Scalable Substrate Development for Aqueous Biological Samples for Atom Probe Tomography

Reliable and consistent preparation of atom probe tomography (APT) specimens from aqueous and hydrated biological specimens remains a significant challenge. One particularly difficult process step is the use of a focused ion beam (FIB) instrument for preparing the required needle-shaped specimen, typically involving a "lift-out" procedure of a small sample of material. Here, two alternative substrate designs are introduced that enable using FIB only for sharpening, along with example APT datasets. The first design is a laser-cut FIB-style half-grid close to those used for transmission-electron microscopy, that can be used in a grid holder compatible with APT pucks. The second design is a larger, standalone self-supporting substrate called a "crown", with several specimen positions that self-aligns in APT pucks, prepared by electrical discharge machining (EDM). Both designs are made nanoporous, to provide strength to the liquid-substrate interface, using chemical and vacuum dealloying. We select alpha brass a simple, widely available, lower-cost alternative to previously proposed substrates. We present the resulting designs, APT data, and provide suggestions to help drive wider community adoption

Enabling near-atomic-scale analysis of frozen water

Transmission electron microscopy has undergone a revolution in recent years with the possibility to perform routine cryo-imaging of biological materials and (bio)chemical systems, as well as the possibility to image liquids via dedicated reaction cells or graphene-sandwiching. These approaches however typically require imaging a large number of specimens and reconstructing an average representation and often lack analytical capabilities. Here, using atom probe tomography we provide atom-by-atom analyses of frozen liquids and analytical sub-nanometre three dimensional reconstructions. The analyzed ice is in contact with, and embedded within, nanoporous gold (NPG). We report the first such data on 2-3 microns thick layers of ice formed from both high purity deuterated water and a solution of 50mM NaCl in high purity deuterated water. We present a specimen preparation strategy that uses a NPG film and, additionally, we report on an analysis of the interface between nanoporous gold and frozen salt water solution with an apparent trend in the Na and Cl concentrations across the interface. We explore a range of experimental parameters to show that the atom probe analyses of bulk aqueous specimens come with their own special challenges and discuss physical processes that may produce the observed phenomena. Our study demonstrates the viability of using frozen water as a carrier for near-atomic scale analysis of objects in solution by atom probe tomography

Functional analysis of four LDLR 5'UTR and promoter variants in patients with familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is an autosomal dominant inherited disease characterised by increased low-density lipoprotein cholesterol (LDL-C) levels. The functionality of four novel variants within the LDLR 5'UTR and promoter located at c.-13A>G, c.-101T>C, c.-121T>C and c.-215A>G was investigated using in silico and in vitro assays, and a systemic bioinformatics analysis of all 36 reported promoter variants are presented. Bioinformatic tools predicted that all four variants occurred in sites likely to bind transcription factors and that binding was altered by the variant allele. Luciferase assay was performed for all the variants. Compared with wild type, the c.-101T>C and c.-121T>C variants showed significantly lower mean (±SD) luciferase activity (64±8 and 72±8%, all PG or c.-215A>G variants (96±15 and 100±12%), suggesting these variants are not FH causing. Similar results were seen for the c.-101T>C and c.-121T>C variants in lipid-depleted serum. However, a significant reduction in luciferase activity was seen in the c.-215A>G variant in lipid-depleted serum. Electrophoretic-mobility shift assays identified allele-specific binding of liver (hepatoma) nuclear proteins to c.-121T>C and suggestive differential binding to c.-101T>C but no binding to c.-215A>G. These data highlight the importance of in vitro testing of reported LDLR promoter variants to establish their role in FH. The functional assays performed suggest that the c.-101T>C and c.-121T>C variants are pathogenic, whereas c.-13A>G variant is benign, and the status of c.-215A>G remains unclear.European Journal of Human Genetics advance online publication, 24 September 2014; doi:10.1038/ejhg.2014.199

Redefining undergraduate nurse teaching during the coronavirus pandemic : use of digital technologies

