Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells

Decitabine (5-aza-2′-deoxycytidine) is a DNA methyltransferase inhibitor and an archetypal epigenetic drug for the therapy of myeloid leukemias. The mode of action of decitabine strictly depends on the incorporation of the drug into DNA. However, DNA incorporation and ensuing genotoxic effects of decitabine have not yet been investigated in human cancer cell lines or in models related to the approved indication of the drug. Here we describe a robust assay for the quantitative determination of decitabine incorporation rates into DNA from human cancer cells. Using a panel of human myeloid leukemia cell lines we show appreciable amounts of decitabine incorporation that closely correlated with cellular drug uptake. Decitabine incorporation was also detectable in primary cells from myeloid leukemia patients, indicating that the assay is suitable for biomarker analyses to predict drug responses in patients. Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level. Interestingly, this approach failed to reveal significant changes in the rates of point mutations and genome rearrangements in myeloid leukemia cell lines. These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.     

Antioxidant properties of a human neuropeptide and its protective effect on free radical‐induced DNA damage

Human catestatin CgA_352–372 (SL21) is an endogenous neuropeptide with multiple biological functions. The present study aimed to evaluate the antioxidant, antibacterial, cytotoxic, and DNA damage protective effects of SL21 neuropeptide. SL21 neuropeptide generated from the C‐terminus of chromogranin A (CgA) was synthesized by solid‐phase method. Synthetic peptide was subjected to various in vitro antioxidant assays including the scavenging of 1,1‐diphenyl‐2‐pycryl‐hydrazyl (DPPH), 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS^·+), and hydroxyl free radicals, metal ion chelation, inhibition of lipid peroxidation, and reducing power. Moreover, protective effect of SL21 on H₂O₂‐induced DNA damage was analyzed using pTZ57/RT plasmid. Methylthiazoltetrazolium assay was also performed to study the cytotoxic effect of SL21 neuropeptide on human peripheral blood mononuclear cells. Furthermore, antibacterial and hemolysis assays were conducted. The results demonstrated high activities of SL21 in scavenging free radicals (DPPH, ABTS^·+, and hydroxyl), chelating of Cu²⁺/Fe²⁺ metal ions, reducing power, and inhibition of lipid peroxidation in a concentration‐dependent manner. SL21 neuropeptide revealed a protective effect on DNA damage caused by hydroxyl radicals. Interestingly, the peptide exhibited no significant cytotoxicity towards peripheral blood mononuclear cells. Furthermore, SL21 peptide displayed antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa without any hemolytic activity on human red blood cells. Conclusively, the present study established SL21 (catestatin) as a novel antioxidative peptide that could further be investigated for its potential use as a pharmaceutical agent. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

Population fluctuation of Ephestia figulilella in Kerman province of Iran

Date palm is one of the most important products in Iran. Kerman province is one of the most important producers of this crop. Major areas of date palm cultivation in the province involve Bam, Jiroft and Shahdad. Date palm acreage of Shahdad is over 3500 hectares. Date palm varieties in Shahdad are Ghasb, Shamsaee and Bazmani. Ghasb date palm is most important in Shahdad that is classified as dry product. Date Palm trees have many pests such as the horned beetle dates, spider mite, leafhopper and moths; and according to the studies done in 2008–2009, Ephestia figulilella is the most important pest on fruit dates in Shadad, sometimes 90% of product is contaminated by this pest. This pest has four to five generations per year and overwintered as all of stages in storage and larvae in garden.     

Association between Farming and Chronic Energy Deficiency in Rural South India

Objective

To examine factors associated with chronic energy deficiency (CED) and anaemia in disadvantaged Indian adults who are mostly involved in subsistence farming.

Design

A cross-sectional study in which we collected information on socio-demographic factors, physical activity, anthropometry, blood haemoglobin concentration, and daily household food intake. These data were used to calculate body mass index (BMI), basal metabolic rate (BMR), daily energy expenditure, and energy and nutrient intake. Multivariable backward stepwise logistic regression was used to assess socioeconomic and lifestyle factors associated with CED (defined as BMI<18 kg/m²) and anaemia.

Setting

The study was conducted in 12 villages, in the Rishi Valley, Andhra Pradesh, India.

Subjects

Individuals aged 18 years and above, residing in the 12 villages, were eligible to participate.

Results

Data were available for 1178 individuals (45% male, median age 36 years (inter quartile range (IQR 27–50)). The prevalence of CED (38%) and anaemia (25%) was high. Farming was associated with CED in women (2.20, 95% CI: 1.39–3.49) and men (1.71, 95% CI: (1.06–2.74). Low income was also significantly associated with CED, while not completing high school was positively associated with anaemia. Median iron intake was high: 35.7 mg/day (IQR 26–46) in women and 43.4 mg/day (IQR 34–55) in men.

Conclusions

Farming is an important risk factor associated with CED in this rural Indian population and low dietary iron is not the main cause of anaemia. Better farming practice may help to reduce CED in this population.     

A two-way communication between microglial cells and angiogenic sprouts regulates angiogenesis in aortic ring cultures

Background

Myeloid cells have been associated with physiological and pathological angiogenesis, but their exact functions in these processes remain poorly defined. Monocyte-derived tissue macrophages of the CNS, or microglial cells, invade the mammalian retina before it becomes vascularized. Recent studies correlate the presence of microglia in the developing CNS with vascular network formation, but it is not clear whether the effect is directly caused by microglia and their contact with the endothelium.

Methodology/Principal Findings

We combined in vivo studies of the developing mouse retina with in vitro studies using the aortic ring model to address the role of microglia in developmental angiogenesis. Our in vivo analyses are consistent with previous findings that microglia are present at sites of endothelial tip-cell anastomosis, and genetic ablation of microglia caused a sparser vascular network associated with reduced number of filopodia-bearing sprouts. Addition of microglia in the aortic ring model was sufficient to stimulate vessel sprouting. The effect was independent of physical contact between microglia and endothelial cells, and could be partly mimicked using microglial cell-conditioned medium. Addition of VEGF-A promoted angiogenic sprouts of different morphology in comparison with the microglial cells, and inhibition of VEGF-A did not affect the microglia-induced angiogenic response, arguing that the proangiogenic factor(s) released by microglia is distinct from VEGF-A. Finally, microglia exhibited oriented migration towards the vessels in the aortic ring cultures.

Conclusions/Significance

Microglia stimulate vessel sprouting in the aortic ring cultures via a soluble microglial-derived product(s), rather than direct contact with endothelial cells. The observed migration of microglia towards the growing sprouts suggests that their position near endothelial tip-cells could result from attractive cues secreted by the vessels. Our data reveals a two-way communication between microglia and vessels that depends on soluble factors and should extend the understanding of how microglia promote vascular network formation.     

Toll-like receptor 4 (TLR4) polymorphisms in Iranian patients with visceral leishmaniasis

The role of Toll-like receptor (TLR) 4 in visceral leishmaniasis (VL), a disease caused by an obligate intracellular protozoan parasites belonging to the genus Leishmania, has been shown in the recent leishmaniasis experimental studies. As genetic host factors play an important role in the susceptibility and/or resistance to VL, the association between TLR4 gene mutations [A896G and C1196T single nucleotide polymorphisms (SNPs)] and VL was investigated. Genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) SNPs was performed in the patients with VL (N?=?122) and ethnically matched controls (N?=?155) using polymerase chain reaction–restriction fragment length polymorphism method. When VL patients and the controls were compared, no statistically significant differences were observed in A896G and C1196T alleles and genotypes (P?>?0.05). The TLR4 A896G and C1196T were in moderate linkage disequilibrium in the controls and patients (r ²?=?0.497, 0.548 and D′?=?0.705, 0.808, respectively), and haplotypes reconstructed from these SNPs were not significantly different between the aforementioned study groups. In conclusion, based on the results, TLR4 gene polymorphisms at the positions 896 and 1196 cannot be regarded as the major contributors to VL susceptibility among the Iranian population.     

The Sphingosine-1-Phosphate Receptor S1PR1 Restricts Sprouting Angiogenesis by Regulating the Interplay between VE-Cadherin and VEGFR2

Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of?vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.