Modeling Contaminant Microbes in Rivers During Both Baseflow and Stormflow.

Rivers transport contaminant microorganisms (including fecal indicator bacteria and human pathogens) long distances downstream of diffuse and point sources, posing a human health risk. We present a mobile-immobile model that incorporates transport as well as immobilization and remobilization of contaminant microbes and other fine particles during baseflow and stormflow. During baseflow conditions, hyporheic exchange flow causes particles to accumulate in streambed sediments. Remobilization of stored particles from streambed sediments occurs slowly during baseflow via hyporheic exchange flow, while remobilization is vastly increased during stormflow. Model predictions are compared to observations over a range of artificial and natural flood events in the dairy contaminated Topehaehae Stream, New Zealand. The model outputs closely matched timing and magnitude of E. coli and turbidity observations through multiple high-flow events. By accounting for both state-of-flow and hyporheic exchange processes, the model provides a valuable framework for predicting particle and contaminant microbe behavior in streams.

Low-level constitutional mosaicism of BRCA1 in two women with young onset ovarian cancer.

Germline pathogenic variants in BRCA1 and BRCA2 cause hereditary breast and ovarian cancer. The vast majority of these variants are inherited from a parent. De novo constitutional pathogenic variants are rare. Even fewer cases of constitutional mosaicism have been reported and these have mostly been described in women with breast cancer. Here we report low-level constitutional mosaicism identified by Next Generation Sequencing in two women with ovarian cancer. A BRCA1 c.5074G > A p.(Asp1692Asn) variant detected in the first female at 42 years, classed as likely pathogenic, was found in ~ 52% of reads in DNA extracted from tumour, ~ 10% of reads in DNA extracted from peripheral blood leukocytes and ~ 10% of reads in DNA extracted from buccal mucosa. The second BRCA1 c.2755_2758dupCCTG p.(Val920AlafsTer6) variant was detected in a female aged 53 years, classed as pathogenic, and was found in ~ 59% of reads in DNA extracted from tumour, ~ 14% of reads in DNA extracted from peripheral blood leukocytes and similarly in ~ 14% of reads in both DNA extracted from buccal mucosa and urine sample. Sanger sequencing confirmed the presence of these variants at a corresponding low level consistent with mosaicism that may not have been detected by this method alone. This report demonstrates the clinical benefit for two women of BRCA1/BRCA2 germline NGS testing at a depth that can detect low-level mosaicism. As well as informing appropriate treatments, tumour sequencing results may facilitate the detection and interpretation of low-level mosaic variants in the germline. Both results have implications for other cancer risks and for relatives when providing a family cancer risk assessment and reproductive risk. The implications for laboratory practice, clinical genetics management and genetic counselling for constitutional mosaicism of BRCA1/BRCA2 are discussed.

Clinical likelihood ratios and balanced accuracy for 44 in silico tools against multiple large-scale functional assays of cancer susceptibility genes.

PURPOSE: Where multiple in silico tools are concordant, the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework affords supporting evidence toward pathogenicity or benignity, equivalent to a likelihood ratio of ~2. However, limited availability of "clinical truth sets" and prior use in tool training limits their utility for evaluation of tool performance. METHODS: We created a truth set of 9,436 missense variants classified as deleterious or tolerated in clinically validated high-throughput functional assays for BRCA1, BRCA2, MSH2, PTEN, and TP53 to evaluate predictive performance for 44 recommended/commonly used in silico tools. RESULTS: Over two-thirds of the tool-threshold combinations examined had specificity of <50%, thus substantially overcalling deleteriousness. REVEL scores of 0.8-1.0 had a Positive Likelihood Ratio (PLR) of 6.74 (5.24-8.82) compared to scores <0.7 and scores of 0-0.4 had a Negative Likelihood Ratio (NLR) of 34.3 (31.5-37.3) compared to scores of >0.7. For Meta-SNP, the equivalent PLR = 42.9 (14.4-406) and NLR = 19.4 (15.6-24.9). CONCLUSION: Against these clinically validated "functional truth sets," there was wide variation in the predictive performance of commonly used in silico tools. Overall, REVEL and Meta-SNP had best balanced accuracy and might potentially be used at stronger evidence weighting than current ACMG/AMP prescription, in particular for predictions of benignity.

Revisiting peak serum cortisol response to insulin-induced hypoglycemia in children.

PURPOSE: To evaluate factors that could potentially affect the hypothalamic-pituitary adrenal (HPA) axis response to insulin-induced hypoglycemia in children without history or symptoms of adrenal insufficiency and to propose a cut-off value to define a normal response in this population. METHODS: Exploratory single-center study involving 78 children that prospectively underwent insulin tolerance test (ITT) for suspected growth hormone (GH) deficiency. METHODS: Glucose, cortisol, GH, adrenocorticotrophic hormone (ACTH), epinephrine and norepinephrine levels were measured at baseline and after insulin-induced hypoglycemia. Serum cortisol was measured using Access automated immunoassay. RESULTS: Mean (range) basal morning serum cortisol of 8 (2.2-19.5) µg/dL/222 (61-542) nmol/L increased after hypoglycemia to 20.5 (14.6-29.5) µg/dL/570 nmol/L (405-819) nmol/L. Peak serum cortisol levels of 14.6 µg/dL (405 nmol/L) and 15.4 µg/dL (428 nmol/L) corresponded to the 2.5th and 5th percentiles, respectively. Peak serum cortisol correlated with peak plasma epinephrine (r = 0.367; P = 0.0014) but did not correlate with age, BMI-SD or peak serum GH. Children with intact and abnormal GH responses presented similar mean peak serum cortisol levels (20.0 vs. 20.6 µg/dL/555 vs. 572 nmol/L; P = 0.21). CONCLUSION: Our data indicate that the current cut-off to define normal HPA axis response in children after insulin-induced hypoglycemia warrants reevaluation to avoid over-diagnosis of adrenal insufficiency. Our results suggest that peak serum cortisol levels ≥ 15.4 µg/dL (428 nmol/L) in children undergoing ITT might represent a normal cortisol response to stress, regardless of age, BMI or GH secretory capacity.

All-Order Amplitudes at any Multiplicity in the Multi-Regge Limit.

We propose an all-loop expression for scattering amplitudes in planar N=4 super Yang-Mills theory in multi-Regge kinematics valid for all multiplicities, all helicity configurations, and arbitrary logarithmic accuracy. Our expression is arrived at from comparing explicit perturbative results with general expectations from the integrable structure of a closely related collinear limit. A crucial ingredient of the analysis is an all-order extension for the central emission vertex that we recently computed at next-to-leading logarithmic accuracy. As an application, we use our all-order formula to prove that all amplitudes in this theory in multi-Regge kinematics are single-valued multiple polylogarithms of uniform transcendental weight.

Evaluation of universal immunohistochemical screening of sebaceous neoplasms in a service setting.

BACKGROUND: Muir-Torre syndrome (MTS) is a subtype of Lynch syndrome, which encompasses the combination of sebaceous skin tumours or keratoacanthomas and internal malignancy, due to mutations in DNA mismatch repair genes. Sebaceous neoplasms (SNs) may occur before other malignancies, and may lead to the diagnosis, which allows testing of other family members, cancer surveillance, risk-reducing surgery or prevention therapies. AIM: To evaluate the efficacy of universal immunohistochemistry (IHC) screening of SNs in a service setting. METHODS: Patients with SNs were ascertained by a regional clinical pathology service over a 3-year period. Results of tumour IHC, clinical genetics notes and germline genetic testing were retrospectively reviewed. RESULTS: In total, 62 patients presented with 71 SNs; 9 (15%) of these patients had previously diagnosed MTS. Tumour IHC was performed for 50 of the 53 remaining patients (94%); 26 (52%) had loss of staining of one or more mismatch repair proteins. Fifteen patients were referred to the Clinical Genetics department, and 10 patients underwent germline genetic testing. Two had a new diagnosis of MTS confirmed, with heterozygous pathogenic mutations detected in the MSH2 and PMS2 genes (diagnostic yield 20%). The PMS2 mutation was identified in a 57-year-old woman with a sebaceous adenoma and history of endometrial cancer; to our knowledge, this is the first time a PMS2 mutation has been reported in MTS. CONCLUSIONS: Universal IHC screening of SNs is an effective method to identify cases for further genetic evaluation. Rates of referral to clinical genetics were only moderate (58%). Increased awareness of MTS could help improve the rate of onward referral.

Cell-specific abnormalities of glutamate transporters in schizophrenia: sick astrocytes and compensating relay neurons?

Excitatory amino-acid transporters (EAATs) bind and transport glutamate, limiting spillover from synapses due to their dense perisynaptic expression primarily on astroglia. Converging evidence suggests that abnormalities in the astroglial glutamate transporter localization and function may underlie a disease mechanism with pathological glutamate spillover as well as alterations in the kinetics of perisynaptic glutamate buffering and uptake contributing to dysfunction of thalamo-cortical circuits in schizophrenia. We explored this hypothesis by performing cell- and region-level studies of EAAT1 and EAAT2 expression in the mediodorsal nucleus of the thalamus in an elderly cohort of subjects with schizophrenia. We found decreased protein expression for the typically astroglial-localized glutamate transporters in the mediodorsal and ventral tier nuclei. We next used laser-capture microdissection and quantitative polymerase chain reaction to assess cell-level expression of the transporters and their splice variants. In the mediodorsal nucleus, we found lower expression of transporter transcripts in a population of cells enriched for astrocytes, and higher expression of transporter transcripts in a population of cells enriched for relay neurons. We confirmed expression of transporter protein in neurons in schizophrenia using dual-label immunofluorescence. Finally, the pattern of transporter mRNA and protein expression in rodents treated for 9 months with antipsychotic medication suggests that our findings are not due to the effects of antipsychotic treatment. We found a compensatory increase in transporter expression in neurons that might be secondary to a loss of transporter expression in astrocytes. These changes suggest a profound abnormality in astrocyte functions that support, nourish and maintain neuronal fidelity and synaptic activity.

Incorporating new materials and techniques into clinical practice.

This article outlines the subjects presented and discussed at the December 2012 IADR Dental Materials Innovation Workshop held at King's College London. Incorporating new materials and techniques into clinical practice was considered from 4 perspectives: (1) Accelerating the "research to regulatory approval" process was presented with current developments in the United States, with the National Institutes of Health/Food and Drug Administration process as a working example; (2) intellectual property and regulatory requirements were discussed across the well-established US and EU frameworks, as well as the more recently developed procedures across Brazil, Russia, India, and China; (3) the challenges and opportunities of incorporating innovations into dental education were considered with reference to the future needs of both students and faculty; and (4) the key but difficult and unpredictable step of translating such innovations into routine dental practice was then explored. Constructive and far-ranging discussion among the broadly based Workshop participants (from dental research, education, practice, and industry, as well as environmental organizations and the World Health Organization) mapped out key issues for the future. The focus was on facilitating the more timely adoption of improvements in both materials and techniques to improve patient health and health systems, while minimizing environmental impact.

How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase.

The crystal structure of a 27-kilodalton methylcobalamin-containing fragment of methionine synthase from Escherichia coli was determined at 3.0 A resolution. This structure depicts cobalamin-protein interactions and reveals that the corrin macrocycle lies between a helical amino-terminal domain and an alpha/beta carboxyl-terminal domain that is a variant of the Rossmann fold. Methylcobalamin undergoes a conformational change on binding the protein; the dimethylbenzimidazole group, which is coordinated to the cobalt in the free cofactor, moves away from the corrin and is replaced by a histidine contributed by the protein. The sequence Asp-X-His-X-X-Gly, which contains this histidine ligand, is conserved in the adenosylcobalamin-dependent enzymes methylmalonyl-coenzyme A mutase and glutamate mutase, suggesting that displacement of the dimethylbenzimidazole will be a feature common to many cobalamin-binding proteins. Thus the cobalt ligand, His759, and the neighboring residues Asp757 and Ser810, may form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group in methionine synthase.

Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells.

A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutS alpha, also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HOT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutS alpha 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.

Anthropometry as a predictor of high speed performance.

To assess anthropometry as a predictor of high-speed performance, subjects performed four seated knee- and hip-extension workouts with their left leg on an inertial exercise trainer (Impulse Technologies, Newnan GA). Workouts, done exclusively in either the tonic or phasic contractile mode, entailed two one-minute sets separated by a 90-second rest period and yielded three performance variables: peak force, average force and work. Subjects provided the following anthropometric data: height, weight, body mass index, as well as total, upper and lower left leg lengths. Via multiple regression, anthropometry attempted to predict the variance per performance variable. Anthropometry explained a modest (R2=0.27-0.43) yet significant degree of variance from inertial exercise trainer workouts. Anthropometry was a better predictor of peak force variance from phasic workouts, while it accounted for a significant degree of average force and work variance solely from tonic workouts. Future research should identify variables that account for the unexplained variance from high-speed exercise performance.

Degradation, fatigue, and failure of resin dental composite materials.

The intent of this article is to review the numerous factors that affect the mechanical properties of particle- or fiber-filler-containing indirect dental resin composite materials. The focus will be on the effects of degradation due to aging in different media, mainly water and water and ethanol, cyclic loading, and mixed-mode loading on flexure strength and fracture toughness. Several selected papers will be examined in detail with respect to mixed and cyclic loading, and 3D tomography with multi-axial compression specimens. The main cause of failure, for most dental resin composites, is the breakdown of the resin matrix and/or the interface between the filler and the resin matrix. In clinical studies, it appears that failure in the first 5 years is a restoration issue (technique or material selection); after that time period, failure most often results from secondary decay.

Analysis of the degradation of a model dental composite.

UNLABELLED: Dental composites undergo material property changes during exposure to the oral environment and may release compounds of potential toxicity, such as bisphenol A. Degradation of dental composites was studied in a simplified overlayer model in which bisphenol A diglycidyl methacrylate (BisGMA) was covalently bound to a porous silicon oxide surface. It was hypothesized that the chemical structure of this overlayer would allow release of bisphenol A, BisGMA, and the decomposition products thereof, upon exposure to water for an extended period. Liquid chromatography mass spectrometry found leaching of intact BisGMA and several degradation products that contained the bisphenol A moiety from the overlayer into distilled water after 2 wks of aging. The absence of bisphenol A release from the overlayer reduces concerns regarding its potential health risk in dental composites. Nevertheless, health concerns might arise with respect to BisGMA and the leached degradation products, since they all contain the bisphenol A moiety. ABBREVIATIONS: BisGMA, bisphenol A diglycidyl methacrylate; HPLC, high-performance liquid chromatography; LCMS, liquid chromatography mass spectrometry; MA, methacrylic acid; MPS, 3-(trimethoxysilyl) propyl methacrylate; m/z, mass-to-charge ratio; and TIC, total ion chromatogram.

Cryptosporidium oocyst persistence in agricultural streams -a mobile-immobile model framework assessment.

Rivers are a means of rapid and long-distance transmission of pathogenic microorganisms from upstream terrestrial sources. Pathogens enter streams and rivers via overland flow, shallow groundwater discharge, and direct inputs. Of concern is the protozoal parasite, Cryptosporidium, which can remain infective for weeks to months under cool and moist conditions, with the infectious stage (oocysts) largely resistant to chlorination. We applied a mobile-immobile model framework to assess Cryptosporidium transport and retention in streams, that also accounts for inactivation. The model is applied to California's Central Valley where Cryptosporidium exposure can be at higher risk due to agricultural and wildlife nonpoint sources. The results demonstrate that hyporheic exchange is an important process to include in models characterizing pathogen dynamics in streams, delaying downstream transmission and allowing for immobilization processes, such as reversible filtration in the sediments, to occur. Although in-stream concentrations decrease relatively quickly (within hours), pathogen accumulation of up to 66% of the inputs due to immobilization processes in the sediments and slower moving surface water could result in long retention times (months to years). The model appropriately estimates baseflow pathogen accumulation and can help predict the potential loads of resuspended pathogens in response to a storm event.

Analysis of two P-element enhancer-trap insertion lines that show expression in the giant fibre neuron of Drosophila melanogaster.

The giant fibre system (GFS) of Drosophila is a simple neural circuit that mediates escape responses in adult flies. Here we report the initial characterization of two genes that are preferentially expressed in the GFS. Two P-element insertion lines, carrying the GAL4 transcriptional activator, were identified that exhibited pronounced expression in elements of the GFS and relatively low levels elsewhere within the adult central nervous system. Genomic DNA flanking the P-element insertion site was recovered from each of these lines, sequenced, and nearby transcripts identified and confirmed to exhibit GFS expression by in situ hybridization. This analysis revealed that these P-elements were in previously characterized genes. Line P[GAL4]-A307 has an insert in the gene short stop for which we have identified a novel transcript, while line P[GAL4]-141 has an insert in the transcription factor ken and barbie. Here we show that ken and barbie mutants have defects in escape behaviour, behavioural responses to visual stimuli and synaptic functions in the GFS. We have therefore revealed a neural role for a transcription factor that previously had no implicated neural function.

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2.

The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals. Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.