During the current coronavirus pandemic, undergraduate nurse teaching is facing many challenges. Universities have had to close their campuses, which means that academics are working from home and may be coping with unfamiliar technology to deliver the theoretical part of the undergraduate nursing curriculum. Emergency standards from the Nursing and Midwifery Council have allowed theoretical instruction to be replaced with distance learning, requiring nursing academics to adapt to providing a completely virtual approach to their teaching. This article provides examples of tools that can be used to deliver the theoretical component of the undergraduate nursing curriculum and ways of supporting students and colleagues in these unprecedented time

Targeted genetic testing for familial hypercholesterolaemia using next generation sequencing:a population-based study

Background<p></p> Familial hypercholesterolaemia (FH) is a common Mendelian condition which, untreated, results in premature coronary heart disease. An estimated 88% of FH cases are undiagnosed in the UK. We previously validated a method for FH mutation detection in a lipid clinic population using next generation sequencing (NGS), but this did not address the challenge of identifying index cases in primary care where most undiagnosed patients receive healthcare. Here, we evaluate the targeted use of NGS as a potential route to diagnosis of FH in a primary care population subset selected for hypercholesterolaemia.<p></p> Methods<p></p> We used microfluidics-based PCR amplification coupled with NGS and multiplex ligation-dependent probe amplification (MLPA) to detect mutations in LDLR, APOB and PCSK9 in three phenotypic groups within the Generation Scotland: Scottish Family Health Study including 193 individuals with high total cholesterol, 232 with moderately high total cholesterol despite cholesterol-lowering therapy, and 192 normocholesterolaemic controls.<p></p> Results<p></p> Pathogenic mutations were found in 2.1% of hypercholesterolaemic individuals, in 2.2% of subjects on cholesterol-lowering therapy and in 42% of their available first-degree relatives. In addition, variants of uncertain clinical significance (VUCS) were detected in 1.4% of the hypercholesterolaemic and cholesterol-lowering therapy groups. No pathogenic variants or VUCS were detected in controls.<p></p> Conclusions<p></p> We demonstrated that population-based genetic testing using these protocols is able to deliver definitive molecular diagnoses of FH in individuals with high cholesterol or on cholesterol-lowering therapy. The lower cost and labour associated with NGS-based testing may increase the attractiveness of a population-based approach to FH detection compared to genetic testing with conventional sequencing. This could provide one route to increasing the present low percentage of FH cases with a genetic diagnosis

Higher Spin Gauge Theory and Holography: The Three-Point Functions

In this paper we calculate the tree level three-point functions of Vasiliev's higher spin gauge theory in AdS4 and find agreement with the correlators of the free field theory of N massless scalars in three dimensions in the O(N) singlet sector. This provides substantial evidence that Vasiliev theory is dual to the free field theory, thus verifying a conjecture of Klebanov and Polyakov. We also find agreement with the critical O(N) vector model, when the bulk scalar field is subject to the alternative boundary condition such that its dual operator has classical dimension 2.Comment: 90 pages, 5 figures; v4, minor changes in the introductio

Phylogeography of Japanese encephalitis virus:genotype is associated with climate

The circulation of vector-borne zoonotic viruses is largely determined by the overlap in the geographical distributions of virus-competent vectors and reservoir hosts. What is less clear are the factors influencing the distribution of virus-specific lineages. Japanese encephalitis virus (JEV) is the most important etiologic agent of epidemic encephalitis worldwide, and is primarily maintained between vertebrate reservoir hosts (avian and swine) and culicine mosquitoes. There are five genotypes of JEV: GI-V. In recent years, GI has displaced GIII as the dominant JEV genotype and GV has re-emerged after almost 60 years of undetected virus circulation. JEV is found throughout most of Asia, extending from maritime Siberia in the north to Australia in the south, and as far as Pakistan to the west and Saipan to the east. Transmission of JEV in temperate zones is epidemic with the majority of cases occurring in summer months, while transmission in tropical zones is endemic and occurs year-round at lower rates. To test the hypothesis that viruses circulating in these two geographical zones are genetically distinct, we applied Bayesian phylogeographic, categorical data analysis and phylogeny-trait association test techniques to the largest JEV dataset compiled to date, representing the envelope (E) gene of 487 isolates collected from 12 countries over 75 years. We demonstrated that GIII and the recently emerged GI-b are temperate genotypes likely maintained year-round in northern latitudes, while GI-a and GII are tropical genotypes likely maintained primarily through mosquito-avian and mosquito-swine transmission cycles. This study represents a new paradigm directly linking viral molecular evolution and climate

The Clinical Genome Resource (ClinGen) Familial Hypercholesterolemia Variant Curation Expert Panel consensus guidelines for LDLR variant classification

PURPOSE: In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published consensus standardized guidelines for sequence-level variant classification in Mendelian disorders. To increase accuracy and consistency, the Clinical Genome Resource Familial Hypercholesterolemia (FH) Variant Curation Expert Panel was tasked with optimizing the existing ACMG/AMP framework for disease-specific classification in FH. In this study, we provide consensus recommendations for the most common FH-associated gene, LDLR, where >2300 unique FH-associated variants have been identified. METHODS: The multidisciplinary FH Variant Curation Expert Panel met in person and through frequent emails and conference calls to develop LDLR-specific modifications of ACMG/AMP guidelines. Through iteration, pilot testing, debate, and commentary, consensus among experts was reached. RESULTS: The consensus LDLR variant modifications to existing ACMG/AMP guidelines include (1) alteration of population frequency thresholds, (2) delineation of loss-of-function variant types, (3) functional study criteria specifications, (4) cosegregation criteria specifications, and (5) specific use and thresholds for in silico prediction tools, among others. CONCLUSION: Establishment of these guidelines as the new standard in the clinical laboratory setting will result in a more evidence-based, harmonized method for LDLR variant classification worldwide, thereby improving the care of patients with FH

Impact of socio-economic deprivation on death rates after surgery for upper gastrointestinal tract cancer

We hypothesised that socio-economic deprivation in England may be a prognostic factor for death after oesophagectomy or gastrectomy for cancer of the upper gastrointestinal tract. We analysed statistical data from hospital records linked to death records for patients who underwent operations for oesophageal and gastric cancer in England from April 1998 to March 2002. The patients were stratified into quintiles according to the index of multiple deprivation (IMD) (2000) for their place (ward) of residence. Age and sex standardised death rates at 30 and 90 days for each deprivation quintile were calculated. Following oesophagectomy, death rates showed a significant association with IMD. They increased with increasing levels of deprivation: the odds ratio for death, comparing highest with lowest quintile for deprivation, was 1.37 (95% confidence interval 1.03–1.85) at 30 days and 1.30 (1.04–1.64) at 90 days. Following gastrectomy, the death rates showed smaller and nonsignificant associations with IMD with odds ratios of 1.16 (0.84–1.62) and 1.10 (0.86–1.41), respectively. There is a significant association between social deprivation and death after oesophagectomy, but less of an association, if any, after gastrectomy in current UK practice

Founder mutations in the Netherlands: geographical distribution of the most prevalent mutations in the low-density lipoprotein receptor and apolipoprotein B genes

Background In the Netherlands, a screening programme was set up in 1994 in order to identify all patients with familial hypercholesterolaemia (FH). After 15 years of screening, we evaluated the geographical distribution, possible founder effects and clinical phenotype of the 12 most prevalent FH gene mutations. Methods Patients who carried one of the 12 most prevalent mutations, index cases and those identified between 1994 and 2009 through the screening programme and whose postal code was known were included in the study. Low-density lipoprotein cholesterol (LDL-C) levels at the time of screening were retrieved. The prevalence of identified patients in each postal code area was calculated and visualised in different maps. Results A total of 10,889 patients were included in the study. Mean untreated LDL-C levels ranged from 4.4 to 6.4 mmol/l. For almost all mutations, a region of high prevalence could be observed. In total, 51 homozygous patients were identified in the Netherlands, of which 13 true homozygous for one of the 12 most prevalent mutations. The majority of them were living in high-prevalence areas for that specific mutation. Conclusions Phenotypes with regard to LDL-C levels varied between the 12 most prevalent FH mutations. For most of these mutations, a founder effect was observed. Our observations can have implications with regard to the efficiency of molecular screening and physician's perception of FH and to the understanding of the prevalence and distribution of homozygous patients in the Netherland