A versatile and reproducible cryo-sample preparation methodology for atom probe studies

Repeatable and reliable site-specific preparation of specimens for atom probe tomography (APT) at cryogenic temperatures has proven challenging. A generalized workflow is required for cryogenic-specimen preparation including lift-out via focused-ion beam and in-situ deposition of capping layers, to strengthen specimens that will be exposed to high electric field and stresses during field evaporation in APT, and protect them from environment during transfer into the atom probe. Here, we build on existing protocols, and showcase preparation and analysis of a variety of metals, oxides and supported frozen liquids and battery materials. We demonstrate reliable in-situ deposition of a metallic capping layer that significantly improve the atom probe data quality for challenging material systems, particularly battery cathode materials which are subjected to delithiation during the atom probe analysis itself. Our workflow designed is versatile and transferable widely to other instruments

In-situ metallic coating of atom probe specimen for enhanced yield, performance, and increased field-of-view

Atom probe tomography requires needle-shaped specimens with a diameter typically below 100 nm, making them both very fragile and reactive, and defects (notches at grain boundaries or precipitates) are known to affect the yield and data quality. The use of a conformal coating directly on the sharpened specimen has been proposed to increase yield and reduce background. However, to date, these coatings have been applied ex-situ and mostly are not uniformly. Here, we report on the controlled focused ion beam in-situ deposition of a thin metal film on specimens immediately after specimen preparation. Different metallic targets e.g. Cr were attached to a micromanipulator via a conventional lift-out method and sputtered using the Ga or Xe ions. We showcase the many advantages of coating specimens from metallic to non-metallic materials. We have identified an increase in data quality and yield, an improvement of the mass resolution, as well as an increase in the effective field-of-view enabling visualization of the entire original specimen, including the complete surface oxide layer. The ease of implementation of the approach makes it very attractive for generalizing its use across a very wide range of atom probe analyses

Role of substrate quality on the performance of semipolar (11 2 - 2) InGaN light-emitting diodes

We compare the optical properties and device performance of unpackaged InGaN/GaN multiple-quantum-well light-emitting diodes (LEDs) emitting at ∼430 nm grown simultaneously on a high-cost small-size bulk semipolar (11 2 - 2) GaN substrate (Bulk-GaN) and a low-cost large-size (11 2 - 2) GaN template created on patterned (10 1 - 2) r-plane sapphire substrate (PSS-GaN). The Bulk-GaN substrate has the threading dislocation density (TDD) of ∼ and basal-plane stacking fault (BSF) density of 0 cm-1, while the PSS-GaN substrate has the TDD of ∼2 × 108cm-2 and BSF density of ∼1 × 103cm-1. Despite an enhanced light extraction efficiency, the LED grown on PSS-GaN has two-times lower internal quantum efficiency than the LED grown on Bulk-GaN as determined by photoluminescence measurements. The LED grown on PSS-GaN substrate also has about two-times lower output power compared to the LED grown on Bulk-GaN substrate. This lower output power was attributed to the higher TDD and BSF density

Extending Miscanthus cultivation with novel germplasm at six contrasting sites

The OPTIMISC project received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 289159.Peer reviewedPublisher PD

Environmental Influences on the Growing Season Duration and Ripening of Diverse Miscanthus Germplasm Grown in Six Countries

The development of models to predict yield potential and quality of a Miscanthus crop must consider climatic limitations and the duration of growing season. As a biomass crop, yield and quality are impacted by the timing of plant developmental transitions such as flowering and senescence. Growth models are available for the commercially grown clone Miscanthus x giganteus (Mxg), but breeding programs have been working to expand the germplasm available, including development of interspecies hybrids. The aim of this study was to assess the performance of diverse germplasm beyond the range of environments considered suitable for a Miscanthus crop to be grown. To achieve this, six field sites were planted as part of the EU OPTIMISC project in 2012 in a longitudinal gradient from West to East: Wales?Aberystwyth, Netherlands?Wageningen, Stuttgart?Germany, Ukraine?Potash, Turkey?Adana, and Russia?Moscow. Each field trial contained three replicated plots of the same 15 Miscanthus germplasm types. Through the 2014 growing season, phenotypic traits were measured to determine the timing of developmental stages key to ripening; the tradeoff between growth (yield) and quality (biomass ash and moisture content). The hottest site (Adana) showed an accelerated growing season, with emergence, flowering and senescence occurring before the other sites. However, the highest yields were produced at Potash, where emergence was delayed by frost and the growing season was shortest. Flowering triggers varied with species and only in Mxg was strongly linked to accumulated thermal time. Our results show that a prolonged growing season is not essential to achieve high yields if climatic conditions are favorable and in regions where the growing season is bordered by frost, delaying harvest can improve quality of the harvested biomasspublishersversionPeer reviewe

SARS-CoV-2 T Cell Response in Severe and Fatal COVID-19 in Primary Antibody Deficiency Patients Without Specific Humoral Immunity

Morbidity and mortality of COVID-19 is increased in patients with inborn errors of immunity (IEI). Age and comorbidities and also impaired type I interferon immunity were identified as relevant risk factors. In patients with primary antibody deficiency (PAD) and lack of specific humoral immune response to SARS-CoV-2, clinical disease outcome is very heterogeneous. Despite extensive clinical reports, underlying immunological mechanisms are poorly characterized and levels of T cellular and innate immunity in severe cases remain to be determined. In the present study, we report clinical and immunological findings of 5 PAD patients with severe and fatal COVID-19 and undetectable specific humoral immune response to SARS-CoV-2. Reactive T cells to SARS-CoV-2 spike (S) and nucleocapsid (NCAP) peptide pools were analyzed comparatively by flow cytometry in PAD patients, convalescents and naive healthy individuals. All examined PAD patients developed a robust T cell response. The presence of polyfunctional cytokine producing activated CD4(+) T cells indicates a memory-like phenotype. An analysis of innate immune response revealed elevated CD169 (SIGLEC1) expression on monocytes, a surrogate marker for type I interferon response, and presence of type I interferon autoantibodies was excluded. SARS-CoV-2 RNA was detectable in peripheral blood in three severe COVID-19 patients with PAD. Viral clearance in blood was observed after treatment with COVID-19 convalescent plasma/monoclonal antibody administration. However, prolonged mucosal viral shedding was observed in all patients (median 67 days) with maximum duration of 127 days. PAD patients without specific humoral SARS-CoV-2 immunity may suffer from severe or fatal COVID-19 despite robust T cell and normal innate immune response. Intensified monitoring for long persistence of SARS-CoV-2 viral shedding and (prophylactic) convalescent plasma/specific IgG as beneficial treatment option in severe cases with RNAemia should be considered in seronegative PAD patients

Cephalopod genomics: a plan of strategies and organization

The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, "Paths to Cephalopod Genomics-Strategies, Choices, Organization," held in Durham, North Carolina, USA on May 24-27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod mollusks. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this white paper

Cephalopod genomics : a plan of strategies and organization

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Standards in Genomic Sciences 7 (2012): 175-188, doi:10.4056/sigs.3136559.The Cephalopod Sequencing Consortium (CephSeq Consortium) was established at a NESCent Catalysis Group Meeting, “Paths to Cephalopod Genomics- Strategies, Choices, Organization,” held in Durham, North Carolina, USA on May 24-27, 2012. Twenty-eight participants representing nine countries (Austria, Australia, China, Denmark, France, Italy, Japan, Spain and the USA) met to address the pressing need for genome sequencing of cephalopod molluscs. This group, drawn from cephalopod biologists, neuroscientists, developmental and evolutionary biologists, materials scientists, bioinformaticians and researchers active in sequencing, assembling and annotating genomes, agreed on a set of cephalopod species of particular importance for initial sequencing and developed strategies and an organization (CephSeq Consortium) to promote this sequencing. The conclusions and recommendations of this meeting are described in this White Paper.The Catalysis Group Meeting was supported by the National Science Foundation through the National Evolutionary Synthesis Center (NESCent) under grant number NSF #EF-0905606

HACE1 deficiency causes an autosomal recessive neurodevelopmental syndrome

Background: The genetic etiology of neurodevelopmental defects is extremely diverse, and the lack of distinctive phenotypic features means that genetic criteria are often required for accurate diagnostic classification. We aimed to identify the causative genetic lesions in two families in which eight affected individuals displayed variable learning disability, spasticity and abnormal gait. Methods: Autosomal recessive inheritance was suggested by consanguinity in one family and by sibling recurrences with normal parents in the second. Autozygosity mapping and exome sequencing, respectively, were used to identify the causative gene. Results: In both families, biallelic loss-of-function mutations in HACE1 were identified. HACE1 is an E3 ubiquitin ligase that regulates the activity of cellular GTPases, including Rac1 and members of the Rab family. In the consanguineous family, a homozygous mutation p.R219* predicted a truncated protein entirely lacking its catalytic domain. In the other family, compound heterozygosity for nonsense mutation p.R748* and a 20-nt insertion interrupting the catalytic HECT domain was present; Western analysis of patient cells revealed an absence of detectable HACE1 protein. Conclusion: HACE1 mutations underlie a new autosomal recessive neurodevelopmental disorder. Previous studies have implicated HACE1 as a tumour suppressor gene; however, since cancer predisposition was not observed either in homozygous or heterozygous mutation carriers, this concept may require re-evaluation

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified HNRNPM as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and back-splicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM dependent linear splicing events using splice-switching-antisense-oligonucleotides (SSOs) was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors