Monochloramine inhibits etoposide-induced apoptosis with an increase in DNA aberration

Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils, and it affects apoptosis signaling. We studied the effects of NH(2)Cl on the cell death induced by etoposide, a widely used anticancer agent that is directed to DNA topoisomerase II. Jurkat T cells, a human acute T cell leukemia cell line, were pretreated with 70 microM of NH(2)Cl for 10 min. After 24 h, 5-30 microM of etoposide was added to the NH(2)Cl pretreated and control cells, and their apoptosis, caspase activity, cell morphology, and cellular DNA contents were measured. NH(2)Cl pretreatment significantly inhibited apoptosis and caspase activation induced by etoposide or camptothecin, a DNA topoisomerase I poison, but not by staurosporine or Fas stimulation. The apoptosis inhibition actually resulted in the proliferation of the survived cells and, notably, the survived cells showed more aberrant morphology, such as variation in nuclear size, nuclear fragments, and multinucleated cells. DNA content analysis of the survived cells showed an increase in aneuploid nuclei. Cell cycle analysis after 24 h of NH(2)Cl treatment showed a significant decrease in S phase cells with a concurrent increase in G(0)/G(1) phase cells, which suggested that NH(2)Cl induced G(1) arrest. Using synchronized Jurkat cells, etoposide and camptothecin were found to be particularly cytotoxic to S phase cells, whereas staurosporine and Fas stimulation were not. Thus NH(2)Cl-induced G(1) arrest was a likely cause of the observed resistance to etoposide. These observations suggested that inflammation-derived oxidants may make the tumor cells more resistant to etoposide and increase the risk of tumor progression and the development of secondary tumors by increasing the survival of DNA damage-bearing cells.     

PP13, maternal ABO blood groups and the risk assessment of pregnancy complications

Background

Placental Protein 13 (PP13), an early biomarker of preeclampsia, is a placenta-specific galectin that binds beta-galactosides, building-blocks of ABO blood-group antigens, possibly affecting its bioavailability in blood.

Methods and Findings

We studied PP13-binding to erythrocytes, maternal blood-group effect on serum PP13 and its performance as a predictor of preeclampsia and intrauterine growth restriction (IUGR). Datasets of maternal serum PP13 in Caucasian (n = 1078) and Hispanic (n = 242) women were analyzed according to blood groups. In vivo, in vitro and in silico PP13-binding to ABO blood-group antigens and erythrocytes were studied by PP13-immunostainings of placental tissue-microarrays, flow-cytometry of erythrocyte-bound PP13, and model-building of PP13 - blood-group H antigen complex, respectively. Women with blood group AB had the lowest serum PP13 in the first trimester, while those with blood group B had the highest PP13 throughout pregnancy. In accordance, PP13-binding was the strongest to blood-group AB erythrocytes and weakest to blood-group B erythrocytes. PP13-staining of maternal and fetal erythrocytes was revealed, and a plausible molecular model of PP13 complexed with blood-group H antigen was built. Adjustment of PP13 MoMs to maternal ABO blood group improved the prediction accuracy of first trimester maternal serum PP13 MoMs for preeclampsia and IUGR.

Conclusions

ABO blood group can alter PP13-bioavailability in blood, and it may also be a key determinant for other lectins'' bioavailability in the circulation. The adjustment of PP13 MoMs to ABO blood group improves the predictive accuracy of this test.     

