OBJECTIVES/GOALS: To develop feasible screening methods for activity of the enzyme Glucose-6-phosphate dehydrogenase (G6PD) with point of care applicability. METHODS/STUDY POPULATION: Current knowledge establishes the relevance of G6PD as a critical therapeutic determinant for effective antimalarial therapy due to the occurrence of mutations that lead to post-treatment severe adverse effects. We present our findings on development of cost effective point-of-care screening methodologies to ascertain G6PD deficiency. RESULTS/ANTICIPATED RESULTS: Using Patient Cohort Explorer and data from the Department of Pathology, we established the prevalence of G6PD deficiency at the University of Mississippi Medical Center, Jackson, MS as high as 11.8% (African-American males in all population, n = 2518). Next, for selection of potential target groups, we set up a protocol for recruitment of volunteers based on ethnic background, parental ethnicity, and medical history. G6PD activity was evaluated using point of care methods [Trinity Biotech test or CareSTART Biosensor], and Gold Standard quantitative spectrophotometric assay (LabCorp). Determinations in >20 subjects have showed comparable concordance. If used with a conservative interpretation of the signal, the Trinity Biotech test showed superior potential for use in the field relative to the CareSTART Biosensor. DISCUSSION/SIGNIFICANCE OF IMPACT: We established the prevalence of G6PD deficiency in our medical center. We have also setup tests for point-of-care assessment of G6PD. Pending evaluation of the relative tests performance, we will be in position to screen individuals and select them for a prospective clinical trial to evaluate the safety of antimalarial agents on scope of G6PD deficiency.

Novel tools for early diagnosis and monitoring of schistosomiasis are urgently needed. This study aimed to validate parasite-derived miRNAs as potential novel biomarkers for the detection of human Schistosoma japonicum infection. A total of 21 miRNAs were initially validated by real-time-polymerase chain reaction (RT-PCR) using serum samples of S. japonicum-infected BALB/c mice. Of these, 6 miRNAs were further validated with a human cohort of individuals from a schistosomiasis-endemic area of the Philippines. RT-PCR analysis showed that two parasite-derived miRNAs (sja-miR-2b-5p and sja-miR-2c-5p) could detect infected individuals with low infection intensity with moderate sensitivity/specificity values of 66%/68% and 55%/80%, respectively. Analysis of the combined data for the two parasite miRNAs revealed a specificity of 77.4% and a sensitivity of 60.0% with an area under the curve (AUC) value of 0.6906 (P = 0.0069); however, a duplex RT-PCR targeting both sja-miR-2b-5p and sja-miR-2c-5p did not result in an increased diagnostic performance compared with the singleplex assays. Furthermore, the serum level of sja-miR-2c-5p correlated significantly with faecal egg counts, whereas the other five miRNAs did not. Targeting S. japonicum-derived miRNAs in serum resulted in a moderate diagnostic performance when applied to a low schistosome infection intensity setting.

In utero exposure to the ubiquitous plasticizer, bisphenol A (BPA) is associated with offspring obesity. As adipogenesis is a critical factor contributing to obesity, we determined the effects of in vivo maternal BPA and in vitro BPA exposure on newborn adipose tissue at the stem-cell level. For in vivo studies, female rats received BPA before and during pregnancy and lactation via drinking water, and offspring were studied for measures of adiposity signals. For in vitro BPA exposure, primary pre-adipocyte cell cultures from healthy newborns were utilized. We studied pre-adipocyte proliferative and differentiation effects of BPA and explored putative signal factors which partly explain adipose responses and underlying epigenetic mechanisms mediated by BPA. Maternal BPA-induced offspring adiposity, hypertrophic adipocytes and increased adipose tissue protein expression of pro-adipogenic and lipogenic factors. Consistent with in vivo data, in vitro BPA exposure induced a dose-dependent increase in pre-adipocyte proliferation and increased adipocyte lipid content. In vivo and in vitro BPA exposure promotes the proliferation and differentiation of adipocytes, contributing to an enhanced capacity for lipid storage. These findings reinforce the marked effects of BPA on adipogenesis and highlight the susceptibility of stem-cell populations during early life with long-term consequence on metabolic homeostasis.

