Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection.

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.

Induction of ROS Overload by Alantolactone Prompts Oxidative DNA Damage and Apoptosis in Colorectal Cancer Cells.

Cancer cells typically display higher than normal levels of reactive oxygen species (ROS), which may promote cancer development and progression but may also render the cancer cells more vulnerable to further ROS insult. Indeed, many of the current anticancer therapeutics kill cancer cells via induction of oxidative stress, though they target both cancer and normal cells. Recently, alantolactone (ATL), a natural sesquiterpene lactone, has been shown to induce apoptosis by increasing ROS levels specifically in cancer cells; however, the molecular mechanisms linking ROS overproduction to apoptosis remain unclear. Here we show that the ATL-induced ROS overload in human SW480 and SW1116 colorectal cancer cells was followed by a prominent accumulation of cellular oxidized guanine (8-oxoG) and immediate increase in the number of DNA strand breaks, indicating that increased ROS resulted in extensive oxidative DNA damage. Consequently, the G1/S-CDK suppresser CDKN1B (p21) and pro-apoptotic proteins Bax and activated caspase-3 were upregulated, while anti-apoptotic Bcl-2 was downregulated, which were followed by cell cycle arrest at G1 and marked apoptosis in ATL-treated cancer but not non-cancer cells. These results suggest that the ATL-induced ROS overload triggers cell death through induction of massive oxidative DNA damage and subsequent activation of the intrinsic apoptosis pathway.

Echinacoside Induces Apoptosis in Human SW480 Colorectal Cancer Cells by Induction of Oxidative DNA Damages.

Echinacoside is a natural compound with potent reactive oxygen species (ROS)-scavenging and anti-oxidative bioactivities, which protect cells from oxidative damages. As cancer cells are often under intense oxidative stress, we therefore tested if Echinacoside treatment would promote cancer development. Surprisingly, we found that Echinacoside significantly inhibited the growth and proliferation of a panel of cancer cell lines. Treatment of the human SW480 cancer cells with Echinacoside resulted in marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 and cleaved PARP, and upregulation of the G1/S-CDK blocker CDKN1B (p21). Interestingly, immunocytochemistry examination of drug-treated cancer cells revealed that Echinacoside caused a significant increase of intracellular oxidized guanine, 8-oxoG, and dramatic upregulation of the double-strand DNA break (DSB)-binding protein 53BP1, suggesting that Echinacoside induced cell cycle arrest and apoptosis in SW480 cancer cells via induction of oxidative DNA damages. These results establish Echinacoside as a novel chemical scaffold for development of anticancer drugs.

A regulatory variant in CYP2E1 affects the risk of lung squamous cell carcinoma.

Cytochrome P450 2E1 (CYP2E1), an ethanol-inducible enzyme, has been shown to metabolically activate various carcinogens, which is critical for the development of cancers. It has been demonstrated that CYP2E1 polymorphisms alter the transcriptional activity. However, studies on the association between CYP2E1 -1239G>C polymorphism and non-small cell lung cancer have reported conflicting results. Thus, the gain of the present study was to investigate whether CYP2E1 -1239G>C polymorphism is associated with the development of non-small cell lung cancer in Chinese population. A case-control study was conducted in which CYP2E1 -1239G>C polymorphism was analyzed in 526 Chinese patients with non-small cell lung cancer and 526 age-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism. Odds ratios were estimated by multivariate logistic regression. A meta-analysis was conducted to evaluate the association of CYP2E1 -1239G>C polymorphism with the risk of lung cancer in Chinese population by calculating pooled odds ratio (OR). For CYP2E1 -1239G>C polymorphism, -1239C allele carriers (OR = 0.67; 95% confidence interval (CI) = 0.51-0.87; P = 0.002) were associated with a decreased risk of non-small cell lung cancer when compared with -1239GG homozygotes. In the group analyses by pathological types, for lung squamous cell carcinoma and other types, the ORs of the C allele carriers were 0.60 (95% CI = 0.41-0.88; P = 0.009) and 0.54 (95% CI = 0.30-0.99; P = 0.045). In the group analysis of smoking status, the OR for the -1239C allele-containing genotype was higher than that for -1239GG genotype (OR = 0.57; 95% CI = 0.40-0.81; P = 0.002) among smokers, but not among nonsmokers. Moreover, when the risk associated with CYP2E1 polymorphism was further evaluated within strata of <25 and ≥25 pack-years smoked, this effect between susceptible genotypes and smoking was mostly evident among light smokers (<25 pack-years) with OR of 0.42 (95% CI 0.23-0.79), but not among heavy smokers with OR of 0.87 (95% CI 0.53-1.43). In the group analyses by TNM stage, there was no significant difference between CYP2E1 -1239G>C polymorphism and the risk of non-small cell lung cancer. Meta-analysis data also showed that the carriers of CYP2E1 -1239C allele had a protect effect on the risk of lung cancer in Chinese with overall OR of 0.77 (95% CI 0.66-0.90). CYP2E1 -1239G>C polymorphism was associated with a decreased risk of development of non-small cell lung cancer in Chinese patients. The association displays a manner of gene-environment interaction between this polymorphism and smoking status.