Kainate receptors mediate regulated exocytosis of secretory phospholipase A(2) in SH-SY5Y neuroblastoma cells

Secretory phospholipase A(2) (sPLA(2)) isoforms are widely expressed in the brain and spinal cord. Group IIA sPLA(2) (sPLA(2)-IIA) has been shown to stimulate exocytosis and release of neurotransmitters in neuroendocrine PC12 cells and neurons, suggesting a role of the enzyme in neuronal signaling and synaptic transmission. However, the mechanisms by which sPLA(2) is itself released, and a possible relation between glutamate receptors and sPLA(2) exocytosis, are unknown. This study was carried out to elucidate the effects of glutamate receptor agonists on exocytosis of sPLA(2)-IIA in transfected SH-SY5Y neuroblastoma cells. sPLA(2)-IIA enzyme was packaged in fusion-competent vesicles and released constitutively or upon stimulation, suggesting regulated secretion. The signal peptide of sPLA(2)-IIA is required for its vesicular localization and exocytosis. External application of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) induced vesicular exocytosis and release of sPLA(2)-IIA. UBP 302, a GluR5-specific KA receptor antagonist, abolished the effect of KA, confirming the role of KA receptors in mediating sPLA(2)-IIA secretion. Moreover, KA-induced sPLA(2)-IIA secretion is dependent on Ca(2+) and protein kinase C. Together, these findings provide evidence of a link between glutamate receptors and regulated sPLA(2) secretion in neurons that may play an important role in synaptic plasticity, pain transmission and neurodegenerative diseases.     

Accuracy and User-Acceptability of HIV Self-Testing Using an Oral Fluid-Based HIV Rapid Test

Background

The United States FDA approved an over-the-counter HIV self-test, to facilitate increased HIV testing and earlier linkage to care. We assessed the accuracy of self-testing by untrained participants compared to healthcare worker (HCW) testing, participants’ ability to interpret sample results and user-acceptability of self-tests in Singapore.

Methodology/Principal Findings

A cross-sectional study, involving 200 known HIV-positive patients and 794 unknown HIV status at-risk participants was conducted. Participants (all without prior self-test experience) performed self-testing guided solely by visual instructions, followed by HCW testing, both using the OraQuick ADVANCE Rapid HIV 1/2 Antibody Test, with both results interpreted by the HCW. To assess ability to interpret results, participants were provided 3 sample results (positive, negative, and invalid) to interpret. Of 192 participants who tested positive on HCW testing, self-testing was positive in 186 (96.9%), negative in 5 (2.6%), and invalid in 1 (0.5%). Of 794 participants who tested negative on HCW testing, self-testing was negative in 791 (99.6%), positive in 1 (0.1%), and invalid in 2 (0.3%). Excluding invalid tests, self-testing had sensitivity of 97.4% (95% CI 95.1% to 99.7%) and specificity of 99.9% (95% CI: 99.6% to 100%). When interpreting results, 96%, 93.1% and 95.2% correctly read the positive, negative and invalid respectively. There were no significant demographic predictors for false negative self-testing or wrongly interpreting positive or invalid sample results as negative. Eighty-seven percent would purchase the kit over-the-counter; 89% preferred to take HIV tests in private. 72.5% and 74.9% felt the need for pre- and post-test counseling respectively. Only 28% would pay at least USD15 for the test.

Conclusions/Significance

Self-testing was associated with high specificity, and a small but significant number of false negatives. Incorrectly identifying model results as invalid was a major reason for incorrect result interpretation. Survey responses were supportive of making self-testing available.     

The design, synthesis, and biological evaluation of PIM kinase inhibitors

A series of substituted benzofuropyrimidinones with pan-PIM activities and excellent selectivity against a panel of diverse kinases is described. Initial exploration identified aryl benzofuropyrimidinones that were potent, but had cell permeability limitation. Using X-ray crystal structures of the bound PIM-1 complexes with 3, 5m, and 6d, we were able to guide the SAR and identify the alkyl benzofuropyrimidinone (6l) with good PIM potencies, permeability, and oral exposure.     

SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.     

Pleistocene genetic connectivity in a widespread,open‐habitat‐adapted mosquito in the Indo‐Oriental region

Aim The environmental effect of Pleistocene climatic change in the Indo‐Oriental region has resulted in allopatric fragmentation and the generation of diversity in forest‐associated species. The aim of this study was to determine the extent to which Pleistocene climatic change has resulted in the fragmentation and speciation of an open‐habitat‐adapted mosquito, Anopheles vagus s.l., across its range. Location Anopheles vagus s.l. was sampled across the Indo‐Oriental region. Methods We generated 116 mitochondrial cytochrome c oxidase subunit I (COI) and 121 nuclear internal transcribed spacer 2 (ITS2) DNA sequences from 18 populations. Relationships between mitochondrial haplotypes were reconstructed using minimum spanning networks, and population structure was examined using analyses of molecular variance. The population history, including lineage divergence times, population expansion and gene flow, was inferred using beast and the isolation with migration (IM) model. Results There was no evidence to support the presence of the endemic Philippines species, A. limosus; instead, Philippine populations were closely related to, and derived from, A. vagus on the eastern Southeast Asian mainland. The most distinct populations were those from Java and East Timor, which differed from all other populations by all individuals having a 4‐bp insertion in the ITS2 sequence. The corresponding mitochondrial haplotypes had an estimated divergence time of 2.6 Ma [95% confidence interval (CI) 1.9–3.6 Ma]. Haplotype networks and analysis of molecular variance for COI supported western (Sri Lanka, India and Myanmar) and eastern (Thailand, Singapore, Cambodia, Vietnam and the Philippines) population groupings. This grouping structure results from the divergence of an eastern and a western mitochondrial lineage, estimated to have occurred 0.37 Ma (95% CI 0.26–0.55 Ma). Subsequent migration from the east to the west (0.16 Ma) is inferred to have created an admixture zone in Myanmar and Thailand. Main conclusions With the possible exception of populations from Java and East Timor, A. vagus appears to be one widespread genetically diverse taxon across its extensive range. The abundance of grassland during long interglacial periods may have facilitated population connectivity and range expansion across the Oriental and western Australasian regions.     

Cloning and expression of <Emphasis Type="Italic">Perilla frutescens FAD2</Emphasis> gene and polymorphism analysis among cultivars

?12 fatty acid desaturase (FAD2) is a key enzyme for linoleic acid and linolenic acid biosynthesis. Perilla frutescens is a special oil plant species with highest linolenic acid content. In this study, based on RACE, two alleles for one FAD2 gene were isolated from P. frutescens cultivar C2: the 3956 bp PfFAD2a and the 3959 bp PfFAD2b, both with a full-length cDNA of 1526 bp, and both encoding a 382aa basic protein. The alleles have identities of over 98%, and their encoded proteins differ only by substitution of a strongly similar residue. Saccharomyces cerevisiae heterologous expression suggested that PfFAD2a/b both encode a bio-functional FAD2 enzyme. Phylogenetic analyses indicated that PfFAD2 shows the highest homologies to FAD2 genes from dicots such as Boraginaceae and Burseraceae. PfFAD2a/b expressions are mainly restricted to developing seeds. PfFAD2a/b expression in the seedling leaf is upregulated by cold (4 °C) and repressed by heat (42 °C). Each of the eight cultivars contains two alleles for one PfFAD2 and 40 SNP sites are found. One allelic gene in cultivars C1 and P1 is pseudogene because of premature stop codon mutation in 5′ coding region. All other normal PfFAD2 genes/allelic genes encode identical or very similar proteins. PfFAD2a/b expression level in developing seeds also varies among the eight cultivars. This study provides systemic molecular and functional features of PfFAD2 and enables its application in the study of plant fatty acids traits.     

Gastrodia putaoensis sp. nov. (Orchidaceae,Epidendroideae) from North Myanmar

Gastrodia putaoensis, a new species from the montane region in northern Myanmar, is described and illustrated. Gastrodia putaoensis is similar to G. dyeriana, but differs from it by having a narrowly triangular lip that is subdivided into two parts, with the apical part densely covered with yellow hairs and the apex obtuse and densely covered with red papillae.     

Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB.