocks the growth of castration-resistant prostate cancer by activating the endoplasmic reticulum stress pathway.

In the original publication of this article [1], there are mistakes in Fig. 4d. The corrected Fig. 4 should be.

The small molecule WNT/ß-catenin inhibitor CWP232291 blocks the growth of castration-resistant prostate cancer by activating the endoplasmic reticulum stress pathway.

BACKGROUND: Androgen receptor (AR)-targeted treatments improve the survival of castration-resistant prostate cancer (CRPC) patients; however, secondary resistance to these agents ultimately occurs in virtually all patients. Therefore, alternative therapeutic targets are urgently needed. Since growing evidence demonstrates that WNT/ß-catenin signaling plays an important role in CRPC, the antitumor activity and mechanism of action of CWP232291, a small molecule ß-catenin inhibitor, were investigated in prostate cancer. METHODS: We assessed the antitumor activity of CWP232291 in prostate cancer cell lines and primary cells derived from CRPC patients. The effect of CWP232291 on apoptotic cell death, endoplasmic reticulum (ER) stress, cell viability, and WNT/ß-catenin signaling was evaluated by flow cytometry, western blotting, luciferase reporter assay, and fluorescence microscopy. Antitumor efficacy was assessed in two CRPC xenograft mouse models. RESULTS: CWP232291 induced ER stress, resulting in upregulation of the proapoptotic protein CHOP and activation of caspase-3-dependent apoptosis. In addition, CWP232291 suppressed the expression of ß-catenin by affecting WNT-dependent transcriptional activity, and downregulated AR and its splice variants in prostate cancer cells. Antitumor activity was observed in prostate cancer cells in vitro and ex vivo, and antitumor efficacy was observed in vivo. CONCLUSIONS: Beyond providing preclinical evidence of therapeutic efficacy for the novel small molecule ß-catenin inhibitor CWP232291 in CRPC, our results show that inducing ER stress and targeting WNT/ß-catenin signaling may be a novel strategy against CRPC.

Expansion of EHR-Based Common Data Model (CDM).

We expanded and constructed a Common Data Model (CDM) based on hospital EHR to enable analysis and comparison of Adverse Drug Reactions(ADRs) integrated with external organizations with different data structures. This is significant in that it is possible to conduct joint research, analysis, and comparisons among institutions with the same type of CDM constructed, and provide the basis for conducting the same research simultaneously on various data sources.

SPSB1 enhances ovarian cancer cell survival by destabilizing p21.

SPRY domain-containing SOCS box protein 1 (SPSB1) is an E3 ligase adaptor protein with unknown functions in cancer cells. In this study, we found that SPSB1 knockdown markedly decreased the viability and migration of ovarian cancer cells, while ectopic SPSB1 overexpression in IL-3-dependent Ba/F3 cells significantly increased their proliferation rate compared with empty vector-transfected cells. SPSB1 knockdown significantly elevated p21 protein and mRNA levels and induced apoptosis in ovarian cancer cells, as evidenced by increased levels of cleaved PARP and decreased levels of Bcl-2. Notably, mechanistic investigations revealed that SPSB1 accelerated p21 destabilization by directly interacting with p21 and promoting its ubiquitin-mediated proteasomal degradation. Taken together, our findings provide novel insights into the role of SPSB1 in ovarian cancer cells.

Anti-inflammatory and antioxidant mechanisms of urolithin B in activated microglia.

BACKGROUND: Urolithin B is one of the gut microbial metabolites of ellagitannins and is found in diverse plant foods, including pomegranates, berries, walnuts, tropical fruits, and medicinal herbs. Although a number of biological activities of urolithin B have been reported, the anti-inflammatory and antioxidant effects of urolithin B in neuroinflammation have not been clearly demonstrated. PURPOSE: The present study aimed to investigate the anti-inflammatory and antioxidant effects of urolithin B in activated microglia and define its underlying molecular mechanisms. STUDY DESIGN: The effects of urolithin B on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and cytokines were examined in BV2 microglial cells using enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Microglial activation in the lipopolysaccharide (LPS)-injected mouse brain was assessed using immunohistochemistry. The detailed molecular mechanisms underlying the anti-inflammatory and antioxidant effects of urolithin B were analyzed using an electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR. RESULTS: Urolithin B inhibited the production of NO and pro-inflammatory cytokines, while increased anti-inflammatory cytokine IL-10 in LPS-stimulated BV2 microglial cells. In addition, urolithin B inhibited NO, TNF-α, and IL-6 production in lipoteichoic acid (LTA) or polyinosinic-polycytidylic acid (poly(I:C))-stimulated BV2 cells, suggesting that the anti-inflammatory effect of urolithin B is not confined to LPS stimulation. Urolithin B also showed an antioxidant effect by reducing intracellular reactive oxygen species (ROS) production and NADPH oxidase subunit expression, and by upregulating the antioxidant hemeoxygenase-1 expression via Nrf2/ARE signaling. More detailed mechanistic studies showed that urolithin B inhibited NF-κB activity by reducing the phosphorylation and degradation of IκBα. In addition, urolithin B suppressed the phosphorylation of JNK, ERK, and Akt, and enhanced the phosphorylation of AMPK, which is associated with anti-inflammatory and antioxidant processes. Finally, we demonstrated that urolithin B suppressed microglia activation in LPS-injected mouse brains. CONCLUSIONS: The strong anti-inflammatory and antioxidant effects of urolithin B may provide therapeutic potential for neuroinflammatory disorders that are associated with oxidative stress and microglial activation.

Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker.

BACKGROUND AND PURPOSE: The pathogenesis of moyamoya disease (MMD) remains poorly understood, and no reliable molecular biomarkers for MMD have been identified to date. The present study aimed to identify epigenetic biomarkers for use in the diagnosis of MMD. METHODS: We performed integrated analyses of gene expression profiles and DNA methylation profiles in endothelial colony forming cells (ECFCs) from three patients with MMD and two healthy individuals. Candidate gene mRNA expression and DNA methylation status were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and pyrosequencing analysis of an expanded ECFC sample set from nine patients with MMD and ten controls. We evaluated the diagnostic accuracy of the potential biomarkers identified here using receiver operating characteristic curve analyses and further measured major angiogenic factor expression levels using a tube formation assay and RT-qPCR. RESULTS: Five candidate genes were selected via integrated analysis; all five were upregulated by hypomethylation of specific promoter CpG sites. After further validation in an expanded sample set, we identified a candidate biomarker gene, sortilin 1 (SORT1). DNA methylation status at a specific SORT1 promoter CpG site in ECFCs readily distinguished patients with MMD from the normal controls with high accuracy (area under the curve 0.98, sensitivity 83.33%, specificity 100%). Furthermore, SORT1 overexpression suppressed endothelial cell tube formation and modulated major angiogenic factor and matrix metalloproteinase-9 expression, implying SORT1 involvement in MMD pathogenesis. CONCLUSION: s Our findings suggest that DNA methylation status at the SORT1 promoter CpG site may be a potential biomarker for MMD.

C16­ceramide and sphingosine 1­phosphate/S1PR2 have opposite effects on cell growth through mTOR signaling pathway regulation.

Recently, sphingolipid derivatives, such as ceramide and sphingosine­1­phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF­7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal­regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF­7 cell proliferation. This effect was partially reversed by co­treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6­mediated decrease in MCF­7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF­7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16­ceramide, which is generated by CerS6.

Peripheral cannabinoid 1 receptor blockade mitigates adipose tissue inflammation via NLRP3 inflammasome in mouse models of obesity.

AIM: To analyze the metabolic parameters and adipose tissue inflammation via NLRP3 inflammasome following chronic treatment of mouse models of obesity with AJ5018 as the peripherally restricted cannabinoid 1 receptor (CB1R) antagonist. MATERIALS AND METHODS: The selectivity for CB1R over CB2R, brain/plasma concentration ratio, and centrally mediated neurobehavioural effects of AJ5018, were assessed. The long-term effects of AJ5018 and rimonabant on the metabolic parameters and adipose tissue inflammation were analyzed in diet-induced obese (DIO) mice and diabetic db/db mice. RESULTS: AJ5018 had a higher degree of selectivity for CB1R over CB2R and markedly reduced brain penetrance, as reflected by the lower brain/plasma concentration ratio and the attenuated centrally mediated neurobehavioural effects, compared with its brain-penetrant parent compound rimonabant. In DIO and db/db mice, AJ5018 exhibited comparable effects to rimonabant in improving metabolic abnormalities and suppressing macrophage infiltration into white adipose tissue, activation of the NLRP3 inflammasome, and production of proinflammatory cytokines. CONCLUSIONS: These results suggest that peripheral CB1R blockade improves obesity-induced insulin resistance by suppressing adipose tissue inflammation via the NLRP3 inflammasome.