Inhibition of Dual/Mixed Tropic HIV-1 Isolates by CCR5-Inhibitors in Primary Lymphocytes and Macrophages

Background

Dual/mixed-tropic HIV-1 strains are predominant in a significant proportion of patients, though little information is available regarding their replication-capacity and susceptibility against CCR5-antagonists in-vitro. The aim of the study was to analyze the replication-capacity and susceptibility to maraviroc of HIV-1 clinical isolates with different tropism characteristics in primary monocyte-derived-macrophages (MDM), peripheral-blood-mononuclear-cells (PBMC), and CD4⁺T-lymphocytes.

Methods

Twenty-three HIV-1 isolates were phenotipically and genotipically characterized as R5, X4 or dual (discriminated as R5⁺/X4, R5/X4, R5/X4⁺). Phenotypic-tropism was evaluated by multiple-cycles-assay on U87MG-CD4⁺-CCR5⁺−/CXCR4⁺-expressing cells. Genotypic-tropism prediction was obtained using Geno2Pheno-algorithm (false-positive-rate [FPR] = 10%). Replication-capacity and susceptibility to maraviroc were investigated in human-primary MDM, PBMC and CD4⁺T-cells. AMD3100 was used as CXCR4-inhibitor. Infectivity of R5/Dual/X4-viruses in presence/absence of maraviroc was assessed also by total HIV-DNA, quantified by real-time polymerase-chain-reaction.

Results

Among 23 HIV-1 clinical isolates, phenotypic-tropism-assay distinguished 4, 17 and 2 viruses with R5-tropic, dual/mixed-, and X4-tropic characteristics, respectively. Overall, viruses defined as R5⁺/X4-tropic were found with the highest prevalence (10/23, 43.5%). The majority of isolates efficiently replicated in both PBMC and CD4⁺T-cells, regardless of their tropism, while MDM mainly sustained replication of R5- or R5⁺/X4-tropic isolates; strong correlation between viral-replication and genotypic-FPR-values was observed in MDM (rho = 0.710;p-value = 1.4e-4). In all primary cells, maraviroc inhibited viral-replication of isolates not only with pure R5- but also with dual/mixed tropism (mainly R5⁺/X4 and, to a lesser extent R5/X4 and R5/X4⁺). Finally, no main differences by comparing the total HIV-DNA with the p24-production in presence/absence of maraviroc were found.

Conclusions

Maraviroc is effective in-vitro against viruses with dual-characteristics in both MDM and lymphocytes, despite the potential X4-mediated escape. This suggests that the concept of HIV-entry through one of the two coreceptors “separately” may require revision, and that the use of CCR5-antagonists in patients with dual/mixed-tropic viruses may be a therapeutic-option that deserves further investigations in different clinical settings.     

Mapping Cortical Degeneration in ALS with Magnetization Transfer Ratio and Voxel-Based Morphometry

Pathological and imaging data indicate that amyotrophic lateral sclerosis (ALS) is a multisystem disease involving several cerebral cortical areas. Advanced quantitative magnetic resonance imaging (MRI) techniques enable to explore in vivo the volume and microstructure of the cerebral cortex in ALS. We studied with a combined voxel-based morphometry (VBM) and magnetization transfer (MT) imaging approach the capability of MRI to identify the cortical areas affected by neurodegeneration in ALS patients. Eighteen ALS patients and 18 age-matched healthy controls were examined on a 1.5T scanner using a high-resolution 3D T1 weighted spoiled gradient recalled sequence with and without MT saturation pulse. A voxel-based analysis (VBA) was adopted in order to automatically compute the regional atrophy and MT ratio (MTr) changes of the entire cerebral cortex. By using a multimodal image analysis MTr was adjusted for local gray matter (GM) atrophy to investigate if MTr changes can be independent of atrophy of the cerebral cortex. VBA revealed several clusters of combined GM atrophy and MTr decrease in motor-related areas and extra-motor frontotemporal cortex. The multimodal image analysis identified areas of isolated MTr decrease in premotor and extra-motor frontotemporal areas. VBM and MTr are capable to detect the distribution of neurodegenerative alterations in the cortical GM of ALS patients, supporting the hypothesis of a multi-systemic involvement in ALS. MT imaging changes exist beyond volume loss in frontotemporal cortices.     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

2-Hydroxy Arachidonic Acid: A New Non-Steroidal Anti-Inflammatory Drug

Background

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a family of COX1 and COX2 inhibitors used to reduce the synthesis of pro-inflammatory mediators. In addition, inflammation often leads to a harmful generation of nitric oxide. Efforts are being done in discovering safer NSAIDs molecules capable of inhibiting the synthesis of pro-inflammatory lipid mediators and nitric oxide to reduce the side effects associated with long term therapies.

Methodology/Principal Findings

The analogue of arachidonic acid (AA), 2-hydroxy-arachidonic acid (2OAA), was designed to inhibit the activities of COX1 and COX2 and it was predicted to have similar binding energies as AA for the catalytic sites of COX1 and COX2. The interaction of AA and 2OAA with COX1 and COX2 was investigated calculating the free energy of binding and the Fukui function. Toxicity was determined in mouse microglial BV-2 cells. COX1 and COX2 (PGH2 production) activities were measured in vitro. COX1 and COX2 expression in human macrophage-like U937 cells were carried out by Western blot, immunocytochemistry and RT-PCR analysis. NO production (Griess method) and iNOS (Western blot) were determined in mouse microglial BV-2 cells. The comparative efficacy of 2OAA, ibuprofen and cortisone in lowering TNF-α serum levels was determined in C57BL6/J mice challenged with LPS. We show that the presence of the –OH group reduces the likelihood of 2OAA being subjected to H* abstraction in COX, without altering significantly the free energy of binding. The 2OAA inhibited COX1 and COX2 activities and the expression of COX2 in human U937 derived macrophages challenged with LPS. In addition, 2OAA inhibited iNOS expression and the production of NO in BV-2 microglial cells. Finally, oral administration of 2OAA decreased the plasma TNF-α levels in vivo.

Conclusion/Significance

These findings demonstrate the potential of 2OAA as a NSAID.     

New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin''s tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin''s tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin''s tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.     

Genome Wide Analysis of Fertility and Production Traits in Italian Holstein Cattle

A genome wide scan was performed on a total of 2093 Italian Holstein proven bulls genotyped with 50K single nucleotide polymorphisms (SNPs), with the objective of identifying loci associated with fertility related traits and to test their effects on milk production traits. The analysis was carried out using estimated breeding values for the aggregate fertility index and for each trait contributing to the index: angularity, calving interval, non-return rate at 56 days, days to first service, and 305 day first parity lactation. In addition, two production traits not included in the aggregate fertility index were analysed: fat yield and protein yield. Analyses were carried out using all SNPs treated separately, further the most significant marker on BTA14 associated to milk quality located in the DGAT1 region was treated as fixed effect. Genome wide association analysis identified 61 significant SNPs and 75 significant marker-trait associations. Eight additional SNP associations were detected when SNP located near DGAT1 was included as a fixed effect. As there were no obvious common SNPs between the traits analyzed independently in this study, a network analysis was carried out to identify unforeseen relationships that may link production and fertility traits.     

Psychiatric disorders and SLC6A4 gene variants: possible effects on alcohol dependence and alzheimer’s disease

Molecular Biology Reports - Serotoninergic system is one of the most important neurotransmission systems investigated in the field of psychiatry. Extensive evidence reveals how alterations of this...     

Insect-associated bacterial communities in an alpine stream

Hydrobiologia - The roles of macroinvertebrate and microbial communities in stream ecosystems are recognized to be important to energy flow and nutrient cycling. While the linkages of these major...     

Probing antioxidant activity of 2′-hydroxychalcones: Crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation

In order to better understand the antioxidant behavior of a series of polyphenolic 2′-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV–vis spectroscopic method indicate that a hydroxyl group in position 5′ induces the highest antioxidant activity. Consequently, 2,2′,5′-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2′,5′-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa⁻/fa⁻) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2′,5′-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.