Prediction of Tacrolimus Dose/Weight-Adjusted Trough Concentration in Pediatric Refractory Nephrotic Syndrome: A Machine Learning Approach.

PURPOSE: Tacrolimus (TAC) is a first-line immunosuppressant for patients with refractory nephrotic syndrome (NS). However, there is a high inter-patient variability of TAC pharmacokinetics, thus therapeutic drug monitoring (TDM) is required. In this study, we aimed to employ machine learning algorithms to investigate the impact of clinical and genetic variables on the TAC dose/weight-adjusted trough concentration (C0/D) in Chinese children with refractory NS, and then develop and validate the TAC C0/D prediction models. PATIENTS AND METHODS: The association of 82 clinical variables and 244 single nucleotide polymorphisms (SNPs) with TAC C0/D in the third month since TAC treatment was examined in 171 children with refractory NS. Extremely randomized trees (ET), gradient boosting decision tree (GBDT), random forest (RF), extreme gradient boosting (XGBoost), and Lasso regression were carried out to establish and validate prediction models, respectively. The best prediction models were validated on a cohort of 30 refractory NS patients. RESULTS: GBDT algorithm performed best in the whole group (R2=0.444, MSE=591.032, MAE=20.782, MedAE=18.980) and CYP3A5 nonexpresser group (R2=0.264, MSE=477.948, MAE=18.119, MedAE=18.771), while ET algorithm performed best in the CYP3A5 expresser group (R2=0.380, MSE=1839.459, MAE=31.257, MedAE=19.399). These prediction models included 3 clinical variables (ALB0, AGE0, and gender) and 10 SNPs (ACTN4 rs3745859, ACTN4 rs56113315, ACTN4 rs62121818, CTLA4 rs4553808, CYP3A5 rs776746, IL2RA rs12722489, INF2 rs1128880, MAP3K11 rs7946115, MYH9 rs2239781, and MYH9 rs4821478). CONCLUSION: The association between the clinical and genetic variables and TAC C0/D was described, and three TAC C0/D prediction models integrating clinical and genetic variables were developed and validated using machine learning, which may support individualized TAC dosing.

EZH2 overexpression dampens tumor-suppressive signals via an EGR1 silencer to drive breast tumorigenesis.

The mechanism underlying EZH2 overexpression in breast cancer and its involvement in tumorigenesis remain poorly understood. In this study, we developed an approach to systematically identify the trans-acting factors regulating the EZH2 expression, and identified more than 20 such factors. We revealed reciprocal regulation of early growth response 1 (EGR1) and EZH2: EGR1 activates the expression of EZH2, and EZH2 represses EGR1 expression. Using CRISPR-mediated genome/epigenome editing, we demonstrated that EHZ2 represses EGR1 expression through a silencer downstream of the EGR1 gene. Deletion of the EGR1 silencer resulted in reduced cell growth, invasion, tumorigenicity of breast cancer cells, and extensive changes in gene expression, such as upregulation of GADD45, DDIT3, and RND1; and downregulation of genes encoding cholesterol biosynthesis pathway enzymes. We hypothesize that EZH2/PRC2 acts as a "brake" for EGR1 expression by targeting the EGR1 silencer, and EZH2 overexpression dampens tumor-suppressive signals mediated by EGR1 to drive breast tumorigenesis.

CBX6 is negatively regulated by EZH2 and plays a potential tumor suppressor role in breast cancer.

Chromobox 6 (CBX6) is a subunit of Polycomb Repressive Complex 1 (PRC1) that mediates epigenetic gene repression and acts as an oncogene or tumor suppressor in a cancer type-dependent manner. The specific function of CBX6 in breast cancer is currently undefined. In this study, a comprehensive analysis of The Cancer Genome Atlas (TCGA) dataset led to the identification of CBX6 as a consistently downregulated gene in breast cancer. We provided evidence showing enhancer of zeste homolog 2 (EZH2) negatively regulated CBX6 expression in a Polycomb Repressive Complex 2 (PRC2)-dependent manner. Exogenous overexpression of CBX6 inhibited cell proliferation and colony formation, and induced cell cycle arrest along with suppression of migration and invasion of breast cancer cells in vitro. Microarray analyses revealed that CBX6 governs a complex gene expression program. Moreover, CBX6 induced significant downregulation of bone marrow stromal cell antigen-2 (BST2), a potential therapeutic target, via interactions with its promoter region. Our collective findings support a tumor suppressor role of CBX6 in breast cancer.

