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Several studies suggest that accelerating technology, increasing product complexity, and an expanding volume of information in the marketplace are changing sales roles, necessitating a review of the current sales skills required for success. Using mixed methods, we examine the skills required of contemporary B2B salespeople. First, we draw on unique data from 3.8 million LinkedIn job postings to examine which skills sales recruiters are seeking in new hires. Whilst confirming the importance of previously researched sales skills, this identified a sales skill largely disregarded by the extant literature: salesperson analytical skills. We triangulated these findings through interviews with 20 sales executives and developed a scale to measure this new analytical skills construct. Then, to test the scale's predictive and nomological validity, we used survey data from 251 business-to-business salespeople. Results reveal that salesperson analytical skills have both a direct and a moderating effect on sales performance across varying selling situations.

IntroductionBreeding for tick resistance is a sustainable alternative to control cattle ticks due to widespread resistance to acaricidal drugs and the lack of a protective vaccine. The most accurate method used to characterise the phenotype for tick resistance in field studies is the standard tick count, but this is labour-intensive and can be hazardous to the operator. Efficient genetic selection requires reliable phenotyping or biomarker(s) for accurately identifying tick-resistant cattle. Although breed-specific genes associated with tick resistance have been identified, the mechanisms behind tick resistance have not yet been fully characterised.MethodsThis study applied quantitative proteomics to examine the differential abundance of serum and skin proteins using samples from naïve tick-resistant and -susceptible Brangus cattle at two-time points following tick exposure. The proteins were digested into peptides, followed by identification and quantification using sequential window acquisition of all theoretical fragment ion mass spectrometry.ResultsResistant naïve cattle had a suite of proteins associated with immune response, blood coagulation and wound healing that were significantly (adjusted P < 10- 5) more abundant compared with susceptible naïve cattle. These proteins included complement factors (C3, C4, C4a), alpha-1-acid glycoprotein (AGP), beta-2-glycoprotein-1, keratins (KRT1 & KRT3) and fibrinogens (alpha & beta). The mass spectrometry findings were validated by identifying differences in the relative abundance of selected serum proteins with ELISA. The proteins showing a significantly different abundance in resistant cattle following early and prolonged tick exposures (compared to resistant naïve) were associated with immune response, blood coagulation, homeostasis, and wound healing. In contrast, susceptible cattle developed some of these responses only after prolonged tick exposure.DiscussionResistant cattle were able to transmigrate immune-response related proteins towards the tick bite sites, which may prevent tick feeding. Significantly differentially abundant proteins identified in this research in resistant naïve cattle may provide a rapid and efficient protective response to tick infestation. Physical barrier (skin integrity and wound healing) mechanisms and systemic immune responses were key contributors to resistance. Immune response-related proteins such as C4, C4a, AGP and CGN1 (naïve samples), CD14, GC and AGP (post-infestation) should be further investigated as potential biomarkers for tick resistance.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of expression of the maternal copy of the UBE3A gene. Individuals with AS have a multifaceted behavioral phenotype consisting of deficits in motor function, epilepsy, cognitive impairment, sleep abnormalities, as well as other comorbidities. Effectively modeling this behavioral profile and measuring behavioral improvement will be crucial for the success of ongoing and future clinical trials. Foundational studies have defined an array of behavioral phenotypes in the AS mouse model. However, no single behavioral test is able to fully capture the complex nature of AS—in mice, or in children. We performed multidimensional analysis (principal component analysis + k-means clustering) to quantify the performance of AS model mice (n = 148) and wild-type littermates (n = 138) across eight behavioral domains. This approach correctly predicted the genotype of mice based on their behavioral profile with ~95% accuracy, and remained effective with reasonable sample sizes (n = ~12–15). Multidimensional analysis was effective using different combinations of behavioral inputs and was able to detect behavioral improvement as a function of treatment in AS model mice. Overall, multidimensional behavioral analysis provides a tool for evaluating the effectiveness of preclinical treatments for AS. Multidimensional analysis of behavior may also be applied to rodent models of related neurodevelopmental disorders, and may be particularly valuable for disorders where individual behavioral tests are less reliable than in AS.

High pressure assisted infusion of nutrients into food was in situ monitored with magnetic resonance imaging (MRI). Modification of an off-the-shelf pressure reactor with an MRI detection circuit provided a large enough volume to accommodate food. The model food used here was peeled apple flesh as it is considered as a good mimic for fibrous food. The nuclear spin relaxation properties of the water surrounding the apple flesh were enhanced by adding paramagnetic manganese cations. In this way, MRI relaxation contrast can be used to monitor the location of doped bulk water in and around the apple flesh during pressurization. This work tracked the efficiency of pressure induced nutrient infusion in situ, demonstrating that pressure gating and ramping offer no nutrient mass transport advantage over operation at constant pressure and that the presence of a peel expectedly disrupts solute transport into the fruit. High pressure assisted infusion, with all pressurization strategies shown here, yielded nearly 100-fold faster infusion times than at ambient pressure.

When gene editing was applied to knockout beta-kafirin, there was a compensatory increase of gamma-kafirin which does not occur in domesticated null varieties, so enhanced grain quality was not achieved. Sorghum bicolor is an important animal feedstock cereal crop throughout Australia and the southern United States, where its use as a food product is limited by issues with low calorific and nutritive value. Qualities such as reduced digestibility and low essential amino acid content are directly attributed to the kafirin grain storage proteins, the major components of protein bodies within the endosperm. Specifically, the β- and γ-kafirins have few protease cleavage sites and high levels of cysteine residues which lead to a highly cross-linked shell of intra- and inter-molecular disulphide linkages that encapsulate the more digestible α- and δ-kafirins in the core of the protein bodies. Naturally occurring β-kafirin mutants exist and are known to have improved grain quality, with enhanced protein contents and digestibility, traits which are often attributed to the lack of this cysteine-rich kafirin in the mature grain. However, when CRISPR/Cas9 editing was used to create β-kafirin knockout lines, there was no improvement to grain quality in the Tx430 background, although they did have unique protein composition and changes to protein body morphology in the vitreous endosperm. One explanation of the divergence in quality traits found the lines lacking β-kafirin are due to a drastic increase of γ-kafirin which was only found in the gene edited lines. This study highlights that in some germplasm, there is a level of redundancy between the peripheral kafirins, and that improvement of grain protein digestibility cannot be achieved by simply removing the β-kafirin protein in all genetic backgrounds.

Sorghum (Sorghum bicolor), a grass native to Africa, is a popular alternative to barley for brewing beer. The importance of sorghum to beer brewing is increasing because it is a naturally gluten-free cereal and because climate change is expected to cause a reduction in the production of barley over the coming decades. However, there are challenges associated with the use of sorghum instead of barley in beer brewing. Here, we used proteomics and metabolomics to gain insights into the sorghum brewing process, to advise processes for efficient beer production from sorghum. We found that during malting, sorghum synthesises the amylases and proteases necessary for brewing. Proteomics revealed that mashing with sorghum malt required higher temperatures than barley malt for efficient protein solubilisation. Both α- and β-amylase were considerably less abundant in sorghum wort than in barley wort, correlating with lower maltose concentrations in sorghum wort. However, metabolomics revealed higher glucose concentrations in sorghum wort than in barley wort, consistent with the presence of an abundant α-glucosidase detected by proteomics in sorghum malt. Our results indicate that sorghum can be a viable grain for industrial fermented beverage production, but that its use requires careful process optimisation for efficient production of fermentable wort and high-quality beer.

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those.

In mammals, sensory signals are transmitted via the thalamus primarily to layer 4 of the primary sensory cortices. While information about average neuronal connectivity in this layer is available, the detailed and higher-order circuit structure is not known. Here, we used 3-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in a layer 4 barrel. We find that thalamic input drives a subset of interneurons with high specificity. These interneurons in turn target spiny stellate and star pyramidal excitatory neurons with subtype specificity. In addition, they create a directed disinhibitory network directly driven by the thalamic input. Together, this circuit can create differential windows of opportunity for activation of the types of excitatory neurons in dependence of strength and timing of thalamic input. With this, we have identified a so-far unknown degree of specialization of the microcircuitry in the main thalamocortical recipient layer of the primary sensory cortex.

Background The study objectives were: provide longitudinal data on upper aerodigestive tract function and late complications following IMRT for nasopharyngeal carcinoma, and elucidate factors that might predict a worse outcome. The hypotheses were: (1) Despite advances such as IMRT, radiation will cause significant functional decline and late complications that often progress or arise years after treatment. (2) Larger radiation volume will be associated with poorer outcomes. Methods Longitudinal, observational cohort study of nasopharyngeal carcinoma patients with retrospective analysis of prospectively collected, population-based data. Late sequelae and validated measures of overall performance, speech, and swallowing were documented pre-treatment and 3,6,12, 24, 36 and ≥ 60-months post-treatment. Results Forty-two patients treated curatively with radiation (N = 9) or chemoradiation (N = 33) were followed for a median 74 months. Functional outcomes showed an initial nadir at 3 months associated with acute effects of treatment, followed by initial recovery. There was subsequent functional decline years post-treatment with advancing dysphagia/aspiration, trismus, muscle spasm, and hypoglossal nerve palsy. Univariable regression analysis revealed that increasing high-dose radiation volumes (PTV 70 Gy) were associated with increased likelihood of less than solid diet (Performance Status Scale (PSS)—Normalcy of Diet score p = 0.04), and reduced PSS—Understandability of Speech (p = 0.005). The probability of poor outcome increased with time. Eleven percent of patients were tube feed dependent at ≥ 5 years. Conclusions Despite improvements in radiation delivery, late effects of radiation remain common. Higher radiation volumes are associated with poorer outcomes that worsen over time. Graphical Abstract

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct. ispartof: PLANT JOURNAL vol:106 issue:1 pages:159-173 ispartof: location:England status: published

© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland. Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup-specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup-specific, tumour-secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup-specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high-risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup-specific therapies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The Rab27 subfamily consists of Rab27a/b isoforms that have similar but not identical functions. Those functions include the regulation of trafficking, docking, and fusion of various lysosome-relat...

The texture evolution of a Zr-2.5wt pctNb alloy pressure tube is characterized after the $$\alpha \rightarrow \beta $$ transformation and the $$\beta \rightarrow \alpha $$ transformation by room temperature EBSD maps and reconstruction of the high temperature $$\beta $$ phase. The $$\beta $$ phase forms a deformation texture which varies along the radial direction of the tube and changes after thermal cycling. Variant selection during the $$\beta \rightarrow \alpha $$ phase transformation is shown to be strongly influenced by residual stress within the material, with a more randomized variant selection for water quenched samples. Slower cooling rates were found to create orientations in addition to the 12 $$\alpha $$ variants expected by the Burger’s orientation relationship in a given $$\beta $$ grain.

Patients with microsatellite stable (MSS) colorectal carcinoma (CRC) do not respond to immune checkpoint inhibitors. Preclinical models suggested synergistic anti-tumour activity combining CXD101 and anti-programmed cell death protein 1 treatment; therefore, we assessed the clinical combination of CXD101 and nivolumab in heavily pre-treated patients with MSS metastatic CRC (mCRC).This single-arm, open-label study enrolled patients aged 18 years or older with biopsy-confirmed MSS CRC; at least two lines of systemic anticancer therapies (including oxaliplatin and irinotecan); at least one measurable lesion; Eastern Cooperative Oncology Group performance status of 0, 1 or 2; predicted life expectancy above 3 months; and adequate organ and bone marrow function. Nine patients were enrolled in a safety run-in study to define a tolerable combination schedule of CXD101 and nivolumab, followed by 46 patients in the efficacy assessment phase. Patients in the efficacy assessment cohort were treated orally with 20 mg CXD101 twice daily for 5 consecutive days every 3 weeks, and intravenously with 240 mg nivolumab every 2 weeks. The primary endpoint was immune disease control rate (iDCR).Between 2018 and 2020, 55 patients were treated with CXD101 and nivolumab. The combination therapy was well tolerated with the most frequent grade 3 or 4 adverse events being neutropenia (18%) and anaemia (7%). Immune-related adverse reactions commonly ascribed to checkpoint inhibitors were surprisingly rare although we did see single cases of pneumonitis, hypothyroidism and hypopituitarism. There were no treatment-related deaths. Of 46 patients assessable for efficacy, 4 (9%) achieved partial response and 18 (39%) achieved stable disease, translating to an immune disease control rate of 48%. The median overall survival (OS) was 7.0 months (95% confidence interval 5.13-10.22 months).The primary endpoint was met in this phase II study, which showed that the combination of CXD101 and nivolumab, at full individual doses in the treatment of advanced or metastatic MSS CRC, was both well tolerated and efficacious.

Short open reading frame (sORF)-encoded peptides (sPEPs) have been found across a wide range of genomic locations in a variety of species. To date, their identification, validation, and characterisation in the human fungal pathogen Cryptococcus neoformans has been limited due to a lack of standardised protocols. We have developed an enrichment process that enables sPEP detection within a protein sample from this polysaccharide-encapsulated yeast, and implemented proteogenomics to provide insights into the validity of predicted and hypothetical sORFs annotated in the C. neoformans genome. Novel sORFs were discovered within the 5' and 3' UTRs of known transcripts as well as in "non-coding" RNAs. One novel candidate, dubbed NPB1, that resided in an RNA annotated as "non-coding", was chosen for characterisation. Through the creation of both specific point mutations and a full deletion allele, the function of the new sPEP, Npb1, was shown to resemble that of the bacterial trans-translation protein SmpB.

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer 4 and layer 6 is differentially modulated by lit and dark conditions during free exploration.

Clinical genetic variant classification science is a growing subspecialty of clinical genetics and genomics. The field's continued improvement is essential for the success of precision medicine in both germline (hereditary) and somatic (oncology) contexts. This review focuses on variant classification for DNA next-generation sequencing tests. We first summarize current limitations in variant discovery and definition, and then describe the current five- and four-tier classification systems outlined in dominant standards and guideline publications for germline and somatic tests, respectively. We then discuss measures of variant classification discordance and the field's bias for positive results, as well as considerations for panel size and population screening in the context of estimates of positive predictive value thatincorporate estimated variant classification imperfections. Finally, we share opinions on the current state of variant classification from some of the authors of the most widely used standards and guideline publications and from other domain experts.

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

Beer is perhaps the oldest biotechnology and continues to this day to be one of the most popular beverages worldwide. Brewing is and always has been dependent on agricultural food sources, primarily barley (Hordeum vulgare). Not only is the brewing process reliant on the agricultural production of grains, it also relies on and encompasses many connected bio-processes: hop farming, germination, malting, yeast propagation, mashing, boiling, fermentation, and maturation. Proteins are key drivers of these bio-processes, facilitating many biochemical changes: allowing germination to begin, breaking down sugars and proteins in the mash, and affecting mouth-feel and flavor in the finished beer, to name a few. Mass spectrometry proteomics allows detailed insights into the identity, abundance, location, modifications, and interactions of proteins across complex proteomes. This chapter will provide an overview of how proteomics has been applied to brewing and its related bio-processes.

Thus far, two Rab27 isoforms (Rab27a and Rab27b) have been identified that interact with their eleven downstream effectors proteins, preferentially in their GTP-bound state. In recent years, a number of studies has suggested roles for Rab27-effector protein interactions in the development of cancer cell invasion and metastasis, and immune and inflammatory responses. Here we develop an in vitro fluorescence resonance energy transfer (FRET)-based protein-protein interaction assay to report Rab27 protein interactions with their effectors. We particularly focus on determining the interaction of mouse (m) Synaptotagmin-like protein (Slp)1 and mSlp2 effector proteins with human (h)Rab27. Green fluorescent protein (GFP)-N-terminus Rab27 binding domains (m-Slp1 and m-Slp2) recombinant proteins were used as donor fluorophores, whereas mCherry-hRab27a/b recombinant proteins were used as acceptor fluorophores. The conditions of this assay were validated and optimized, and the specificity of the assay was confirmed. Accordingly, this assay can be used to assess and identify key determinants and/or candidate inhibitors of Rab27-effector interactions.

We report the result from a search for charged-current coherent pion production induced by muon neutrinos with a mean energy of 1.3 GeV. The data are collected with a fully active scintillator detector in the K2K long-baseline neutrino oscillation experiment. No evidence for coherent pion production is observed and an upper limit of $0.60 \times 10^{-2}$ is set on the cross section ratio of coherent pion production to the total charged-current interaction at 90% confidence level. This is the first experimental limit for coherent charged pion production in the energy region of a few GeV., 5 pages, 4 figures

OBJECTIVE: To identify the source of ongoing coronavirus disease 2019 (COVID-19) infections after 4 weeks of lockdown and to characterise the presentation of COVID-19 in the elderly, who represent the highest risk group. DESIGN: Retrospective observational cohort study of 115 patients at one acute district general hospital with a catchment population of approximately 500,000 people, during weeks 5 and 6 of the UK lockdown. RESULTS: More than 2 in 3 of the overall cohort had had contacts with the health and social care system prior to diagnosis. This figure rose to 85% in those 70 years and over. In the older cohort, the most common reasons for presentation were shortness of breath or falls, and 1 in 3 had neither cough nor fever. CONCLUSION: COVID-19 can present differently in the elderly, overlapping with many common presentations, so focusing testing on those with a cough or fever will miss at least 1 in 3 cases in those over the age of 70. A high degree of vigilance, suspicion and repeated testing is required if streaming into high and low risk areas is to succeed, allowing safe restarting of services such as elective surgery and cancer care.

INTRODUCTION: Breakthroughs in cancer immunotherapy have spurred interest in the development of vaccines to mediate prophylactic protection and therapeutic efficacy against primary tumors or to prevent relapse. However, immunosuppressive mechanisms employed by cancer cells to generate effective resistance has hampered clinical translation of therapeutic cancer vaccines. To enhance vaccine efficacy, the immunomodulatory properties of cytoreductive therapies such as chemotherapy and radiation could amplify a cancer-specific immune response. AREAS COVERED: Herein, the authors discuss therapeutic cancer vaccines that harness whole cells and antigen targeted vaccines. First, recent advancements in both autologous and allogenic whole cell vaccines and combinations with checkpoint blockade and chemotherapy are reviewed. Next, tumor antigen targeted vaccines using peptide-based vaccines and DNA-vaccines are discussed. Finally, combination therapies using antigen targeted vaccines are reviewed. EXPERT OPINION: A deeper understanding of the immunostimulatory properties of cytoreductive therapies has supported their utility in combination therapies involving cancer vaccines as a potential strategy to induce a durable anti-tumor immune response for multiple types of cancers. Based on current evidence, combination therapies may have synergies which depend on the identity of the cytotoxic agent, vaccine target, dosing schedule, and cancer type. Together, these observations suggest that combining cancer vaccines with immunomodulatory cytoreductive therapy is a promising strategy for cancer therapy.

Structural data on ABCG5/G8 and ABCG2 reveal a unique molecular architecture for subfamily G ATP-binding cassette (ABCG) transporters and disclose putative substrate-binding sites. ABCG5/G8 and ABCG2 appear to use several unique structural motifs to execute transport, including the triple helical bundles, the membrane-embedded polar relay, the re-entry helices, and a hydrophobic valve. Interestingly, ABCG2 shows extreme substrate promiscuity, whereas ABCG5/G8 transports only sterol molecules. ABCG2 structures suggest a large internal cavity, serving as a binding region for substrates and inhibitors, while mutational and pharmacological analyses support the notion of multiple binding sites. By contrast, ABCG5/G8 shows a collapsed cavity of insufficient size to hold substrates. Indeed, mutational analyses indicate a sterol-binding site at the hydrophobic interface between the transporter and the lipid bilayer. In this review, we highlight key differences and similarities between ABCG2 and ABCG5/G8 structures. We further discuss the relevance of distinct and shared structural features in the context of their physiological functions. Finally, we elaborate on how ABCG2 and ABCG5/G8 could pave the way for studies on other ABCG transporters.

Members of the ATP binding cassette (ABC) transporter family perform a critical function in maintaining lipid homeostasis in cells as well as the transport of drugs. In this review, we provide an update on the ABCG-transporter subfamily member proteins, which include the homodimers ABCG1, ABCG2 and ABCG4 as well as the heterodimeric complex formed between ABCG5 and ABCG8. This review focusses on progress made in this field of research with respect to their function in health and disease and the recognised transporter substrates. We also provide an update on post-translational regulation, including by transporter substrates, and well as the involvement of microRNA as regulators of transporter expression and activity. In addition, we describe progress made in identifying structural elements that have been recognised as important for transport activity. We furthermore discuss the role of lipids such as cholesterol on the transport function of ABCG2, traditionally thought of as a drug transporter, and provide a model of potential cholesterol binding sites for ABCG2.

Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used Sequential Window Acquisition of all THeoretical ions Mass Spectrometry (SWATH-MS) proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We verified our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy.

Changes in brain metabolism are a hallmark of alcohol use disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in prefrontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.

Regulatory T cells (T(regs)) are critical mediators of peripheral immune tolerance. T(regs) suppress immune activation against self-antigens and are the focus of cell-based therapies for autoimmune diseases. However, T(regs) circulate at a very low frequency in blood, limiting the number of cells that can be isolated by leukapheresis. To effectively expand T(regs) ex vivo for cell therapy, we report the metabolic modulation of T cells using mono-(6-amino-6-deoxy)-β-cyclodextrin (βCD-NH(2)) encapsulated rapamycin (Rapa). We used a computational model to qualitatively describe the kinetics of T(reg) expansion. Encapsulating Rapa in β-cyclodextrin increased its aqueous solubility ~154-fold and maintained bioactivity for at least 30 days. βCD-NH(2)-Rapa complexes (CRCs) enriched the fraction of CD4(+)CD25(+)FoxP3(+) mouse T (mT) cells and human T (hT) cells up to 6-fold and up to 2-fold respectively and suppressed the overall expansion of effector T cells by 5-fold in both species. Combining CRCs and transforming growth factor beta-1 (TGF-β1) synergistically promoted the expansion of CD4(+)CD25(+)FoxP3(+) T cells. CRCs significantly reduced the fraction of pro-inflammatory interferon-gamma (IFN-γ) expressing CD4(+) T cells, suppressing this Th1-associated cytokine while enhancing the fraction of IFN-γ(−) tumor necrosis factor-alpha (TNF-α) expressing CD4(+) T cells. The computational model describes the influence of the composition of the initial cell population on the enrichment of T(regs) in vitro and reveals how differences in the expansion kinetics of mT and hT cells result in differences in their susceptibility to immunophenotypic modulation. CRCs may be an effective and potent means for phenotypic modulation of T cells and the enrichment of T(regs) in vitro and our findings contribute to the development of experimental and analytical techniques for manufacturing T(reg) based immunotherapies.

Despite great genetic diversity, sorghum grain consistently suffers from poor protein digestibility. The physicochemical packaging of protein bodies which consist of protease-resistant β- and γ-kafirin is considered a major obstacle. A synthetic β-kafirin gene, which shares the endosperm-specific promoter and signal peptide with the native β-kafirin gene (Sobic.009G001600.1), was transformed into sorghum inbred line Tx430. The gene was modified with ten additional proteolytic sites. These sites were designed to be amenable to cleavage by pepsin and/or chymotrypsin proteinases. Five independent transgenic lines were regenerated by microprojectile transformation. Notably, considerably more protein was observed in the peripheral endosperm of transgenic lines under scanning electron microscopy. Microscopy revealed invaginated or irregularly shaped protein bodies in the endosperm of transgenic lines. Grains of transgenic lines contained 11–37% more protein, which was 11–21% more pepsin digestible and 7–25% more chymotrypsin digestible than Tx430. Additionally, the abundant synthetic β-kafirin protein (5.6% of total protein) was detected by mass spectrometry data analysis in the transgenic line 9-1. Field-grown homozygous transgenics retained higher protein content, larger seed size and no reduction in grain number per plant. The results illustrated that plant synthetic biology could play an important role in improving sorghum nutritional value.

Granular protein is an important structural feature in determining starch digestibility. High-amylose wheat starch (HAWS) with80% amylose content contains more granular protein than wild-type starch. As analyzed by mass spectrometry-based proteomics, granular-bound starch synthase (GBSS) is the major granular protein in isolated starch materials. GBSS content increases with amylose content (Spearman's correlation, p 0.05), whereas the abundance relative to other proteins is similar among starches. Multiple amylase inhibitors were also identified. From Michaelis-Menten analysis, HAWS has a similar K

ABCG2 is one of a few human membrane transporters which display the amazing ability to transport multiple different chemicals out of cells. These multidrug pumps, which have orthologues in all organisms, are important in humans in the context of drug pharmacokinetics, especially with respect to resistance to chemotherapy. In 2016, we presented a mini-review on ABCG2 which identified many areas of exciting research progress as well as many areas of frustrating ignorance. Just 2 years on the field has advanced, particularly with respect to structural biology as the cryo-electron microscopy revolution has brought us new insights into the structure and mechanism of ABCG2. In this update, we evaluate the degree to which new data have enhanced our understanding of the structure and mechanism of ABCG2 and whether we are now in a position to translate some of these findings into inhibitor design and development.

The widespread expression and polyspecificity of the multidrug ABCG2 efflux transporter make it an important determinant of the pharmacokinetics of a variety of substrate drugs. Null ABCG2 expression has been linked to the Junior blood group. Polymorphisms affecting the expression or function of ABCG2 may have clinically important roles in drug disposition and efficacy. The most well-studied single nucleotide polymorphism (SNP), Q141K (421C>A), is shown to decrease ABCG2 expression and activity, resulting in increased total drug exposure and decreased resistance to various substrates. The effect of Q141K can be rationalized by inspection of the ABCG2 structure, and the effects of this SNP on protein processing may make it a target for pharmacological intervention. The V12M SNP (34G>A) appears to improve outcomes in cancer patients treated with tyrosine kinase inhibitors, but the reasons for this are yet to be established, and this residue's role in the mechanism of the protein is unexplored by current biochemical and structural approaches. Research into the less-common polymorphisms is confined to in vitro studies, with several polymorphisms shown to decrease resistance to anticancer agents such as SN-38 and mitoxantrone. In this review, we present a systematic analysis of the effects of ABCG2 polymorphisms on ABCG2 function and drug pharmacokinetics. Where possible, we use recent structural advances to present a molecular interpretation of the effects of SNPs and indicate where we need further in vitro experiments to fully resolve how SNPs impact ABCG2 function.

Mallinckrodt Chemical Works was the earliest uranium processing facility in the Manhattan Project, beginning in 1942. Even then, concern existed about possible health effects resulting from exposure to radiation and pitchblende dust. This concern was well founded as the facility processed Belgian Congo pitchblende ore that was up to 60% pure uranium with high U content and up to 100 mg of radium per ton. Workers were exposed to external gamma radiation plus internal radiation from inhalation and ingestion of pitchblende dust (uranium, radium, and silica). Multiple sources of exposure were available for organ dose reconstruction to a degree unique for an epidemiologic study. Personal film badge measures available from 1945 captured external exposures. Additional external exposure included 15,518 occupational medical x-rays and 210 radiation exposure records from other facilities outside of Mallinckrodt employment. Organ dose calculations considered organ-specific coefficients that account for photon energy and job-specific orientation of workers to the radiation source during processing. Intakes of uranium and radium were based on 39,451 uranium urine bioassays and 2,341 breath radon measurements, and International Commission on Radiological Protection (ICRP) Publication 68 biokinetic models were used to estimate organ-specific radiation absorbed dose. Estimates of exposure to airborne radon and its short-lived progeny were based on radon measurements in work areas where radium-containing materials were handled or stored, together with estimated exposure times in these areas based on job titles. Dose estimates for radon and its short-lived progeny were based on models and methods recently recommended in ICRP Publication 137. This comprehensive dosimetric approach follows methods outlined by the National Council on Radiation Protection Scientific Committee 6-9 for the Million Worker Study. Annual doses were calculated for six organs: lung, brain, heart, kidney, colon and red bone marrow. Evaluation and adjustment for individual cumulative measures of pitchblende dust inhalation were made for lung and kidney diseases.

In this paper, we describe the development of ADEPT, a pioneering project of collaboration in developing clinical leadership training in an apprenticeship model through 1 year out of programme for postgraduate medical trainees in Northern Ireland, involving Northern Ireland Medical and Dental Training Agency, the health and social care Leadership Centre and relevant host organisations.

Litoria cyclorhyncha is a hylid frog native to southwest Western Australia (WA). It was first recorded in South Australia (SA) in 2000 and has established a breeding population in Streaky Bay on the western Eyre Peninsula since at least 2011. L. cyclorhyncha is a relatively large predatory frog that presents a potential threat to fauna and ecosystem processes in SA and eastern Australia. This study examines the invasion history and current and potential future distributions of this frog using historical records, field surveys and species distribution modelling (SDM). The historical records in SA suggest human-assisted dispersal through transport networks. Field surveys throughout the Eyre Peninsula during 2016–2017 detected three native species of frogs but no additional breeding populations of L. cyclorhyncha outside of Streaky Bay. Within Streaky Bay, frog abundance appears to be concentrated around permanent water bodies, but the species is also well established in urban habitats. SDM suggests that suitable environmental conditions exist for L. cyclorhyncha in southern and eastern Australia. This, and the frog’s ability to disperse over long distances, suggests considerable potential for the species to become invasive. As L. cyclorhyncha has potential to significantly impact biodiversity and ecosystems, management strategies are urgently needed. Refereed/Peer-reviewed

This work presents a detailed characterization of the microstructural and crystallographic texture changes observed in the transition region in a weld between two Zircaloy-4 cold rolled and recrystallized plates. The microstructural study was performed by optical microscopy under polarized light and scanning electron microscopy (SEM). Texture changes were characterized at different lengthscales: in the micrometric size, orientation imaging maps (OIM) were constructed by electron backscatter diffraction (EBSD), in the millimetre scale, high energy XRD experiments were done at the Advanced Photon Source (USA) and compared to neutron diffraction texture determinations performed in the HIPPO instrument at Los Alamos National Laboratory. In the heat affected zone (HAZ) we observed the development of Widmanstatten microstructures, typical of the α( hcp ) to β( bcc ) phase transformation. Associated with these changes a rotation of the c -poles is found in the HAZ and fusion zone. While the base material shows the typical texture of a cold rolled plate, with their c -poles pointing 35° apart from the normal direction of the plate in the normal-transversal line, in the HAZ, c -poles align along the transversal direction of the plate and then re-orient along different directions, all of these changes occurring within a lengthscale in the order of mm. The evolution of texture in this narrow region was captured by both OIM and XRD, and is consistent with previous measurements done by Neutron Diffraction in the HIPPO diffractometer at Los Alamos National Laboratory, USA. The microstructural and texture changes along the HAZ were interpreted as arising due to the effect of differences in the cooling rate and β grain size on the progress of the different α variants during transformation. Fast cooling rates and large β grains are associated to weak variant selection during the β−>α transformation, while slow cooling rates and fine β grains result in strong variant selection.