Lipidomic Approaches to Study HDL Metabolism in Patients with Central Obesity Diagnosed with Metabolic Syndrome

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated “omics” approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the “lysophosphatidylcholines to phosphatidylcholines” and “cholesteryl ester to free cholesterol” ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated “omics” approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.     

Effects of Different Types of Chronic Training on Bioenergetic Profile and Reactive Oxygen Species Production in LHCN-M2 Human Myoblast Cells

Human skeletal muscle contains three different types of fibers, each with a different metabolism. Exercise differently contributes to differentiation and metabolism in human myoblast cells. The aims of the present study were to investigate the effects of different types of chronic training on the human LHCN-M2 myoblast cell bioenergetic profile during differentiation in real time and on the ROS overproduction consequent to H₂O₂ injury. We demonstrated that exercise differently affects the myoblast bioenergetics: aerobic exercise induced the most efficient glycolytic and oxidative capacity and proton leak reduction compared to untrained or anaerobic trained sera-treated cells. Similarly, ROS overproduction after H₂O₂ stress was lower in cells treated with differently trained sera compared to untrained sera, indicating a cytoprotective effect of training on the reduction of oxidative stress, and thus the promotion of longevity. In conclusion, for the first time, this study has provided knowledge regarding the modifications induced by different types of chronic training on human myoblast cell bioenergetics during the differentiation process in real time, and on ROS overproduction due to stress, with positive implications in terms of longevity.     

Chiral Discrimination Mechanisms by Silylated-Acetylated Cyclodextrins: Superficial Interactions vs. Inclusion

Cyclodextrin derivatives constitute a powerful class of auxiliary agents for the discrimination of apolar chiral substrates. Both host–guest inclusion phenomena and interactions with the derivatizing groups located on the surface of the macrocycle could drive the enantiodiscrimination; thus, it is important to understand the role that these processes play in the rational design of new chiral selectors. The purpose of this study is to compare via nuclear magnetic resonance (NMR) spectroscopy the efficiency of silylated-acetylated α-, β-, and γ-cyclodextrins in the chiral discrimination of 1,1,1,3,3-pentafluoro-2-(fluoromethoxy)-3-methoxypropane (compound B) and methyl 2-chloropropionate (MCP). NMR DOSY (Diffusion Ordered SpectroscopY) experiments were conducted for the determination of the bound molar fractions and the association constants, whereas ROESY (Rotating-frame Overhauser Enhancement SpectroscopY) measurements provided information on the hosts’ conformation and on the interaction phenomena with the guests. Compound B, endowed with fluorinated moieties, is not deeply included due to attractive Si-F interactions occurring at the external surface of the cyclodextrins. Therefore, a low selectivity toward the size of cyclodextrin cavity is found. By contrast, enantiodiscrimination of MCP relies on the optimal fitting between the size of the guest and that of the cyclodextrin cavity.     

The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and conserved water molecules

CK2 is a highly pleiotropic Ser/Thr protein kinase that is able to promote cell survival and enhance the tumour phenotype under specific circumstances. We have determined the crystal structure of three new complexes with tetrabromobenzimidazole derivatives that display K(i) values between 0.15 and 0.30 microM. A comparative analysis of these data with those of four other inhibitors of the same family revealed the presence of some highly conserved water molecules in the ATP-binding site. These waters reside near Lys68, in an area with a positive electrostatic potential that is able to attract and orient negatively charged ligands. The presence of this positive region and two unique bulky residues that are typical of CK2, Ile66 and Ile174, play a critical role in determining the ligand orientation and binding selectivity.     

Platelet lysate and adipose mesenchymal stromal cells on silk fibroin nonwoven mats for wound healing

In this work platelet lysate (PL) and adipose‐derived mesenchymal stromal cells (ASCs) seeded on nonwoven fibroin mats were in vitro and in vivo evaluated for tissue regenerative applications. Nonwoven mats obtained by a large scale water entanglement technique were characterized for their physico‐chemical properties. Results indicated a high purity of fibroin fibers, their stability after sterilization process and appropriate technological properties suitable for tissue engineering. Moreover, the scaffolds in vitro supported adhesion and migration of ASCs and the presence of PL improved the cell proliferation. The products were then applied on epithelial/dermal wounds carried out on the dorsal surface of rabbit: the skin reparative process was solved in 9 days, with a completely restitutio ad integrum of the epithelium in animals treated with PL alone; ASCs did not further improve the wound healing. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 42942.     

