bluuebunny/crossref_metadata_2025 huggingface dataset:
Single cell and single molecule resolution of herpesvirus genome transport, condensation state and transcriptional output
Authors: Sekine Eiki , O’ Hare Peter
Published: April 2019

Events controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.
Biodiversity and biochemical characterization of bacteria from terrestrial subsurface environment
Authors: Rahamathullah Nazeerullah , Nooruddin Thajuddin
Published: April 2019

Forty-one aerotolerant subsurface bacterial colonies were isolated from terrestrial subsurface environment at various depth from 5 feet to 50 feet at 5 feet intervals. Out of 41 colonies, 32 morphologically distinct bacterial colonies were selected for gram staining reaction, various sugar utilization pattern and scanning electron microscopic studies. Most of the bacterial colonies were gram-positive rods; some are gram-negative rods, gram-positive cocci and coccobacilli, and gram-negative coccobacilli respectively. Seven bacterial colonies NTN33, NTN34, NTN35, NTN36, NTN01, NTN02 and NTN03 isolated from 40 to 50 feet depth were neither gram positive nor gram negative, and no distinct morphological shape but gigantic structure have identified in that bacteria. The bacterial size were identified on the bacteria NTN36, NTN35, NTN01, NTN30, NTN34, NTN02 as 4.5 µm, 4.1 µm, 3.5 µm, 3.4 µm, 3.2 µm and 2.2 µm. Gigantic rod surrounded by capsular sheath and gigantic rod with footprint like appearance were identified in the bacteria NTN02 and NTN36; those are isolated from 45 and 50 feet depth. Intracellular granules were also found in bacteria NTN34, NTN35 and NTN01. In total, 24 different sugars had used to analyse the sugar utilization pattern of that 32 bacteria. Most of the bacteria have utilized only 03 different types of sugars either maltose, galactose and glycerol or lactose, trehalose and mannose. However, the bacteria NTN16, NTN12, NTN06, NTN24, NTN27, NTN28, NTN34 and NTN02 had utilized 14 different sugars. Nevertheless, very few bacteria have utilized the l-arabinose, α-methyl-d-gluconate and inulin. None of the bacteria had utilized the sugar sodium gluconate, salicin, glucosamine and dulcitol.
Host response of Agaricus bisporus to mushroom virus X infection
Authors: O’Connor Eoin , Fitzpatrick David , Grogan Helen
Published: April 2019

Commercial mushroom crops (Agaricus bisporus) are susceptible to a disease causedby a complex of 18 viruses known collectively as mushroom virus X (MVX). Symptoms of MVX infection vary from bare patches in crops to mushroom discolouration (browning). To understand the dynamic interaction between A. bisporus and MVX, we have studied five strains; including the globally-cultivated commercial strain, one wild strain, and one commercial-wild hybrid strain. Our transmission experiments using ‘healthy’ mycelium challenged with MVX-infected mycelium, detected MVX in the first day of hyphal anastomosis in all five strains. However, our commercial-scale crop experiment revealed varying degrees of disease tolerance in the fruiting body, the commercial strain being most susceptible and the hybrid strain most resistant to MVX. LC-MS/MS proteomics and RNA-seq analyses have elucidated key differences in response to both early and late crop inoculation of MVX. Quantitative shotgun proteomics of the susceptible commercial strain at late MVX inoculation revealed striking levels of proteins relating to mechanical membrane damage via detection of myo-inositol-associated biosynthetic proteins. Defense proteins relating to chalcone isomerase activity were also detected exclusively in the MVX-infected commercial strain. MVX-infected wild strain isolates showed significantly greater abundance of proteins related to fundamental cellular processes such as RNA polymerase activity and cell-redox homeostasis. Our findings show that although vegetative transmission of MVX is prevalent in all five strains, the fruiting body may oppose the infection in certain strains. Our findings of dynamic host response of A. bisporus to MVX, provides novel insights for this economically important, globally cultivated crop.
Molecular approaches to understand the effect of acetic acid in uropathogenic E. coli
Authors: Alatar Fatemah , Bushell Francesca , Sannasiddappa Thippesh , Herbert John , Falciani Francesco , Lund Peter
Published: April 2019

Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigate the molecular mechanisms by which acetic acid acts as an antibacterial agent. To do this, we grew a high-density transposon library in uropathogenic E. coli EO499 serotype 131 in M9 media at pH 7 and pH 5.5 with acetic acid concentrations of 40 mM and 4 mM, respectively, or without added acetic acid. Sequencing libraries were generated from total bacterial populations after growth, and sequenced using a transposon-specific primer to generate positions and frequencies for each transposon. By comparing numbers of reads before and after the stress, we identified candidate genes where transposon inserts led to a decrease of fitness under acetic acid stress. Eight of these were chosen for further study: nuoM, nuoG, sucA, sthA, pitA, apaH, rssB and ytfP. Because of the difficulties of constructing gene deletions in the uropathogenic strain for validating the TraDIS results, we tested the relative fitness of the corresponding gene deletion mutants from the Keio library (in strain BW25113), with the growth conditions used for EO499. Interestingly, only a few knockouts showed a reduction in relative fitness in time course competitions at pH 5.5 with acetic acid. This may due to the differences between strains used in TraDIS and competition. To overcome this issue, we have also isolated transposon mutants from E. coli EO499 transposon library for the determination of relative fitness. The results will be presented.
Infectious bursal disease virus (IBDV) replicates in the gut associated lymphoid tissue and alters the gut microbiome of chickens
Authors: Daines Rebecca , Leng Joy , Horton Dan , Ragione Roberto La , Broadbent Andrew
Published: April 2019

Infectious bursal disease virus (IBDV) is a member of the Birnaviridae family that infects B cells in chickens, leading to immunosuppression and mortality. Immunosuppression is known to exacerbate the colonisation and shedding of zoonotic gut bacteria, for example Campylobacter jejuni, Salmonella Enteritidis and Escherichia coli, for reasons that are poorly understood. In order to address this, we infected groups of chickens (n=6) with either a classical strain (F52-70) or a very virulent strain (UK661) of IBDV. At 3 days post-infection, both strains were found to replicate in the gut-associated lymphoid tissue of thecaecaltonsils. 16 s rRNA sequencing revealed that in birds infected with IBDV, regardless of strain, there was a decrease in bacterial diversity in the caecal tonsils, but an increase in diversity of bacteria shed from the cloaca. Secretary IgA binding to commensal bacteria is known to influence the composition of the microbiome, and we speculate that IBDV alters the repertoire of sIgA thereby altering the microbiome composition. Interestingly, we found the number of clostridial species was reduced following IBDV infection. Clostridial species have been shown to induce Treg populations in mice and we speculate that IBDV-mediated changes in the microbiome affect the population of different immune cells in the mucosa. Taken together, we hypothesise that IBDV infection directly affects the B cell population and indirectly affects other immune cell populations in the gut and alters the gut microbiome, which leads to a more favourable environment for zoonotic bacterial infections to colonise.
Visualising influenza virus replication using a click chemistry approach
Authors: Swann Olivia , Peacock Thomas , O’Hare Peter , Barclay Wendy
Published: April 2019

Influenza A viruses (IAVs) have a significant impact on public health through seasonal epidemics. Moreover, the sporadic emergence of zoonotic and pandemic strains represents an additional global threat. Understanding how novel IAV strains adapt to infect and transmit between humans is essential for effective surveillance and may provide rationale for anti-viral therapeutics. IAVs replicate their RNA genomes in the host cell nucleus. Host proteins, such as the proviral factor ANP32, are co-opted to support viral replication and transcription, whilst other inhibitory factors act to restrict. Ultimately the outcome of infection in different species depends on compatibility with these factors, however the precise nuclear localisation of these interactions is not clearly defined. It is not known whether viral RNA synthesis takes place at discrete sites, or whether the virus modifies the cell to produce viral factories in a similar manner to other viruses. We are utilising a click chemistry approach to investigate the spatial details of IAV replication and to probe the differences in nuclear localisation between human-adapted vs poorly-adapted avian origin IAVs, to gain insight into mechanisms of host restriction. Influenza genomes labelled with the click reagent 5-ethynyl uridine (5-EU) remain infectious and can be visualised through the cycloaddition of azide-tagged fluorescent dyes. Utilising this tool, the co-localisation of incoming viral particles with nuclear sub-domains, as well as key host factors, are being examined. Moreover, the localisation of human- vs avian-adapted virus in human and avian cells, as well as cell lines lacking key host factors, are being compared.
Functionalised liposomal formulations for delivery of antibiotic agents
Authors: Catania Rosa , Maguire Oran , Moore Chris , Falcone Franco H. , Chan Weng , Mantovani Giuseppe , Stolnik Snow , Huett Alan
Published: April 2019

Antimicrobial resistance is a major global healthcare challenge. Beyond the discovery of novel antimicrobial agents, the development of novel formulations for enhancing current antibiotics is a promising strategy to reduce the rate of treatment failure. Drug delivery nano-carriers can achieve high local concentration of antimicrobial agents, reduce toxicity, and improve biodistribution and pharmacokinetics. We have taken two approaches to enhance antibiotic delivery and effectiveness. Firstly, we used a bespoke targeted liposomal system for intracellular antibiotic delivery to phagocytic cells. This enables treatment of an intracellular Gram-negative infection with a cell-impermeable antibiotic. Targeted liposomes were found to significantly enhance uptake compared to uncoated liposome control formulations both in in vitro and in vivo (zebrafish model). Secondly, liposomal nanoformulations were utilised to deliver peptide antibiotics, where liposomes protect peptides from degradation, and allow potential co-delivery of combination therapeutics. We investigated different liposomal formulations and drug combinations against E. coli and S. aureus. We show that peptide-loaded liposomes are more efficient compared to free drug in inhibiting the growth of both Gram-negative and Gram-positive bacteria. These initial results suggest that liposome-mediated delivery can be utilised for the repositioning and repurposing of existing antibiotics, potentially allowing for the treatment of diverse infections in a more effective manner.
Continuous culture of Escherichia coli, under selective pressure by a novel antimicrobial complex, does not result in the development of resistance
Authors: Tonoyan Lilit , Friel Ruairi , Fleming Gerard , O’Flaherty Vincent
Published: April 2019

Antibiotic resistance is a major global health problem. Preservation of antibiotics, underpinned by the availability of novel antimicrobials that avoid the emergence of antimicrobial resistance and antibiotic cross-resistance is an important societal goal. We have developed a novel biocidal complex (iodo-thiocyanate complex or ITC), drawing the inspiration from naturally occurring peroxidase-catalysed systems. This study was aimed to reveal the potential of ITC for induction of resistance and cross-resistance, and, thus, different aspects of resistance were explored. We show that the repeated exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus to sub-inhibitory levels of ITC during serial passage of batch cultures did not generate ITC resistance. By comparison, E. coli and S. aureus developed low-level and high-level resistance to levofloxacin (LVX), respectively. Further, we attempted to generate de novo resistance in antimicrobial-sensitive E. coli during 20 days of continuous culturing when exposed to gradually increasing concentrations of ITC and LVX. The exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based analysis of ITC-challenged bacterial populations. By contrast, the resistance to LVX was rapidly induced, selected for high-level and enriched with a distinctly characteristic genome mutational pattern. WGS of LVX-challenged population revealed that the majority of mutations appeared in the genes of LVX target proteins and drug influx. This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.
Development of advanced corrosion-resistant coatings with synthetic biology-inspired protein technologies
Authors: Allan Craig , Van Keulen Geertje , Penney Dave , Allan Gethin , Harold Alex , Griffin Sholeem
Published: April 2019

The purpose of this research is to further develop existing technology using engineered functional amyloid proteins for corrosion resistance by developing a coating which provides both adhesion to substrate and corrosion resistance. Pathogenic amyloid proteins have been attributed a role in certain diseases such as Alzheimer’s Disease. However, the research undertaken here involves non-pathogenic functional bacterial amyloids, using coelicolor hydrophobic aerial proteins (chaplins or Chp) produced by Streptomyces for improving corrosion resistance. Chaplins have been proven conceptually to provide corrosion resistance properties on metals and, also provide strong adhesive properties in a multi-composite systems. The composition, application and curing of the better bonding chaplin composite will now be optimised for development of a better performing functional coating, which includes inclusion of small metabolites or natural products acting as corrosion inhibitors. This would then allow for benchmarking against existing corrosion-resistant coatings. Upon determining the most relevant formulae, samples will be analysed by using microscopic techniques such as Scanning Electron Microscopy and Atomic Force Microscopy, while corrosion will be tested using immersion tests, weathering via salt-spray and in-situ scanning vibrating electrode.
Developing a universal strategy for cloning and assembly of the genomes of diverse Epstein – Barr virus strains
Authors: Bayoumy Amr , Abdullah Mohammed Ba , White Rob
Published: April 2019

Epstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.
Gut microbiome of poison arrow frogs and their potential role in toxin sequestration
Authors: Siu-Ting Karen , Friedersdorff Jess , Carreño David , Thomas Ben , Newbold Jamie , O’Connell Mary , Creevey Christopher
Published: April 2019

Poison frogs secrete alkaloid toxins in their skin as defence mechanisms against predators. Numerous studies have shown that the origin of alkaloid toxins in the skin is through ‘sequestration from diet’, i.e. uptake and storage of toxins or their chemical precursors, mostly from consumed arthropods. There exists the intriguing possibility that the gut microbiome of these frogs may play a role in this process. We address this question by looking at the organism together with its associated microbial communities, an effective symbiotic relationship between host and microbiome that could have allowed phenotypic adaptation of the host to a toxic diet. We sequenced the Bacterial and Archaeal 16S rRNA regions of the gut microbiome of 7 Poison frog species and 9 outgroup frog species caught in the rainforest of Eastern Peru. Frog species were selected based on sharing similar microhabitats and comparable individual sizes. A comparative analysis of the microbiome composition across all our samples allowed us to identify a core group of abundant symbiotic microbes unique to poison frogs in spite of intrinsic variation within species. We speculate this group could be associated to their ability to sequester toxins and we carried further metagenomic sequencing to allow us to determine possible functions that may be involved in toxin processing in these frogs.
Applying Next Generation Sequencing Technology to Hepatitis C genotyping in a routine diagnostic laboratory
Authors: May Sophie
Published: April 2019

Background

To meet the target of Hepatitis C elimination in the UK, a rapid and high throughput method of genotyping is desirable. Current methods target the NS5B region with a nested PCR followed by Sanger sequencing. Drawbacks include insufficient information to guide second line treatment in the event of failure and poor sensitivity compared to assays targeting the 5’UTR.

Method

Previously tested HCV positive patient samples were selected and RNA extracted using an ultra-centrifugation manual extraction method. A two-step reverse transcription long range PCR generated amplicons which were quantified using the Qubit fluorometer and libraries prepared using the Nextera XT library preparation kit. Sequencing was performed using an Illumina Miseq and genomes were assembled using a Linex-based bioinformatics pipeline.

Results

A total of 150 samples were tested, with full HCV genomes obtained for genotypes 1–6 and the sensitivity of the assay corresponded to around 1000 IU  ml−1. Where previous sequence data for resistance associated variants (RAVs) was available, mutations were consistent. Furthermore, the assay identified a 2 k/1b recombinant and allowed the typing of previously unassigned genotypes, demonstrating its utility in the diverse local population.

Conclusion

This study displayed the utility of a whole genome sequencing approach to HCV. A major benefit of whole genome analysis was the inclusion of regions targeted by antiviral therapy such as NS5A and NS3. This allowed the identification of RAVs in patients experiencing treatment failure. Its application to dried blood spots is currently being investigated as part of a wider screening programme.
Analysis of phenotypic traits which may impact long term survival of different Escherichia coli pathotypes
Authors: Gray Jennifer , Rani Sonia , Duffy Geraldine , Fanning Seamus , Burgess Catherine
Published: April 2019

Shiga toxin producing E. coli (STEC) is a foodborne pathogen which causes severe, debilitating, and sometimes fatal, illness. Ireland consistently has one of the highest incidence rates of human STEC infection in Europe. Cattle are one of the primary reservoirs for STEC, excreting the pathogen in their faeces. The amount of pathogen excreted varies greatly, with animals shedding >log104 c.f.u. g−1 faeces being termed ‘super-shedders’. Human infection can occur from faecal contamination of meat, dairy, fresh produce or drinking water. STEC can survive for extended periods in soil, slurry and water, although the exact means is unknown. The objective of this study was to examine phenotypic traits potentially relevant to extended environmental survival in two strain banks: (1) clinical and bovine STEC, in comparison with non-STEC, isolated from the production environment and (2) E. coli O157:H7 of known shedding status. The strain banks were assessed for biofilm-forming abilities and the ability to adhere to the muscle component collagen-I, using a 96-well crystal violet assay, where the absorbance of bound cells indicated biofilm formation levels or adherence to collagen. Extracellular components involved in attachment were assessed using Congo red agar and pellicle formation was also examined. Phenotypic traits potentially related to extended environmental persistence were observed more frequently in non-STEC. However, these traits were also observed in some STEC isolates, showing phenotype is strain dependent indicating a risk for enhanced environmental survival of some STEC isolates. The shedding status of E. coli O157:H7 is not dictated by the investigated characteristics alone.
Unravelling the features of Influenza as entry mechanism
Authors: Desbki-Antoniak Oliver , Hover Samantha , Tiede Christian , Tomlinson Darren , Mankouri Jamel , Fontana Juan
Published: April 2019

Influenza virus is the causative agent of the ‘flu’. According to the World Health Organisation, Influenza causes up to 5 million cases of severe flu and 500 000 deaths annually. To release its genome inside the cell and start an infection, Influenza virus must fuse its envelope with the endosomal membrane of the host, making this process an excellent drug target. In order to attempt to control, or better yet, eradicate this pathogen, a greater understanding of its entry mechanism, a fundamental aspect of the viral life cycle, is required. It is well established that Influenza A virus (IAV) fusion is driven by the viral glycoprotein hemagglutinin (HA), which when exposed to low pH transitions from a meta-stable pre-fusion to post-fusion state. However, it is becoming increasingly apparent that the ionic balance of the endosomes also has a significant role in the entry of enveloped viruses. Specifically, our preliminary studies on Influenza virus suggest that K+ concentrations within the endocytic pathway play a significant role in Influenza A fusion events. Through the use of broad-spectrum K+ channel inhibitors, a dose-dependant reduction in Influenza infectivity in tissue culture can be observed. Further to these studies, we have isolated Affimers (novel antibody-like proteins that can be produced in large quantities in E. coli) that recognise HAs from different Influenza subtypes (H3N2 and H1N1). Using these Affimers, we aim to develop an early detection method to distinguish between bacterial respiratory infections and ‘the flu’ to alleviate mounting pressures on an already diminishing antibiotic treatment system.
Biotechnological approach to produce riboflavin enriched iru – using riboflavin overproducing Bacillus subtilis
Authors: Olanbiwoninu Afolake , Greppi Anna , Lacroix Christophe , Jans Christoph
Published: April 2019

Dietary deficiencies are major cause of malnutrition in the developing world, particularly vitamins deficiencies such as riboflavin, which lead to various health disorders. Traditional plant fermentation and their indigenous starter cultures such as Bacillus subtilis might provide solutions to bioenrich riboflavin. We aimed to investigate this idea on the example of B. subtilis derived from iru an alkaline fermented condiment playing an important role in rural Nigeria. After initial isolation, identification and safety assessment, roseoflavin exposure was used to obtain riboflavin overproducing mutants. These were further analysed for functional characteristics. Bàcillus species’ (n- 123) were isolated from iru. Initial riboflavin production in supportive growth medium ranged from 50.3 to 479.0 µg l−1 for 27 out of 123 strains evaluated. Subsequent gràdual exposure to 200 mg l−1 roseoflavin increased riboflavin production in the three best producing strains from 350 µg l−1 to 542 µg l−1, 479 µg l−1 to 580 µg l−1 and 362 µg l−1 to 618 µg l−1. This increased riboflavin in lab-scàle iru fermentation by over 150 percent to 0.12–0.14 mg g−1 and near the recommended daily intake while retaining desired proteolytic and esterase activity. This research provides important proof of concept for the bioenrich the of traditional B. subtilis based plant fermentations used across sub-Saharan Africa and possibly other areas globally.
Evolution and spread of bacterial transposons
Authors: Blackwell Grace , Iqbal Zamin , Thomson Nick
Published: April 2019

Transposons are mobile genetic elements (MGE) that can carry additional genetic cargo nonessential for their own transposition. This cargo can include antibiotic or heavy metal resistance genes or those increasing metabolic plasticity. In addition to transposing across or between the chromosome and other replicons in a single cell, they can transfer between bacteria as passengers on conjugative plasmids that are capable of intercellular transfer. Transposons can be grouped on mechanisms of movement and by lengths of the bounding inverted repeats or of target site duplication created by transposition. Class II transposons, such as Tn3 and Tn501 utilise two-step replicative transposition involving a transposase, tnpA, and a resolvase, tnpR, gene. The Tn402/Tn5053 family has four genes, tniABQR, required for transposition, while Tn7 and relatives contain five (tnsABCDE). Despite their role in antimicrobial resistance dissemination and a detailed mechanistic view of transposition there have been no studies aimed at revealing the evolutionary history of transposon families by interrogating global genome sequence datasets. This is largely because the ability to search all existent bacterial sequences is non-trivial and only recently realised. We took a selection of 37 representative variants of the above transposon families and queried their nature and distribution across sequence space using bigsi (http://www.bigsi.io/), a searchable index of the bacterial ENA (455 632 datasets, Dec 2016) and Shovill (https://github.com/tseemann/shovill). Constrained by the sequence data that exists and the biases this may engender, this analysis provides broad insights into the prevalence and spread of important MGE.
Early life innate immune responses to RSV in cystic fibrosis airway epithelium
Authors: Coulter Patricia J. , Groves Helen , Broadbent Lindsay , Coey Jonathan Dean , Shields Michael D. , Power Ultan F.
Published: April 2019

Respiratory syncytial virus (RSV) infection is the major cause of severe lower respiratory tract disease in young infants. Evidence suggests that cystic fibrosis (CF) is associated with significant morbidity following RSV infection. We and others previously demonstrated that airway epithelium is the primary target of RSV infection. However, little is known about the impact of RSV infection on CF airway epithelium. To address this, we established and characterised well-differentiated primary nasal epithelial cell cultures (WD-PNECs) from recently diagnosed CF infants to study RSV cytopathogenesis in CF airway epithelium. CF WD-PNECs were successfully generated from 11 infants and characterised by light and fluorescent microscopy. CF WD-PNECs secreted thick dry apical mucous, consistent with in vivo observations. Extensive cilia coverage was also evident, although cilia beat frequencies appeared lower than those evident in WD-PNECs from healthy neonates. Quantification of goblet and ciliated cells in the CF WD-PNEC cultures were similar to those of healthy cultures. Viral growth kinetics were similar for CF WD-PNECs and healthy WD- PNECs, with peak virus titres evident at 72–96 hpi. CXCL8/IL-8 and CXCL10/IP-10 secretions were upregulated following RSV infection of CF WD-PNECs, while IL-6 secretions did not change. Interestingly, our data suggested that duoxa2 and duox2 expression post-infection were reduced in WD-PNECs from CF compared to healthy infants. Our data suggest that this model provides an exciting opportunity to elucidate the cytopathogenic, inflammatory and molecular consequences of RSV infection of airway epithelium derived from very young CF infants.
Investigating the impact of influenza point-of-care testing – what are the benefits?
Authors: Goldstein Emily , Harvey William , Maycock Matthew , Bellhouse Emilie , Gunson Rory , Choo-Kang Brian
Published: April 2019

Point-of-care testing (POCT) for influenza viruses is being used increasingly in NHS hospitals. In the 2017/18 influenza season, NHS Greater Glasgow and Clyde implemented the Cepheid GeneXpert System for the detection of influenza A, B and respiratory syncytial virus in 7 wards across 5 hospital sites. To evaluate the impact of influenza POCT in Glasgow Royal Infirmary during the 2017/18 influenza season, we retrospectively compared data from 150 influenza A infected patients. We analysed data from 100 patients who were diagnosed using a laboratory based in-house respiratory PCR assay (50 patients from 2016/17 and 50 patients from the 2017/18 season) and 50 patients who were diagnosed using POCT in the 2017/18 season. The aim of the study was to investigate whether POCT impacted patient management and outcome. General linear models were used to test for an association between POCT and a number of outcomes, whilst accounting for host factors. These outcomes included: (1) time from review to influenza result (2) admission status (3) length of hospital stay (4) patient outcome (5) duration of antibiotics (6) antiviral treatment (7) patient isolation and (8) the number of duplicate influenza tests performed. In addition to investigating the impact of POCT, we used this opportunity to explore additional factors that may affect the outcomes of patients infected with influenza A, including: age, sex, imaging results and underlying risk factors.
A multiplatform approach to investigate the structure and architecture of the biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in response to antimicrobial treatment
Authors: Brown James , Alexander Morgan , Williams Paul , Hardie Kim
Published: April 2019