[Association of CYP1A1 Ile462Val polymorphisms with susceptibility to small cell lung cancer].

OBJECTIVE: To investigate the association of Ile462Val polymorphisms of cytochrome P450 1A1 (CYP1A1) gene with small cell lung cancer susceptibility. METHODS: A case-control study was done in 275 patients with small cell lung cancer and 406 control subjects. Genotypes of CYP1A1 Ile462Val were determined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). Logistic regression analysis was performed to estimate odd ratios (ORs) and 95% confidence intervals (95% CI) associated with genetic variants of CYP1A1. RESULTS: A case-control analysis showed ORs of 0.65 (95% CI 0.48 - 0.91) and 0.60 (95% CI 0.32 - 0.97) for small cell lung cancer in the CYP1A1 462 Ile/Val and 462 Val/Val genotype carriers respectively, compared with 462 Ile/Ile carriers. When stratified by smoking status, the ORs of 462 Ile/Val or Val/Val genotype for nonsmokers and smokers were 0.99 (95% CI 0.62 - 1.51) and 0.42 (95% CI 0.26 - 0.65), respectively. Furthermore, for light and heavy smokers, the ORs of 462 Ile/ Val or Val/Val genotype were 0.42 (95% CI 0.21 - 0.85) and 0.44 (95% CI 0.24 - 0.82), respectively. CONCLUSION: CYP1A1 Ile462Val polymorphisms may contribute to the decreased susceptibility of small cell lung cancer.

Drug-drug co-amorphous systems: An emerging formulation strategy for poorly water-soluble drugs.

Overcoming the poor water solubility of small-molecule drugs is a major challenge in the development of clinical pharmaceuticals. Amorphization of crystalline drugs is a highly effective strategy to improve their aqueous solubility. However, amorphous drugs are thermodynamically unstable and likely to crystallize during manufacturing and storage. Recently, drug-drug co-amorphous systems have emerged as a novel strategy to not only enable enhanced dissolution and physical stability of the individual drugs within the system but also to provide a strategy for combination therapy of the same or different clinical indications. This review serves to highlight advances in the methods used to manufacture and characterize drug-drug co-amorphous systems, summarize drug-drug co-amorphous applications reported in recent decades, and provide an outlook on future possibilities and perspectives.

Fabrication of Vascular Grafts Using Poly(ε-Caprolactone) and Collagen-Encapsuled ADSCs for Interposition Implantation of Abdominal Aorta in Rhesus Monkeys.

The production of small-diameter artificial vascular grafts continues to encounter numerous challenges, with concerns regarding the degradation rate and endothelialization being particularly critical. In this study, porous PCL scaffolds were prepared, and PCL vascular grafts were fabricated by 3D bioprinting of collagen materials containing adipose-derived mesenchymal stem cells (ADSCs) on the internal wall of the porous PCL scaffold. The PCL vascular grafts were then implanted in the abdominal aorta of Rhesus monkeys for up to 640 days to analyze the degradation of the scaffolds and regeneration of the aorta. Changes in surface morphology, mechanical properties, crystallization property, and molecular weight of porous PCL revealed a similar degradation process of PCL in PBS at pH 7.4 containing Thermomyces lanuginosus lipase and in situ in the abdominal aorta of rhesus monkeys. The contrast of in vitro and in vivo degradation provided valuable reference data for predicting in vivo degradation based on in vitro enzymatic degradation of PCL for further optimization of PCL vascular graft fabrication. Histological analysis through hematoxylin and eosin (HE) staining and fluorescence immunostaining demonstrated that the PCL vascular grafts successfully induced vascular regeneration in the abdominal aorta over the 640-day period. These findings provided valuable insights into the regeneration processes of the implanted vascular grafts. Overall, this study highlights the significant potential of PCL vascular grafts for the regeneration of small-diameter blood vessels.

Preparation and characterization of photosensitive methacrylate-grafted sodium carboxymethyl cellulose as an injectable material to fabricate hydrogels for biomedical applications.

Injectable materials have attracted great attention in the manufacture of in situ forming hydrogels for biomedical applications. In this study, a facile method to prepare methacrylic anhydride (MA)-modified sodium carboxymethyl cellulose (CMC) as an injectable material for the fabrication of hydrogels with controllable properties is reported. The chemical structure of the series of MA-grafted CMC (CMCMAs) with different MA contents was confirmed by Fourier transform infrared and nuclear magnetic resonance spectroscopy, and the properties of CMCMAs were characterized. Then, the CMCMAs gel (CMCMAs-G) was fabricated by crosslinking of MA under blue light irradiation. The gelation performances, swelling behaviors, transmittance, surface porous structures and mechanical properties of CMCMAs-G can be controlled by varying the content of MA grafted on the CMC. The compressive strength of CMCMAs-G was measured by mechanical compressibility tests and up to 180 kPa. Furthermore, the in vitro cytocompatibility evaluation results suggest that the obtained CMCMAs-G exhibit good compatibility for cell proliferation. Hence, our strategy provides a facile approach for the preparation of light-sensitive and an injectable CMC-derived polymer to fabricate hydrogels for biomedical applications.

Precision co-composting of multi-source organic solid wastes provide a sustainable waste management strategy with high eco-efficiency: a life cycle assessment.

With the increase of organic solid wastes (OSWs), current waste management practices, such as landfill, incineration, and windrow composting, have shown weaknesses in both resource recycling and environmental protection. Co-composting has been used to achieve nutrient and carbon recycling but is accused of high ammonia emission and low degradation efficiency. Therefore, this study developed a precision co-composting strategy (S3, which adds functional bacteria generated from food processing waste to a co-composting system) and compared it with the current OSW treatment strategy (S1) and traditional co-composting strategy (S2) from a life cycle assessment (LCA) perspective. The results showed that compared with S1, the eco-efficiency increased by 31.3% due to the higher economic profit of S2 but did not directly reduce the environmental cost. The addition of bacterial agents reduced ammonia emissions and shortened composting time, so compared with S1 and S2, the environmental cost of S3 was reduced by 37.9 and 43.6%, while the economic profit increased by 79.8 and 24.4%, respectively. The changes in environmental costs and economic benefits resulted in a huge improvement of S3's eco-efficiency, which was 189.6 and 121.7% higher than S1 and S2. Meanwhile, the adoption of S3 at a national scale in China could reduce the emission of 1,4-dichlorobenzene by 99.9% compared with S1 and increase profits by 6.58 billion USD per year. This study proposes a novel approach that exhibits high eco-efficiency in the treatment of OSWs.

Meta-analysis addressing the potential of antibiotic resistance gene elimination through aerobic composting.

The significant increase in antibiotic resistance genes (ARGs) in organic solid wastes (OSWs) has emerged as a major threat to the food chain. Aerobic composting is a widely used technology for OSW management, with the potential to influence the fate of AGRs. However, the variability of the ARG elimination effects reported in different studies has highlighted the uncertainty regarding the effects of composting on ARGs. To identify the potential of composting in reducing ARG and the factors (e.g., composting technologies and physiochemical properties) influence ARG changes, a meta-analysis was conducted with a database including 4,232 observations. The abundances of ARGs and mobile genetic elements (MGEs) can be substantially reduced by 74.3% and 78.8%, respectively, via aerobic composting. During composting, the ARG levels in chicken and swine manure tended to be reduced more significantly (81.7% and 78.0%) compared to those in cattle manure (52.3%) and sewage sludge (32.6%). The reduction rate of sulfonamide resistant genes was only 35.3%, which was much lower than those of other types. MGEs and composting duration (CD) were identified as the most important factors driving ARG changes during composting. These findings provide a comprehensive insight into the effects of composting on ARG reduction, which may help prevent the transmission in food systems.

Cylindrical granules in the development of mesalazine solid formulations (â ¡): The contribution of high aspect ratio to favorable tabletability.

Recently, cylindrical granules have been applied in pharmaceutical fields and their aspect ratio (AR) is considered an important factor in the manufacturing process. However, the relationships between AR and the tableting process were seldom reported. This study aims to clarify the role of AR in the tableting process of cylindrical granules. First, mesalazine cylindrical granules with different AR were extruded, and their physical attributes were then comprehensively characterized. Subsequently, their compression behaviors and tableting performances were systematically assessed. Notably, it was found that the cylindrical granules with high AR possessed good anti-deformation capacity and favorable tabletability. Finally, the dissolution test suggested that tablets compressed from cylindrical granules with higher AR showed lower dissolution rates. Collectively, findings in this study identified that the AR of cylindrical granules was a critical factor in the tableting process and provided valuable guidance for the application of these granules in oral solid formulations.

Implantation of Adipose-Derived Mesenchymal Stromal Cells (ADSCs)-Lining Prosthetic Graft Promotes Vascular Regeneration in Monkeys and Pigs.

BACKGROUND: Current replacement procedures for stenosis or occluded arteries using prosthetic grafts have serious limitations in clinical applications, particularly, endothelialization of the luminal surface is a long-standing unresolved problem. METHOD: We produced a cell-based hybrid vascular graft using a bioink engulfing adipose-derived mesenchymal stromal cells (ADSCs) and a 3D bioprinting process lining the ADSCs on the luminal surface of GORE-Tex grafts. The hybrid graft was implanted as an interposition conduit to replace a 3-cm-long segment of the infrarenal abdominal aorta in Rhesus monkeys. RESULTS: Complete endothelium layer and smooth muscle layer were fully developed within 21 days post-implantation, along with normalized collagen deposition and crosslinking in the regenerated vasculature in all monkeys. The regenerated blood vessels showed normal functionality for the longest observation of more than 1650 days. The same procedure was also conducted in miniature pigs for the interposition replacement of a 10-cm-long right iliac artery and showed the same long-term effective and safe outcome. CONCLUSION: This cell-based vascular graft is ready to undergo clinical trials for human patients.

CYP1A2 rs762551 polymorphism contributes to cancer susceptibility: a meta-analysis from 19 case-control studies.

BACKGROUND: Genetic polymorphism (rs762551A>C) in gene encoding cytochrome P450 1A2 (CYP1A2) has been shown to influence the inducibility of CYP1A2 expression and thus might be associated with risk of several types of human cancer. However, the results of previous studies on the associations of this polymorphism with risk of cancer are not all consistent. To clarify the potential contribution of CYP1A2 rs762551 to cancer risk, we performed a meta-analysis of the published case-control studies. METHODS: We used PubMed, Embase, OVID, ScienceDirect, and Chinese National Knowledge Infrastructure databases to identify the related publications for this meta-analysis. The pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using random effect model to evaluate the association of rs762551 with cancer risk. A χ(2)-based Q-test was used to examine the heterogeneity assumption and the funnel plot and Egger's test were used to examine the potential publication bias. The leave-one-out sensitivity analysis was conducted to determine whether our assumptions or decisions have a major effect on the results of the review. RESULTS: Our analysis of 19 eligible case-control studies showed a significant association between rs762551C variant with risk of cancer in the genetic model of CC versus AA (OR = 1.30, 95% CI = 1.02-1.64) and the dominant model (OR = 1.19, 95% CI = 1.04-1.36). In subgroup analysis based on ethnicity, the rs762551CC genotype was associated with increased cancer risk (OR = 1.29, 95% CI = 1.27-1.63 in co-dominate model and OR = 1.17, 95% CI = 1.02-1.34 in dominant model in Caucasians, but not in Asians and the mixed population. CONCLUSION: These results suggested that CYP1A2 rs762551 polymorphism is likely to be associated with susceptibility to cancer in Caucasians.

Mechanism of Erastin-Induced Ferroptosis in MDA-MB-231 Human Breast Cancer Cells: Evidence for a Critical Role of Protein Disulfide Isomerase.

Ferroptosis is a form of regulated cell death resulting predominantly from catastrophic accumulation of lipid reactive oxygen species (ROS). While the antioxidant systems that counter ferroptosis have been well characterized, the mechanism underlying ferroptosis-associated accumulation of lipid ROS remains unclear. In this study, we demonstrated that protein disulfide isomerase (PDI) is a novel mediator of ferroptosis, which is responsible for the accumulation of lipid ROS and ultimately ferroptosis in MDA-MB-231 human breast cancer cells. Treatment with erastin led to a significant increase in inducible nitric oxide synthase (iNOS)-mediated nitric oxide production, which contributes to the accumulation of the death-inducing cellular lipid ROS. Small interfering RNA (siRNA)-mediated PDI knockdown or pharmacological inhibition of PDI's isomerase activity with cystamine strongly suppressed iNOS dimerization and its catalytic activation, subsequently prevented lipid ROS accumulation, and conferred strong protection against erastin-induced ferroptosis. Remarkably, PDI knockdown in MDA-MB-231 cells also largely abrogated the protective effect of cystamine against erastin-induced ferroptotic cell death. Together, these experimental observations demonstrate a noncanonical role of PDI in ferroptosis, which may serve as a potential therapeutic target for ferroptosis-related diseases.

The effects of electric field assisted composting on ammonia and nitrous oxide emissions varied with different electrolytes.

Enhancing electron transfer through directly elevating electric potential has been verified to reduce gaseous emissions from composting. Reducing electric resistance of composting biomass might be a choice to further strengthening electron transfer. Here, the effects of chemical electrolytes addition on gaseous Nitrogen emission in electric field assistant composting were investigated. Results suggest that adding acidic electrolyte (ferric chloride) significantly reduced ammonia (NH3) emission by 72.1% but increased nitrous oxide (N2O) emission (by 24-fold) (P < 0.05), because of a dual effect on nitrifier activity: i) an elevated abundance and proportion of ammonia oxidizing bacteria Nitrosomonadaceae, and ii) delayed growth of nitrite oxidizing bacteria. Neutral and alkaline electrolytes had no negative or positive effect on N2O or NH3 emission. Hence, there is a potential trade-off between NH3 and N2O mitigation if using ferric chloride as acidic electrolyte, and electrolyte addition should aim to enhance electron production promote N2O mitigation.

An Improved Monkey Model of Myocardial Ischemic Infarction for Cardiovascular Drug Development.

Non-human primate monkey model of myocardial ischemic infarction is precious for translational medicine research. Ligation of the left anterior descending (LAD) artery is a common procedure to induce myocardial ischemic infarction. However, the consistency of the myocardial infarction thus generated remains problematic. The present study was undertaken to critically evaluate the monkey model of myocardial ischemic infarction to develop a procedure for a consistent cross-study comparison. Forty male Rhesus monkeys were divided into 4 groups and subjected to LAD artery ligation at different levels along the artery. In addition, the major diagonal branch was selectively ligated parallel to the ligation site of the LAD artery according to the diagonal branch distribution. Analyses of MRI, echocardiography, cardiac hemodynamics, electrocardiography, histopathology, and cardiac injury biomarkers were undertaken to characterize the monkeys with myocardial infarction. Ligation at 40% of the total length of the artery, measured from the apex end, produced variable infarct areas with inconsistent functional alterations. Ligation at 60% or above coupled with selective ligation of diagonal branches produced a consistent myocardial infarction with uniform dysfunction. However, ligation at 70% caused a lethal threat. After a thorough analysis, it is concluded that ligation at 60% of the total length coupled with selective ligation of diagonal branches, enables standardization of the location of occlusion and the subsequent ischemic area, as well as avoids the influence of the diagonal branches, are ideal to produce a consistent monkey model of myocardial ischemic infarction.

Evaluation of Neurofilament Light Chain as a Biomarker of Neurodegeneration in X-Linked Childhood Cerebral Adrenoleukodystrophy.

Cerebral adrenoleukodystrophy (CALD) is a devastating, demyelinating neuroinflammatory manifestation found in up to 40% of young males with an inherited mutation in ABCD1, the causative gene in adrenoleukodystrophy. The search for biomarkers which correlate to CALD disease burden and respond to intervention has long been sought after. We used the Olink Proximity Extension Assay (Uppsala, Sweden) to explore the cerebral spinal fluid (CSF) of young males with CALD followed by correlative analysis with plasma. Using the Target 96 Neuro Exploratory panel, we found that, of the five proteins significantly increased in CSF, only neurofilament light chain (NfL) showed a significant correlation between CSF and plasma levels. Young males with CALD had a 11.3-fold increase in plasma NfL compared with controls. Importantly, 9 of 11 young males with CALD who underwent HCT showed a mean decrease in plasma NfL of 50% at 1 year after HCT compared with pre-HCT levels. In conclusion, plasma NfL could be a great value in determining outcomes in CALD and should be scrutinized in future studies in patients prior to CALD development and after therapeutic intervention.

Vascular endothelial growth factor gene polymorphisms and risk of neovascular age-related macular degeneration in a Chinese cohort.

AIMS: To evaluate the association between vascular endothelial growth factor (VEGF) gene polymorphism and the risk of neovascular age-related macular degeneration (AMD) in a case-control study in a Chinese cohort. METHODS: We genotyped 4 common single-nucleotide polymorphisms (SNPs), namely -460T/C (rs833061), +405C/G (rs2010963), +674C/T (rs1413711) and +936C/T (rs3025039), simultaneously detected 7 tag SNPs (tSNPs) in the VEGF gene, in 159 neovascular AMD patients and 140 age- and sex-matched control subjects. Genetic analyses for an additive, dominant and recessive model were performed on all available genotype data. All the possible haplotypes of these 11 SNPs were detected. RESULTS: No evident association was found in the allele frequencies of any individual SNP between patients and controls; the combined p values in each genotype group were greater than 0.05. Haplotype analyses of these SNPs did not provide any evidence for an association with the risk of neovascular AMD in this Chinese cohort (p > 0.05). CONCLUSIONS: Detection of 4 common SNPs and 7 tSNPs in the VEGF gene did not find any statistically significant association with neovascular AMD in the Chinese cohort. Further studies of comprehensive VEGF gene variations are required to characterize the susceptibility of the VEGF gene in the pathogenesis of AMD.

Atherosclerotic lesion-specific copper delivery suppresses atherosclerosis in high-cholesterol-fed rabbits.

Dietary cholesterol supplements cause hypercholesterolemia and atherosclerosis along with a reduction of copper concentrations in the atherosclerotic wall in animal models. This study was to determine if target-specific copper delivery to the copper-deficient atherosclerotic wall can block the pathogenesis of atherosclerosis. Male New Zealand white rabbits, 10-weeks-old and averaged 2.0 kg, were fed a diet containing 1% (w/w) cholesterol or the same diet without cholesterol as control. Twelve weeks after the feeding, the animals were injected with copper-albumin microbubbles and subjected to ultrasound sonication specifically directed at the atherosclerotic lesions (Cu-MB-US) for target-specific copper delivery, twice a week for four weeks. This regiment was repeated 3 times with a gap of two weeks in between. Two weeks after the last treatment, the animals were harvested for analyses of serum and aortic pathological changes. Compared to controls, rabbits fed cholesterol-rich diet developed atherosclerotic lesion with a reduction in copper concentrations in the lesion tissue. Cu-MB-US treatment significantly increased copper concentrations in the lesion, and reduced the size of the lesion. Furthermore, copper repletion reduced the number of apoptotic cells as well as the content of cholesterol and phospholipids in the atherosclerotic lesion without a disturbance of the stability of the lesion. The results thus demonstrate that target-specific copper supplementation suppresses the progression of atherosclerosis at least in part through preventing endothelial cell death, thus reducing lipid infiltration in the atherosclerotic lesion.

MicroRNAs as Biomarkers of Charcot-Marie-Tooth Disease Type 1A.

OBJECTIVE: To determine whether microRNAs (miRs) are elevated in the plasma of individuals with the inherited peripheral neuropathy Charcot-Marie-Tooth disease type 1A (CMT1A), miR profiling was employed to compare control and CMT1A plasma. METHODS: We performed a screen of CMT1A and control plasma samples to identify miRs that are elevated in CMT1A using next-generation sequencing, followed by validation of selected miRs by quantitative PCR, and correlation with protein biomarkers and clinical data: Rasch-modified CMT Examination and Neuropathy Scores, ulnar compound muscle action potentials, and motor nerve conduction velocities. RESULTS: After an initial pilot screen, a broader screen confirmed elevated levels of several muscle-associated miRNAs (miR1, -133a, -133b, and -206, known as myomiRs) along with a set of miRs that are highly expressed in Schwann cells of peripheral nerve. Comparison to other candidate biomarkers for CMT1A (e.g., neurofilament light) measured on the same sample set shows a comparable elevation of several miRs (e.g., miR133a, -206, -223) and ability to discriminate cases from controls. Neurofilament light levels were most highly correlated with miR133a. In addition, the putative Schwann cell miRs (e.g., miR223, -199a, -328, -409, -431) correlate with the recently described transmembrane protease serine 5 (TMPRSS5) protein biomarker that is most highly expressed in Schwann cells and also elevated in CMT1A plasma. CONCLUSIONS: These studies identify a set of miRs that are candidate biomarkers for clinical trials in CMT1A. Some of the miRs may reflect Schwann cell processes that underlie the pathogenesis of the disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that a set of plasma miRs are elevated in patients with CMT1A.