Performance of the CareStart? G6PD Deficiency Screening Test,a Point-of-Care Diagnostic for Primaquine Therapy Screening

Development of reliable, easy-to-use, rapid diagnostic tests (RDTs) to detect glucose-6-phosphate dehydrogenase (G6PD) deficiency at point of care is essential to deploying primaquine therapies as part of malaria elimination strategies. We assessed a kit under research and development called CareStart™ G6PD deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method (‘gold standard’). Blood samples (n = 903) were collected from Cambodian adults living in Pailin province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median 12.0 U/g Hg). Based on a normal haemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population was 3.6 to 20.5 U/g Hg (95^th percentiles from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was analyzed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang genotype. The sensitivity and specificity of the CareStart™ G6PD deficiency screening test was 0.68 and 1.0, respectively. Its detection threshold was <2.7 U/g Hg, well within the range of moderate and severe enzyme deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities <2 U/g Hg were falsely classified as “normal” by RDT. This experimental RDT test here evaluated outside of the laboratory for the first time shows real promise, but safe application of it will require lower rates of falsely “normal” results.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Differential Metabolite Profiles during Fruit Development in High-Yielding Oil Palm Mesocarp

To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.     

Effects of the Neurogranin Variant rs12807809 on Thalamocortical Morphology in Schizophrenia

Although the genome wide supported psychosis susceptibility neurogranin (NRGN) gene is expressed in human brains, it is unclear how it impacts brain morphology in schizophrenia. We investigated the influence of NRGN rs12807809 on cortical thickness, subcortical volumes and shapes in patients with schizophrenia. One hundred and fifty six subjects (91 patients with schizophrenia and 65 healthy controls) underwent structural MRI scans and their blood samples were genotyped. A brain mapping algorithm, large deformation diffeomorphic metric mapping, was used to perform group analysis of subcortical shapes and cortical thickness. Patients with risk TT genotype were associated with widespread cortical thinning involving frontal, parietal and temporal cortices compared with controls with TT genotype. No volumetric difference in subcortical structures (hippocampus, thalamus, amygdala, basal ganglia) was observed between risk TT genotype in patients and controls. However, patients with risk TT genotype were associated with thalamic shape abnormalities involving regions related to pulvinar and medial dorsal nuclei. Our results revealed the influence of the NRGN gene on thalamocortical morphology in schizophrenia involving widespread cortical thinning and thalamic shape abnormalities. These findings help to clarify underlying NRGN mediated pathophysiological mechanisms involving cortical-subcortical brain networks in schizophrenia.     

Murine gallbladder epithelial cells can differentiate into hepatocyte-like cells in vitro

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4alpha, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, alpha-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.     

Localized Traps Limited Recombination in Lead Bromide Perovskites

Traps exert an omnipotent influence over the performance of halide perovskite optoelectronic devices. A clear understanding of the origin and nature of the traps in halide perovskites is the key to controlling them and realizing optimal devices. Herein, the role of localized traps on the optical properties of lead bromide perovskite films is investigated. In the low‐temperature orthorhombic phase of CH₃NH₃PbBr₃ perovskite, band‐edge carrier dynamics exhibit a power‐law decay due to the presence of structural‐disorder‐induced localized traps, which has a depth of ≈40 meV. The continuous distribution of these localized traps gives rise to a broad sub‐band‐gap emission that becomes more prominent in thicker films with a larger trap density. The presence of this emission only from the hybrid organic–inorganic perovskites points to the vital role of organic dipoles in localized trap states formation. This study explicates the nature of these localized traps as well as their nontrivial role in carrier recombination kinetics, which is of fundamental importance in perovskites optoelectronics.     

Prevalence and Associated Risk Factors of Toxocara vitulorum Infections in Buffalo and Cattle Calves in Three Provinces of Central Cambodia

The prevalence and associated risk factors of Toxocara vitulorum infection in buffalo and cattle calves was studied in 3 provinces in central Cambodia. Fecal samples were collected from 517 calves between the age of 1-15 weeks and processed for nematode egg counts by a modified McMaster method. A total of 64 calves were found to excrete T. vitulorum eggs in their feces (12.4%; 95% exact CI: 9.7-15.5). The mean fecal egg count was 2,798 EPG (SD=16,351; range=0-224,400). A multivariable generalized linear mixed model showed higher odds of T. vitulorum infection for buffalo versus cattle, for animals aged 4-8 weeks versus younger and older ones, and for animals with strongyle infection. There was no association with fecal consistency. Farmers should be aware of the potential impact of T. vitulorum, and treat their calves at the age of 2-3 weeks with anthelmintics such as benzimidazoles or pyrantel.     

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile¹⁶⁵ and Met³³¹) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala⁴⁷) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme''s k_cat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.     

Genome-wide association study in east Asians identifies novel susceptibility loci for breast cancer

Genetic factors play an important role in the etiology of both sporadic and familial breast cancer. We aimed to discover novel genetic susceptibility loci for breast cancer. We conducted a four-stage genome-wide association study (GWAS) in 19,091 cases and 20,606 controls of East-Asian descent including Chinese, Korean, and Japanese women. After analyzing 690,947 SNPs in 2,918 cases and 2,324 controls, we evaluated 5,365 SNPs for replication in 3,972 cases and 3,852 controls. Ninety-four SNPs were further evaluated in 5,203 cases and 5,138 controls, and finally the top 22 SNPs were investigated in up to 17,423 additional subjects (7,489 cases and 9,934 controls). SNP rs9485372, near the TGF-β activated kinase (TAB2) gene in chromosome 6q25.1, showed a consistent association with breast cancer risk across all four stages, with a P-value of 3.8×10⁻¹² in the combined analysis of all samples. Adjusted odds ratios (95% confidence intervals) were 0.89 (0.85–0.94) and 0.80 (0.75–0.86) for the A/G and A/A genotypes, respectively, compared with the genotype G/G. SNP rs9383951 (P = 1.9×10⁻⁶ from the combined analysis of all samples), located in intron 5 of the ESR1 gene, and SNP rs7107217 (P = 4.6×10⁻⁷), located at 11q24.3, also showed a consistent association in each of the four stages. This study provides strong evidence for a novel breast cancer susceptibility locus represented by rs9485372, near the TAB2 gene (6q25.1), and identifies two possible susceptibility loci located in the ESR1 gene and 11q24.3, respectively.     

A new multiplex PCR test for the determination of Vrn-B1 alleles in bread wheat (Triticum aestivum L.)

Winter wheat requires vernalization, a long exposure to low but non-freezing temperatures, to promote reproductive development. The vernalization requirement in bread wheat (Triticum aestivum L.) is mainly controlled by the Vrn-1 genes that are located on chromosomes 5A, 5B and 5D. Dominant alleles confer spring habit and are epistatic to the recessive winter alleles which means that spring varieties carry at least one dominant allele. To date, two dominant and one recessive Vrn-B1 alleles have been described. Vrn-B1a (formerly designated as Vrn-B1) differs from the winter vrn-B1 allele by a large deletion in intron 1. Vrn-B1b has an additional small deletion and is probably derived from Vrn-B1a. The novel allele described here and designated as Vrn-B1c also has a large deletion within intron 1 but with different breakpoints from Vrn-B1a or b, and sequence duplication, showing that this is an independently derived spring allele. By combining an exon 1 primer with previously published PCR primers it was possible to develop a multiplex PCR that distinguished all four alleles simultaneously. The multiplex PCR was validated by testing 320 winter wheat and 137 spring wheat varieties. This demonstrated that the novel Vrn-B1c allele was present in 25 spring varieties of diverse origin, showing this allele to be widely distributed.