Histone H3.3K27M Mobilizes Multiple Cancer/Testis (CT) Antigens in Pediatric Glioma.

Lysine to methionine mutations at position 27 (K27M) in the histone H3 (H3.3 and H3.1) are highly prevalent in pediatric high-grade gliomas (HGG) that arise in the midline of the central nervous system. H3K27M perturbs the activity of polycomb repressor complex 2 and correlates with DNA hypomethylation; however, the pathways whereby H3K27M drives the development of pediatric HGG remain poorly understood. To understand the mechanism of pediatric HGG development driven by H3.3K27M and discover potential therapeutic targets or biomarkers, we established pediatric glioma cell model systems harboring H3.3K27M and performed microarray analysis. H3.3K27M caused the upregulation of multiple cancer/testis (CT) antigens, such as ADAMTS1, ADAM23, SPANXA1, SPANXB1/2, IL13RA2, VCY, and VCX3A, in pediatric glioma cells. Chromatin immunoprecipitation analysis from H3.3K27M cells revealed decreased H3K27me3 levels and increased H3K4me3 levels on the VCX3A promoter. Knockdown of VCX3A by siRNA significantly inhibited the growth of pediatric glioma cells harboring H3.3K27M. Overexpression of VCX3A/B genes stimulated the expression of several HLA genes, including HLA-A, HLA-B, HLA-E, HLA-F, and HLA-G The expression of VCX3A in pediatric HGG was confirmed using a tissue microarray. Gene set enrichment analysis revealed that CT antigens are enriched in pediatric HGG clinical specimens with H3.3K27M, with the upregulation of IL13RA2 contributing to the enrichment significantly. These results indicate that the upregulation of CT antigens, such as VCX3A and IL13RA2, correlates with pediatric gliomagenesis. Mol Cancer Res; 16(4); 623-33. ©2018 AACR.

Do different reperfusion methods affect the outcomes of stroke induced by MCAO in adult rats?

There are two patterns of ischemia/reperfusion (I/R) models used in rat middle cerebral artery occlusion (MCAO) I/R models, which differ in the use of unilateral or bilateral carotid artery reperfusion. The primary difference between the two patterns of I/R models is the complexity of the surgery procedure. However, researchers in this field have no idea whether there are any differences in outcomes of these two methods. In this study, we investigated the effects of the two methods on neurological deficits, infarct volume, blood-brain barrier (BBB) integrity and brain derived neurotrophic factor (BDNF) expression. Through evaluating the current way of bilateral common carotid artery reperfusion, we tried to find whether it could be replaced by an easier way. We found that there were no statistical significant differences between the different methods in infarct volume, neurological deficits, BBB integrity, and the level of BDNF (P > 0.05). These data demonstrated that different methods did not affect the neurological deficits, infarct volume, BBB integrity, and the BDNF protein level, which provides reference when we use an experimental stroke. These results suggest that the two methods have similar capability for inducing cerebral I/R injury and can be interchanged.

α-Lipoic acid protects against hypoxia/reoxygenation-induced injury in human umbilical vein endothelial cells through suppression of apoptosis and autophagy.

α-Lipoic acid (ALA) is known as a powerful antioxidant, which has been reported to have protective effects against various cardiovascular diseases. The present study aimed to determine whether ALA pre- or post-treatment induced protective effects against hypoxia/reoxygenation-induced injury via inhibition of apoptosis and autophagy in human umbilical vein endothelial cells (HUVECs). In order to simulate the conditions of hypoxia/reoxygenation, HUVECs were subjected to 4 h of oxygen-glucose deprivation (OGD) followed by 12 h of reoxygenation. For the pre-treatment, ALA was added to the buffer 12 h prior to OGD, whereas for the post-treatment, ALA was added at the initiation of reoxygenation. The results demonstrated that ALA pre- or post-treatment significantly reduced lactate dehydrogenase (LDH) release induced through hypoxia/reoxygenation in HUVECs in a dose-dependent manner; of note, 1 mM ALA pre- or post-treatment exhibited the most potent protective effects. In addition, ALA significantly reduced hypoxia/reoxygenation-induced loss of mitochondrial membrane potential, apoptosis and the expression of cleaved caspase-3 in HUVECs. In the presence of the specific autophagy inhibitor 3-methyladenine, hypoxia/reoxygenation-induced apoptosis was significantly reduced. Furthermore, the formation of autophagosomes, cytosolic microtubule-associated protein 1A/1B-light chain 3 ratio and beclin1 levels significantly increased following hypoxia/reoxygenation injury; however, all of these effects were ameliorated following pre- or post-treatment with ALA. The results of the present study suggested that ALA may provide beneficial protection against hypoxia/reoxygenation-induced injury via attenuation of apoptosis and autophagy in HUVECs.

Α­lipoic acid protects against cerebral ischemia/reperfusion-induced injury in rats.

It is well established that the brain is sensitive to ischemia/reperfusion (I/R)­induced injury. α­lipoic acid (LA), a free radical scavenger and antioxidant, has a neuroprotective effect against cerebral I/R­induced injury, however, the underlying mechanisms remain to be elucidated. Therefore, the present study was undertaken to evaluate whether LA was able to protect against cerebral I/R­induced injury and to examine the potential mechanisms. The neuroprotective effects of LA were investigated in a rat model of transient focal ischemia induced by middle cerebral artery occlusion (MCAO) followed by reperfusion. Adult male Sprague­Dawley rats were randomly assigned into the sham, cerebral I/R injury model and model plus LA groups. Cerebral I/R injury was induced by 90 min MCAO followed by reperfusion for 24 h. Cerebral infarct size was detected by 2,3,5­triphenyltetrazolium chloride staining. Neurological deficit score (NDS), brain water content and oxidative parameters, including malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T­AOC) and superoxide dismutase (SOD) were measured. The expression of cleaved caspase­3, brain­derived neurotrophic factor (BDNF), phosphatidylinositol­4,5­bisphosphate 3­kinase (PI3K), p­Akt and phosphorylated extracellular signal­regulated kinase 1/2 (p­ERK1/2) were also analyzed using western blotting. The present study demonstrated that pretreatment with LA significantly decreased the infarction size, brain water content and improved NDS. LA reversed the levels of oxidative parameters, including MDA, NO, T­AOC and SOD to their normal state in rat brains following cerebral I/R. Furthermore, the expression of cleaved caspase­3 markedly decreased and the expression of BDNF, PI3K, p­Akt and p­ERK1/2 significantly increased following administration of LA. On the basis of these findings, it was concluded that LA protected the brain from cerebral I/R damage by attenuation of oxidative stress and caspase­dependent apoptosis. Furthermore, LA exerts its neuroprotective effects potentially through activation of the BDNF­PI3K/Akt­ERK1/2 pathway.

Regulation of lncRNA expression.

Long non-coding RNAs (lncRNAs) are series of transcripts with important biological functions. Various diseases have been associated with aberrant expression of lncRNAs and the related dysregulation of mRNAs. In this review, we highlight the mechanisms of dynamic lncRNA expression. The chromatin state contributes to the low and specific expression of lncRNAs. The transcription of non-coding RNA genes is regulated by many core transcription factors applied to protein-coding genes. However, specific DNA sequences may allow their unsynchronized transcription with their location-associated mRNAs. Additionally, there are multiple mechanisms involved in the post-transcriptional regulation of lncRNAs. Among these, microRNAs might have indispensible regulatory effects on lncRNAs, based on recent discoveries.

Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA.

RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvß3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvß3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.

Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy.

The clinical application of small interfering RNA (siRNA) has been restricted by their poor intracellular uptake, low serum stability, and inability to target specific cells. During the last several decades, a great deal of effort has been devoted to exploring materials for siRNA delivery. In this study, biodegradable, tumor-targeted, self-assembled peptide nanoparticles consisting of cyclo(Arg-Gly-Asp-d-Phe-Lys)-8-amino-3,6-dioxaoctanoic acid-ß-maleimidopropionic acid (hereafter referred to as RPM) were found to be an effective siRNA carrier both in vitro and in vivo. The nanoparticles were characterized based on transmission electron microscopy, circular dichroism spectra, and dynamic light scattering. In vitro analyses showed that the RPM/VEGFR2-siRNA exhibited negligible cytotoxicity and induced effective gene silencing. Delivery of the RPM/VEGFR2 (zebrafish)-siRNA into zebrafish embryos resulted in inhibition of neovascularization. Administration of RPM/VEGFR2 (mouse)-siRNA to tumor-bearing nude mice led to a significant inhibition of tumor growth, a marked reduction of vessels, and a down-regulation of VEGFR2 (messenger RNA and protein) in tumor tissue. Furthermore, the levels of IFN-α, IFN-γ, IL-12, and IL-6 in mouse serum, assayed via enzyme-linked immunosorbent assay, did not indicate any immunogenicity of the RPM/VEGFR2 (mouse)-siRNA in vivo. In conclusion, RPM may provide a safe and effective delivery vector for the clinical application of siRNAs in tumor therapy.

Something between the amazing functions and various morphologies of self-assembling peptides materials in the medical field.

In recent years, there are a growing number of researches in the field of self-assembling peptides. Due to their diversity structures and the promising applications, self-assembling peptides have already become the focus of studies in the fields of materials and biological science. Some amazing functions of these peptides in the medical field caught our attention, such as tissue repair and regeneration, therapeutic delivery, haemostasis, antimicrobial and so on. There are different morphologies of self-assembling peptides in different functions. This review provides an overview of the relationship between some amazing functions and various morphologies of self-assembling peptides principally. Furthermore, the mechanisms of peptide self-assembly are also discussed.

Comparison of repaglinide and metformin versus metformin alone for type 2 diabetes: a meta-analysis of randomized controlled trials.

We conducted a meta-analysis to compare the efficacy and safety of repaglinide plus metformin with metformin alone on type 2 diabetes. Twenty-two studies were included in this meta-analysis. Results showed combination therapy was safe and could gain better outcomes in glycemic control. Well-designed studies are required to confirm this conclusion.

Hugan Qingzhi medication ameliorates hepatic steatosis by activating AMPK and PPARα pathways in L02 cells and HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hugan Qingzhi tablet (HQT), a lipid- lowering traditional Chinese medicine formula, has been used for the prevention and treatment of nonalcoholic fatty liver (NAFLD). AIM OF THE STUDY: This study was realized to evaluate the effects of HQT-medicated serum on hepatic steatosis using in vitro experiments with cells and explore the relevant mechanisms with method of serum pharmacology. MATERIALS AND METHODS: A model of hepatic steatosis in the L02 and HepG2 cells was induced by free fatty acid (FFA). The components in the HQT-medicated serum were assayed by high-performance liquid chromatography. Intracellular lipid droplets were detected by Oil Red O staining, and their ultrastructure was examined by transmission electron microscope. The biochemical parameters, including triglyceride (TG), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH), were measured with commercial kits. Furthermore, the expression of adiponectin, AMP-activated protein kinase (AMPK) phosphorylation, sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator activated receptor-α (PPARα), carnitine palmitoyltransferase 1 (CPT-1), and acetyl-CoA oxidase 1 (ACOX1) was analyzed by Western blot and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Moderate- and high-dose HQT-medicated serum reduced (P<0.05 or P<0.01) the accumulation of lipid droplets and the cellular TG content in L02 and HepG2 cells. They caused significant reductions (P<0.01) in LDH, AST, ALT and MDA and significant increase (P<0.05 or P<0.01) in T-AOC in the culture medium. They also caused increase (P<0.05 or P<0.01) in GSH level and SOD activity in FFA-induced steatotic L02 and HepG2 cells. Furthermore, moderate- and high-dose HQT-medicated serum enhanced (P<0.01) adiponectin expression in a concentration-dependent manner and increased (P<0.05 or P<0.01) the phosphorylation of AMPK and the expression of PPARα, CPT-1, and ACOX1, and reduced (P<0.05 or P<0.01) the expression of SREBP-1. CONCLUSION: The results suggested that HQT-medicated serum exerts a preventive effect against hepatic steatosis, and the potential mechanism might be activation of AMPK and PPARα pathways.

