Rapid quantification of underivatized alloisoleucine and argininosuccinate using mixed-mode chromatography with tandem mass spectrometry

Plasma elevations of the amino acids alloisoleucine and argininosuccinic acid (ASA) are pathognomonic for maple syrup urine disease and argininosuccinate lyase deficiency, respectively. Reliable detection of these biomarkers is typically achieved using methods with tedious sample preparations or long chromatographic separations, and many published amino acid assays report poor specificity and/or sensitivity for one or both of these compounds. This report describes a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method that provides rapid quantification of alloisoleucine and ASA in human plasma. The basis of this method is a mixed-mode solid phase separation that achieves baseline resolution of alloisoleucine from isobaric interferents without the use of derivatization or ion pairing agents. The inject-to-inject time is 6 min including elution, column washing and re-equilibration. Validation studies demonstrate excellent limits of quantification (1 μmol/L), linearity (r = 0.999 from 1 to 250 μmol/L), accuracy (bias = −3.8% and −10.1%), and inter-assay imprecision (CV < 8.06%) for plasma analyses. Data from long-term clinical application confirms chromatographic consistency equivalent to more traditional reversed-phase or HILIC based columns. Additional matrix studies indicate low suppression (<10%) for a wide range of amino acids and compatibility with other matrixes such as blood spot analyses. Finally, analysis of our first 257 clinical specimens demonstrates high analytic specificity and sensitivity, allowing the detection of subtle but clinically relevant elevations of alloisoleucine and ASA that may be missed by other less sensitive methods. In conclusion, the novel LC-MS/MS method reported here overcomes a number of the challenges associated with alloisoleucine and ASA quantification. Combining this approach with published incomplete amino acid quantification methods allows, for the first time, a rapid and comprehensive LC-MS/MS analysis of underivatized amino acids without the use of ion pairing agents

Comprehensive U.S. Cyber Framework: Key Aspects of Critical Infrastructure, Private Sector, and Personally Identifiable Information

his project is a product of the Class of 2019 Bush School of Government and Public Service,Texas A&M University Capstone Program. The project lasted one academic year and involved eight second-year master students. It intends to synthesize and provide clarity in the realm of issues pertaining to U.S. Internet Protocol Space by demonstrating natural partnerships and recommendations for existing cyber incident response. The project was produced at the request of PointStream Inc., a private cybersecurity contractor. This capstone team analyzed existing frameworks for cyber incident response for PointStream, Inc. in order to propose a comprehensive and efficient plan for U.S. cybersecurity, critical infrastructure, and private sector stakeholders.Pointstream, Inc

Low-Enriched Uranium Fuel Conversion Activities for the High Flux Isotope Reactor, Annual Report for FY 2011

This report describes progress made during FY11 in ORNL activities to support converting the High Flux Isotope Reactor (HFIR) from high-enriched uranium (HEU) fuel to low-enriched uranium (LEU) fuel. Conversion from HEU to LEU will require a change in fuel form from uranium oxide to a uranium-molybdenum (UMo) alloy. With both radial and axial contouring of the fuel foil and an increase in reactor power to 100 MW, calculations indicate that the HFIR can be operated with LEU fuel with no degradation in performance to users from the current levels achieved with HEU fuel. Studies are continuing to demonstrate that the fuel thermal safety margins can be preserved following conversion. Studies are also continuing to update other aspects of the reactor steady state operation and accident response for the effects of fuel conversion. Technical input has been provided to Oregon State University in support of their hydraulic testing program. The HFIR conversion schedule was revised and provided to the GTRI program. In addition to HFIR conversion activities, technical support was provided directly to the Fuel Fabrication Capability program manager

Effects of vitamin D-2 or D-3 supplementation on glycaemic control and cardiometabolic risk among people at risk of type 2 diabetes: results of a randomized double-blind placebo-controlled trial

This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1111/dom.12625$\textbf{Aims:}$ To investigate the effect of short-term vitamin D supplementation on cardiometabolic outcomes among individuals with an elevated risk of diabetes. $\textbf{Methods:}$ In a double-blind placebo-controlled randomized trial, 340 adults who had an elevated risk of type 2 diabetes (non-diabetic hyperglycaemia or positive diabetes risk score) were randomized to either placebo, 100 000 IU vitamin D$_2$ (ergocalciferol) or 100 000 IU vitamin D$_3$ (cholecalciferol), orally administered monthly for 4 months. The primary outcome was change in glycated haemoglobin (HbA1c) between baseline and 4 months, adjusted for baseline. Secondary outcomes included: blood pressure; lipid levels; apolipoprotein levels; C-reactive protein levels; pulse wave velocity (PWV); anthropometric measures; and safety of the supplementation. $\textbf{Results:}$ The mean [standard deviation (s.d.)] 25-hydroxyvitamin D [25(OH)D]$_2$ concentration increased from 5.2 (4.1) to 53.9 (18.5) nmol/l in the D$_2$ group, and the mean (s.d.) 25(OH)D$_3$ concentration increased from 45.8 (22.6) to 83.8 (22.7) nmol/l in the D$_3$ group. There was no effect of vitamin D supplementation on HbA1c: D$_2$ versus placebo: −0.05% [95% confidence interval (CI) −0.11, 0.02] or −0.51 mmol/mol (95% CI −1.16, 0.14; p=0.13); D$_3$ versus placebo: 0.02% (95% CI −0.04, 0.08) or 0.19 mmol/mol (95% CI −0.46, 0.83; p=0.57). There were no clinically meaningful effects on secondary outcomes, except PWV [D$_2$ versus placebo: −0.68 m/s (95% CI −1.31, −0.05); D$_3$ versus placebo −0.73 m/s (95% CI −1.42, −0.03)]. No important safety issues were identified. $\textbf{Conclusions:}$ Short-term supplementation with vitamin D$_2$ or D$_3$ had no effect on HbA1c. The modest reduction in PWV with both D$_2$ and D$_3$ relative to placebo suggests that vitamin D supplementation has a beneficial effect on arterial stiffness.The trial was jointly sponsored by Queen Mary University of London and the Medical Research Council Epidemiology Unit at Cambridge. The trial was funded from a block grant from the NHS Tower Hamlets Primary Care NHS Trust and East London CLRN, and from MRC Epidemiology Unit core funding (MC_UP_A100_1003, MC_U106179474, MC_UU_12015/5 and MC_UU_12015/4)

Fungal entomopathogens: new insights on their ecology

An important mechanism for insect pest control should be the use of fungal entomopathogens. Even though these organisms have been studied for more than 100 y, their effective use in the field remains elusive. Recently, however, it has been discovered that many of these entomopathogenic fungi play additional roles in nature. They are endophytes, antagonists of plant pathogens, associates with the rhizosphere, and possibly even plant growth promoting agents. These findings indicate that the ecological role of these fungi in the environment is not fully understood and limits our ability to employ them successfully for pest management. In this paper, we review the recently discovered roles played by many entomopathogenic fungi and propose new research strategies focused on alternate uses for these fungi. It seems likely that these agents can be used in multiple roles in protecting plants from pests and diseases and at the same time promoting plant growth

Rapid prenatal diagnosis using targeted exome sequencing: a cohort study to assess feasibility and potential impact on prenatal counseling and pregnancy management.

Purpose Unexpected fetal abnormalities occur in 2-5% of pregnancies. While traditional cytogenetic and microarray approaches achieve diagnosis in around 40% of cases, lack of diagnosis in others impedes parental counseling, informed decision making, and pregnancy management. Postnatally exome sequencing yields high diagnostic rates, but relies on careful phenotyping to interpret genotype results. Here we used a multidisciplinary approach to explore the utility of rapid fetal exome sequencing for prenatal diagnosis using skeletal dysplasias as an exemplar. Methods Parents in pregnancies undergoing invasive testing because of sonographic fetal abnormalities, where multidisciplinary review considered skeletal dysplasia a likely etiology, were consented for exome trio sequencing (both parents and fetus). Variant interpretation focused on a virtual panel of 240 genes known to cause skeletal dysplasias. Results Definitive molecular diagnosis was made in 13/16 (81%) cases. In some cases, fetal ultrasound findings alone were of sufficient severity for parents to opt for termination. In others, molecular diagnosis informed accurate prediction of outcome, improved parental counseling, and enabled parents to terminate or continue the pregnancy with certainty. Conclusion Trio sequencing with expert multidisciplinary review for case selection and data interpretation yields timely, high diagnostic rates in fetuses presenting with unexpected skeletal abnormalities. This improves parental counseling and pregnancy management.Genetics in Medicine advance online publication, 29 March 2018; doi:10.1038/gim.2018.30

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Chronic Alcohol Exposure Alters Behavioral and Synaptic Plasticity of the Rodent Prefrontal Cortex

In the present study, we used a mouse model of chronic intermittent ethanol (CIE) exposure to examine how CIE alters the plasticity of the medial prefrontal cortex (mPFC). In acute slices obtained either immediately or 1-week after the last episode of alcohol exposure, voltage-clamp recording of excitatory post-synaptic currents (EPSCs) in mPFC layer V pyramidal neurons revealed that CIE exposure resulted in an increase in the NMDA/AMPA current ratio. This increase appeared to result from a selective increase in the NMDA component of the EPSC. Consistent with this, Western blot analysis of the postsynaptic density fraction showed that while there was no change in expression of the AMPA GluR1 subunit, NMDA NR1 and NRB subunits were significantly increased in CIE exposed mice when examined immediately after the last episode of alcohol exposure. Unexpectedly, this increase in NR1 and NR2B was no longer observed after 1-week of withdrawal in spite of a persistent increase in synaptic NMDA currents. Analysis of spines on the basal dendrites of layer V neurons revealed that while the total density of spines was not altered, there was a selective increase in the density of mushroom-type spines following CIE exposure. Examination of NMDA-receptor mediated spike-timing-dependent plasticity (STDP) showed that CIE exposure was associated with altered expression of long-term potentiation (LTP). Lastly, behavioral studies using an attentional set-shifting task that depends upon the mPFC for optimal performance revealed deficits in cognitive flexibility in CIE exposed mice when tested up to 1-week after the last episode of alcohol exposure. Taken together, these observations are consistent with those in human alcoholics showing protracted deficits in executive function, and suggest these deficits may be associated with alterations in synaptic plasticity in the mPFC