Exposure to maternal over-nutrition in utero is linked with developmental programming of obesity, metabolic syndrome and cardiovascular disease in offspring, which may be exacerbated by postnatal high-fat (HF) diet. Skeletal muscle mitochondrial function contributes to substrate metabolism and is impaired in metabolic disease. We examined muscle mitochondrial respiration in male and female mice exposed to maternal HF diet in utero, followed by postweaning HF diet until middle age. After in utero exposure to maternal control (Con) or HF diet (45% kcal fat; 39.4% lard, 5.5% soybean oil), offspring were weaned to Con or HF, creating four groups: Con/Con (male/female (m/f), n=8/8), Con/HF (m/f, n=7/4), HF/Con (m/f, n=9/6) and HF/HF (m/f, n=4/4). Oxidative phosphorylation (OXPHOS) and electron transfer system (ETS) capacity were measured in permeabilized gastrocnemius bundles. Maternal HF diet increased fasting glucose and lean body mass in males and body fat percentage in both sexes (P⩽0.05). Maximal adenosine diphosphate-stimulated respiration (complex I OXPHOS) was decreased by maternal HF diet in female offspring (−21%, P=0.053), but not in male (−0%, P>0.05). Sexually divergent responses were exacerbated in offspring weaned to HF diet. In females, OXPHOS capacity was lower (−28%, P=0.041) when weaned to high-fat (HF/HF) v. control diet (HF/Con). In males, OXPHOS (+33%, P=0.009) and ETS (+42%, P=0.016) capacity increased. Our data suggest that maternal lard-based HF diet, rich in saturated fat, affects offspring skeletal muscle respiration in a sex-dependent manner, and these differences are exacerbated by HF diet in adulthood.

Glioblastoma is considered among the most aggressive cancers, dismal prognosis and overall survival is only 14 months, 80% of primary low grade gliomas and seccondary GBMs that progress from low grade to grade II or III WHO classification have isocitrate dehydrogenase, (IDH1) or IDH2, mutations [1]. IDH1 mutant glioma is characterized by impaired glycolysis activity resulting in an abnormal production of 2-hydroxyglutarate (2-HG) [2] resulting in an undifferentiated phenotype with permanent hyper-methylation status and enhanced proliferation and invasion[3]. Interestingly, the IDH1 mutant phenotype of U87MG glioma cells shows resistance to autophagy induced cell death even in starving and low oxygen conditions [4]. Recent evidence has demonstrated increased autophagy activity on IDH1 mt cytotoxic activity when cells are exposed to autophagy inhibitors such as chloroquine [5]. To this date it remains elusive whether increased autophagy phenotype is the result of IDH1 mutation or if it either represents a secondary co-existing condition in the setting of the IDH1 mutation. Autophagy is also a mechanism of detoxification induced by chronic heavy metal exposure in both normal and cancer cells. Thus, we hypothesized that autophagy activity in IDH1 mt glioma is partially induced by chronic heavy metal exposure, leading to increased cell survival and abnormal DNA repair. Our approach included characterization and quantification of metal content on IDH1 mt glioma cell lines and tissues, in addition to correlation analyses of the celullar metallome with autophagy markers, ROS and DNA repair of IDH1 mt glioma cells allowed us to explore targets responsible for cell survival and DNA repair response. Furthermore, we evaluated the potential therapeutic value of Chloroquine (CQ) and bafylomicin for IDH1 mutant gliomas targeting autophagy pathway in combination with TMZ and radiation. We demonstrated that 2-HG induces autophagy activity via LC3B activation, and autophagy inhibiton by beclin gene silencing results in a reduction of 2-HG leading to cell starvation and apoptosis. Remarkably, we observed a positive correlation on at least six different metals with autophagy induced LC3B and beclin1 expression that significantly differed between the mutant and the wt genotype in glioma cell lines. ROS and DNA repair were also positively associated with at least 6 different metals and only seen in the IDH1 mt cell lines, then suggesting a possible explanation for the increase on autophagy, analysis of both LC3B and beclin 1 expression demonstrated a positive correlation with Mo98, Fe54, and Zn 66 on IDH1 mt cell lines and a positively correlation with Mo98 and V concentrations in relation to H2AX expression. Co, SeO Mo, V and Mg were positively correlated to ROS expression. TMZ and CQ induced autophagy pathway activation as measured by LC3B, Beclin, Atg expression. Silencing beclin in IDH1 mutant glioma cell lines induced apoptosis and reduction on 2-HG production after treatment with TMZ and radiation. Overall the results contained in this study 1) identify cellular metal content in relationship to mechanisms leading to increased autophagy on IDH1 mt glioma cells. 2) evaluate the combination of CQ and TMZ to potentially target and inhibit autophagy as a mechanism downstream the 2-HG production in IDH1 mt glioma cells.

n-3 PUFA are lipids that play crucial roles in immune-regulation, cardio-protection and neurodevelopment. However, little is known about the role that these essential dietary fats play in modulating caecal microbiota composition and the subsequent production of functional metabolites. To investigate this, female C57BL/6 mice were assigned to one of three diets (control (CON), n-3 supplemented (n3+) or n-3 deficient (n3−)) during gestation, following which their male offspring were continued on the same diets for 12 weeks. Caecal content of mothers and offspring were collected for 16S sequencing and metabolic phenotyping. n3− male offspring displayed significantly less % fat mass than n3+ and CON. n-3 Status also induced a number of changes to gut microbiota composition such that n3− offspring had greater abundance of Tenericutes, Anaeroplasma and Coriobacteriaceae. Metabolomics analysis revealed an increase in caecal metabolites involved in energy metabolism in n3+ including α-ketoglutaric acid, malic acid and fumaric acid. n3− animals displayed significantly reduced acetate, butyrate and total caecal SCFA production. These results demonstrate that dietary n-3 PUFA regulate gut microbiota homoeostasis whereby n-3 deficiency may induce a state of disturbance. Further studies are warranted to examine whether these microbial and metabolic disturbances are causally related to changes in metabolic health outcomes.

We study the dynamics of a domain wall under the influence of applied magnetic fields in a one-dimensional ferromagnetic nanowire, governed by the Landau–Lifshitz–Gilbert equation. Existence of travelling-wave solutions close to two known static solutions is proven using implicit-function-theorem-type arguments.

Background: Cerebral palsy (CP) is a debilitating disorder (1). Based on neuromotor impairments it is divided to spastic, dyskinetic and ataxic types (2). Inborn Errors of Metabolism (IEMs), monogenic and chromosomal disorders mimic CP (3). We aimed to identify causal genetic variants in patients with atypical dyskinetic CP in whom known IEMs were ruled out. Timely diagnosis is essential for proper management, especially in conditions that mimic CP and are treatable. Methods: We enrolled 23 patients with unexplained atypical dyskinetic CP, for whole exome sequencing. Variants were filtered against public and in-house databases to identify variants predicted as damaging (in silico tools and ACMG criteria). We applied a virtual gene panel of known and suspected CP and movement disorder genes and investigated each sample. Results: The participants presented with symptoms including: spasticity, dystonia, choera-athetosis, ataxia and cognitive delays. We identified 23 diagnoses: 13 dominant,6 recessive and 4 X-linked. 12 patients had movement disorders. In 4, the diagnoses enabled targeted treatment (neurotransmitter supplements in Unverricht Lundborg diseases (CSTB) and PAK3 deficiency, deep brain stimulation in GNAO1 deficiency, medical diet in Glutaric Aciduria (GCDH). Conclusions: Whole Exome Sequencing contributes to establishing diagnosis in patients with atypical dyskinetic CP resulting in precision medicine and improved health outcomes.

This study aimed to assess the merit and suitability of individual functional units (FU) in expressing greenhouse gas emissions intensity in different dairy production systems. An FU provides a clearly defined and measurable reference to which input and output data are normalised. This enables the results from life-cycle assessment (LCA) of different systems to be treated as functionally equivalent. Although the methodological framework of LCA has been standardised, selection of an appropriate FU remains ultimately at the discretion of the individual study. The aim of the present analysis was to examine the effect of different FU on the emissions intensities of different dairy production systems. Analysis was based on 7 years of data (2004 to 2010) from four Holstein-Friesian dairy systems at Scotland’s Rural College’s long-term genetic and management systems project, the Langhill herd. Implementation of LCA accounted for the environmental impacts of the whole-farm systems and their production of milk from ‘cradle to farm gate’. Emissions intensity was determined as kilograms of carbon dioxide equivalents referenced to six FU: UK livestock units, energy-corrected milk yield, total combined milk solids yield, on-farm land used for production, total combined on- and off-farm land used for production, and the proposed new FU–energy-corrected milk yield per hectare of total land used. Energy-corrected milk was the FU most effective for reflecting differences between the systems. Functional unit that incorporated a land-related aspect did not find difference between systems which were managed under the same forage regime, despite their comprising different genetic lines. Employing on-farm land as the FU favoured grazing systems. The proposed dual FU combining both productivity and land use did not differentiate between emissions intensity of systems as effectively as the productivity-based units. However, this dual unit displayed potential to quantify in a simple way the positive or negative outcome of trade-offs between land and production efficiencies, in which improvement in emissions intensity using one FU may be accompanied by deterioration using another FU. The perceived environmental efficiencies of different dairy production systems in terms of their emissions intensities were susceptible to change based upon the FU employed, and hence the FU used in any study needs to be taken into account in the interpretation of results.

Accurate models of X-ray absorption and re-emission in partly stripped ions are necessary to calculate the structure of stars, the performance of hohlraums for inertial confinement fusion and many other systems in high-energy-density plasma physics. Despite theoretical progress, a persistent discrepancy exists with recent experiments at the Sandia Z facility studying iron in conditions characteristic of the solar radiative–convective transition region. The increased iron opacity measured at Z could help resolve a longstanding issue with the standard solar model, but requires a radical departure for opacity theory. To replicate the Z measurements, an opacity experiment has been designed for the National Facility (NIF). The design uses established techniques scaled to NIF. A laser-heated hohlraum will produce X-ray-heated uniform iron plasmas in local thermodynamic equilibrium (LTE) at temperatures ${\geqslant}150$  eV and electron densities ${\geqslant}7\times 10^{21}~\text{cm}^{-3}$ . The iron will be probed using continuum X-rays emitted in a ${\sim}200$  ps, ${\sim}200~\unicode[STIX]{x03BC}\text{m}$ diameter source from a 2 mm diameter polystyrene (CH) capsule implosion. In this design, $2/3$ of the NIF beams deliver 500 kJ to the ${\sim}6$  mm diameter hohlraum, and the remaining $1/3$ directly drive the CH capsule with 200 kJ. Calculations indicate this capsule backlighter should outshine the iron sample, delivering a point-projection transmission opacity measurement to a time-integrated X-ray spectrometer viewing down the hohlraum axis. Preliminary experiments to develop the backlighter and hohlraum are underway, informing simulated measurements to guide the final design.

The collective response of electrons in an ultrathin foil target irradiated by an ultraintense ( ${\sim}6\times 10^{20}~\text{W}~\text{cm}^{-2}$ ) laser pulse is investigated experimentally and via 3D particle-in-cell simulations. It is shown that if the target is sufficiently thin that the laser induces significant radiation pressure, but not thin enough to become relativistically transparent to the laser light, the resulting relativistic electron beam is elliptical, with the major axis of the ellipse directed along the laser polarization axis. When the target thickness is decreased such that it becomes relativistically transparent early in the interaction with the laser pulse, diffraction of the transmitted laser light occurs through a so called ‘relativistic plasma aperture’, inducing structure in the spatial-intensity profile of the beam of energetic electrons. It is shown that the electron beam profile can be modified by variation of the target thickness and degree of ellipticity in the laser polarization.

The aim of this study was to examine the population structure, transmission and spatial relationship between genotypes of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni, on 20 dairy farms in a defined catchment. Pooled faecal samples (n = 72) obtained from 288 calves were analysed by real-time polymerase chain reaction (rtPCR) for E. coli serotypes O26, O103, O111, O145 and O157. The number of samples positive for E. coli O26 (30/72) was high compared to E. coli O103 (7/72), O145 (3/72), O157 (2/72) and O111 (0/72). Eighteen E. coli O26 and 53 C. jejuni isolates were recovered from samples by bacterial culture. E. coli O26 and C. jejuni isolates were genotyped using pulsed-field gel electrophoresis and multilocus sequence typing, respectively. All E. coli O26 isolates could be divided into four clusters and the results indicated that E. coli O26 isolates recovered from calves on the same farm were more similar than isolates recovered from different farms in the catchment. There were 11 different sequence types of C. jejuni isolated from the cattle and 22 from water. An analysis of the population structure of C. jejuni isolated from cattle provided evidence of clustering of genotypes within farms, and among groups of farms separated by road boundaries.

Atrazine has been used for control of many weeds, primarily broadleaf weeds, in U.S. corn fields since 1957. Recently, the adoption of glyphosate-resistant corn hybrids have led to glyphosate eclipsing atrazine as the most commonly used herbicide in corn production. However, the evolution and spread of glyphosate-resistant weeds is a major concern. Atrazine use in Wisconsin is prohibited in 102 areas encompassing 0.49 million ha where total chlorinated residues were found in drinking water wells at concentrations > 3 μg L−1. Atrazine has been prohibited in many of those areas for > 10 yr, providing an opportunity to evaluate weed community composition differences due to herbicide regulation. In question, has the abundance of broadleaf weeds increased, coupled with an increased reliance on glyphosate, where atrazine use has been discontinued? To answer this, an online questionnaire was distributed to Wisconsin growers in June and then weeds present in 343 fields in late July through mid-September in 2012 and 2013 were counted. Data were summarized for frequency, uniformity, density, and relative abundance to compare weed community composition in fields with discontinued vs. recent atrazine use. Growers used glyphosate in 70 vs. 54% of fields with discontinued vs. recent atrazine use, respectively (P = 0.021). Moreover, broadleaf weeds were found more frequently, (73 vs. 61%; P = 0.03), they had 50% greater in-field uniformity (P = 0.002), and density was 0.4 vs. 0.19 plants m−2 (i.e., twofold greater; P < 0.0001) in discontinued vs. recent atrazine-use fields. Changes were most evident with troublesome glyphosate-resistant broadleaf weeds such as Amaranthus species and giant ragweed. In conclusion, weed community composition consisted of more broadleaf weeds in fields where atrazine has not been used in the recent decade coupled with greater glyphosate use. These results provide evidence of negative long-term implications for glyphosate resistance where growers increased reliance on glyphosate in place of atrazine.