Dihydrotanshinone,a Natural Diterpenoid,Preserves Blood-Retinal Barrier Integrity via P2X7 Receptor

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1β, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.     

Mutual Prodrugs of 5-Fluorouracil: From a Classic Chemotherapeutic Agent to Novel Potential Anticancer Drugs

The development of potent antitumor agents with a low toxicological profile against healthy cells is still one of the greatest challenges facing medicinal chemistry. In this context, the “mutual prodrug” approach has emerged as a potential tool to overcome undesirable physicochemical features and mitigate the side effects of approved drugs. Among broad-spectrum chemotherapeutics available for clinical use today, 5-fluorouracil (5-FU) is one of the most representative, also included in the World Health Organization model list of essential medicines. Unfortunately, severe side effects and drug resistance phenomena are still the primary limits and drawbacks in its clinical use. This review describes the progress made over the last ten years in developing 5-FU-based mutual prodrugs to improve the therapeutic profile and achieve targeted delivery to cancer tissues.     

The Age-Sensitive Efficacy of Calorie Restriction on Mitochondrial Biogenesis and mtDNA Damage in Rat Liver

Calorie restriction (CR) is the most efficacious treatment to delay the onset of age-related changes such as mitochondrial dysfunction. However, the sensitivity of mitochondrial markers to CR and the age-related boundaries of CR efficacy are not fully elucidated. We used liver samples from ad libitum-fed (AL) rats divided in: 18-month-old (AL-18), 28-month-old (AL-28), and 32-month-old (AL-32) groups, and from CR-treated (CR) 28-month-old (CR-28) and 32-month-old (CR-32) counterparts to assay the effect of CR on several mitochondrial markers. The age-related decreases in citrate synthase activity, in TFAM, MFN2, and DRP1 protein amounts and in the mtDNA content in the AL-28 group were prevented in CR-28 counterparts. Accordingly, CR reduced oxidative mtDNA damage assessed through the incidence of oxidized purines at specific mtDNA regions in CR-28 animals. These findings support the anti-aging effect of CR up to 28 months. Conversely, the protein amounts of LonP1, Cyt c, OGG1, and APE1 and the 4.8 Kb mtDNA deletion content were not affected in CR-28 rats. The absence of significant differences between the AL-32 values and the CR-32 counterparts suggests an age-related boundary of CR efficacy at this age. However, this only partially curtails the CR benefits in counteracting the generalized aging decline and the related mitochondrial involvement.     

Regulatory Noncoding and Predicted Pathogenic Coding Variants of CCR5 Predispose to Severe COVID-19

Genome-wide association studies (GWAS) found locus 3p21.31 associated with severe COVID-19. CCR5 resides at the same locus and, given its known biological role in other infection diseases, we investigated if common noncoding and rare coding variants, affecting CCR5, can predispose to severe COVID-19. We combined single nucleotide polymorphisms (SNPs) that met the suggestive significance level (P ≤ 1 × 10⁻⁵) at the 3p21.31 locus in public GWAS datasets (6406 COVID-19 hospitalized patients and 902,088 controls) with gene expression data from 208 lung tissues, Hi-C, and Chip-seq data. Through whole exome sequencing (WES), we explored rare coding variants in 147 severe COVID-19 patients. We identified three SNPs (rs9845542, rs12639314, and rs35951367) associated with severe COVID-19 whose risk alleles correlated with low CCR5 expression in lung tissues. The rs35951367 resided in a CTFC binding site that interacts with CCR5 gene in lung tissues and was confirmed to be associated with severe COVID-19 in two independent datasets. We also identified a rare coding variant (rs34418657) associated with the risk of developing severe COVID-19. Our results suggest a biological role of CCR5 in the progression of COVID-19 as common and rare genetic variants can increase the risk of developing severe COVID-19 by affecting the functions of CCR5.