Meta-analysis of methylcobalamin alone and in combination with prostaglandin E1 in the treatment of diabetic peripheral neuropathy.

This study aimed to compare the efficacy and safety of prostaglandin E1 plus methylcobalamin (PGE1-MC) with that of methylcobalamin alone (MC) on diabetic peripheral neuropathy (DPN). We searched published randomized controlled trials (RCTs) of PGE1 combined with MC for DPN up to June 1, 2013. Data were extracted to evaluate methodological quality and describe characteristics of studies in duplicate. A random or a fixed effect model was used to analyze outcomes which were expressed as relative risk (RR) or mean difference with a 95 % confidence interval (CI). All data were analyzed using Review Manager 5.2 software. Twenty-six RCTs involving 2,107 individuals were included. Meta-analysis showed that PGE1-MC combination therapy was significantly better than MC monotherapy (RR = 1.40; 95 % CI 1.33-1.48) on efficacy. The weighted mean differences in nerve conduction velocities (NCVs) were 6.72 (95 % CI: 5.42-8.02) for median motor nerve conduction velocity (MNCV), 5.13 (CI 4.13-6.13) for median sensory nerve conduction velocity (SNCV), 5.74 (CI 4.87-6.61) for peroneal MNCV and 4.62 (CI 3.89-5.34) for peroneal SNCV in favor of the PGE1 + MC combination group. Moreover, there were no serious adverse events in both groups during the treatment period. The results of the meta-analysis show that treatment with PGE1-MC is safe and can gain better outcomes in neuropathic symptoms and NCVs compared with MC alone. However, the conclusion may not be strong because most of the studies included in this meta-analysis have poor methodological quality.

Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases.

Long noncoding RNAs (lncRNAs) have been attracting immense research interest, while only a handful of lncRNAs have been characterized thoroughly. Their involvement in the fundamental cellular processes including regulate gene expression at epigenetics, transcription, and post-transcription highlighted a central role in cell homeostasis. However, lncRNAs studies are still at a relatively early stage, their definition, conservation, functions, and action mechanisms remain fairly complicated. Here, we give a systematic and comprehensive summary of the existing knowledge of lncRNAs in order to provide a better understanding of this new studying field. lncRNAs play important roles in brain development, neuron function and maintenance, and neurodegenerative diseases are becoming increasingly evident. In this review, we also highlighted recent studies related lncRNAs in central nervous system (CNS) development and neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS), and elucidated some specific lncRNAs which may be important for understanding the pathophysiology of neurodegenerative diseases, also have the potential as therapeutic targets.

Meta-analysis of methylcobalamin alone and in combination with lipoic acid in patients with diabetic peripheral neuropathy.

AIMS: To compare the efficacy and safety of daily lipoic acid (300-600 mg i.v.) plus methylcobalamin (500-1000 mg i.v. or im.) (LA-MC) with that of methylcobalamin alone (MC) on diabetic peripheral neuropathy (DPN). METHODS: Electronic database were searched for studies published up to November 1, 2012 and study quality was assessed in duplicate. A random or a fixed effect model was used to analyse outcomes which were expressed as risk ratios (RRs) or mean difference (MD). I(2) statistic was used to assess heterogeneity. RESULTS: Seventeen studies were included. Combined data from all studies showed that the LA-MC combination therapy was significantly superior to MC monotherapy (RR=1.47; 95% CI: 1.37-1.58). Superiority of the LA-MC combination was shown in nerve conduction velocity (NCV) with WMDs of 6.89 (95% CI: 4.24-9.73) for median motor nerve conduction velocity (MNCV), 5.24 (4.14-6.34) for median sensory nerve conduction velocity (SNCV), 4.34 (3.03-5.64) for peroneal MNCV, and 4.53 (3.2-5.85) for peroneal SNCV. There were no serious adverse events associated with treatment. CONCLUSIONS: The results of the meta-analysis show that treatment with LA-MC for 2-4 weeks is associated with better outcomes in NCV and neuropathic symptoms relative to MC treatment. However larger well-designed studies are required to confirm this conclusion.