Bacterial biofilms are highly complex communities, composed of highly structured extracellular polymers and subpopulations of differentiated cells, such as persisters. These contribute to the resistance of bacterial biofilms to antibiotics, creating a significant issue in the treatment of infections and resulting in elevated levels of mortality and morbidity. Here, we use a microfluidic system coupled with time-lapse microscopy and fluorescent dyes for exopolysaccharide (EPS) and extracellular DNA (eDNA) to investigate how the architecture of the growing biofilm is ordered. We show that in Pseudomonas aeruginosa EPS is deposited first in the initial stages of microcolony development, but that eDNA then acts as a leading edge for further microcolony expansion. We explore how this assembly is perturbed by the introduction of different antimicrobial agents. Further, working with partners in the European Association of National Metrology Institutes (EURAMET) we are developing cross platform methods for the label-free localisation of antimicrobial agents and bacterial components within the biofilm. These platforms include 3D OrbiSIMS (secondary ion mass spectrometry) and Raman spectroscopy. Here we demonstrate the localisation of key biofilm components such as Pseudomonas Quinolone Signal (PQS) molecules in a 3D chemical map of the biofilm.
Cerebellar Modulation of Mesolimbic Dopamine Transmission is Functionally Asymmetrical
Authors: Holloway Zade R. , Paige Nick B. , Comstock Josiah F. , Nolen Hunter G. , Sable Helen J. , Lester Deranda B.
Published: April 2019

Cerebral and cerebellar hemispheres are known to be asymmetrical in structure and function, and previous literature supports that asymmetry extends to the neural dopamine systems. Using in vivo fixed-potential amperometry with carbon-fiber microelectrodes in anesthetized mice, the current study assessed hemispheric lateralization of stimulation-evoked dopamine in the nucleus accumbens (NAc) and the influence of the cerebellum in regulating this reward-associated pathway. Our results suggest that cerebellar output can modulate mesolimbic dopamine transmission, and this modulation contributes to asymmetrically lateralized dopamine release. Dopamine release did not differ between hemispheres when evoked by medial forebrain bundle (MFB) stimulation; however, dopamine release was significantly greater in the right NAc relative to the left when evoked by electrical stimulation of the cerebellar dentate nucleus (DN). Furthermore, cross-hemispheric talk between the left and right cerebellar DN does not seem to influence mesolimbic release given that lidocaine infused into the DN opposite to the stimulated DN did not alter release. These studies may provide a neurochemical mechanism for studies identifying the cerebellum as a relevant node for reward, motivational behavior, saliency, and inhibitory control. An increased understanding of the lateralization of dopaminergic systems may reveal novel targets for pharmacological interventions in neuropathology of the cerebellum and extending projections.
Graph-Based Method for Anomaly Detection in Functional Brain Network using Variational Autoencoder
Authors: Mirakhorli Jalal , Mirakhorli Mojgan
Published: April 2019

Functional neuroimaging techniques using resting-state functional MRI (rs-fMRI) have accelerated progress in brain disorders and dysfunction studies. Since, there are the slight differences between healthy and disorder brains, investigation in the complex topology of human brain functional networks is difficult and complicated task with the growth of evaluation criteria. Recently, graph theory and deep learning applications have spread widely to understanding human cognitive functions that are linked to gene expression and related distributed spatial patterns. Irregular graph analysis has been widely applied in many brain recognition domains, these applications might involve both node-centric and graph-centric tasks. In this paper, we discuss about individual Variational Autoencoder and Graph Convolutional Network (GCN) for the region of interest identification areas of brain which do not have normal connection when apply certain tasks. Here, we identified a framework of Graph Auto-Encoder (GAE) with hyper sphere distributer for functional data analysis in brain imaging studies that is underlying non-Euclidean structure, in learning of strong rigid graphs among large scale data. In addition, we distinguish the possible mode correlations in abnormal brain connections.
Carryover effects of temperature and pCO2across multiple Olympia oyster populations
Authors: Spencer Laura H , Venkataraman Yaamini R , Crim Ryan , Ryan Stuart , Horwith Micah J , Roberts Steven B
Published: April 2019

Predicting how populations will respond to ocean change across generations is critical to effective conservation of marine species. One emerging factor is the influence of parental exposures on offspring phenotype, known as intergenerational carryover effects. Parental exposure may deliver beneficial or detrimental characteristics to offspring that can influence larval recruitment patterns, thus shaping how populations and community structure respond to ocean change. Impacts of adult exposure to elevated winter temperature and pCO2on reproduction and offspring viability were examined in the Olympia oyster (Ostrea lurida) using three populations of adult, hatchery-rearedO. lurida,plus an additional cohort spawned from one of the populations. Oysters were sequentially exposed to elevated temperature (+4°C, at 10°C), followed by elevated pCO2(+2204 µatm, at 3045 µatm) during winter months. Male gametes were more developed after elevated temperature exposure and less developed after high pCO2exposure, but there was no impact on female gametes or sex ratios. Oysters previously exposed to elevated winter temperature released larvae earlier, regardless of pCO2exposure. Those exposed to elevated winter temperature as a sole treatment released more larvae on a daily basis, but when also exposed to high pCO2there was no effect. These combined results indicate that elevated winter temperature acceleratesO. luridaspermatogenesis, resulting in earlier larval release and increased production, with elevated pCO2exposure negating effects of elevated temperature. Altered recruitment patterns may therefore follow warmer winters due to precocious spawning, but these effects may be masked by coincidental high pCO2. Offspring were reared in common conditions for one year, then deployed for three months in four estuarine bays with distinct environmental conditions. Offspring of parents exposed to elevated pCO2had higher survival rates in two of the four bays. This carryover effect demonstrates that parental conditions can have substantial ecologically relevant impacts that should be considered when predicting impacts of environmental change. Furthermore, Olympia oysters may be more resilient in certain environments when progenitors are pre-conditioned in stressful conditions. Combined with other recent studies, our work suggests that the Olympia may be more equipped than other oysters for the challenge of a changing ocean.
The splicing factor hnRNP M is a critical regulator of innate immune gene expression in macrophages
Authors: West K.O. , Scott H.M. , Torres-Odio S. , West A.P. , Patrick K.L. , Watson R.O.
Published: April 2019

While transcriptional control mechanisms of innate immune gene expression are well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M leads to hyperinduction of a unique regulon of inflammatory and antimicrobial genes, including IL6, Mx1, and Gbp5, following a variety of innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of “housekeeping” genes in resting macrophages, it greatly impacted the protein’s ability to dampen induction of specific innate immune transcripts following activation of pathogen sensing cascades. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.
Separation and detection of minimal length isomeric glycopeptide neoantigen epitopes centering GSTA region of MUC1 by LC-MS
Authors: Zhou Dapeng , Xiao Kaijie , Tian Zhixin
Published: April 2019

MUC1 ranks No.2 on the list of targets for cancer immunotherapy. We previously reported monoclonal antibodies binding to glycopeptide neoantigen epitopes centering GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering GSTA sequence are also predicted by NetMHC programs to bind to MHC molecules, although empirical data are lacking. Detecting isomeric MUC1 glycopeptide epitopes by mass spectrometry (MS) remains a technical challenge since antigenic epitopes are often shorter than 10 amino acids. MUC1 digests by Arg-C-specific endopeptidase clostripain could generate heterogenous icosapeptides, but isomeric 20-residue glycopetides could not be separated by liquid chromatography. In this study, we used pronase fromStreptomyces griseus, which has no amino acid sequence preference for enzymatic cleavage sites, to digest a pair of synthetic glycopeptide isomers RPAPGST(Tn)APPAHG and RPAPGS(Tn)TAPPAHG, and analyzed the digests by LC-MS using electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) methods. The results showed that glycopeptide isomers containing 8 to 11 amino acids could be efficiently generated by pronase digestion. Such glycopeptide isomers of minimal epitope lengths were clearly distinguished by characteristic MS/MS ion patterns and elution profiles of liquid chromatography. A glycopeptide library was generated which may serve as standards for measuring neoantigen epitopes centering GSTA sequence.
Tacit knowledge as a multilayer phenomenon: the “onion” model
Authors: Asher Dan , Popper Micha
Published: April 2019

Purpose

This paper aims to clarify the term “tacit knowledge” and suggests the “onion model” as a way to explore conceptually linked layers of tacit knowledge. The model allows the application of different methodologies to elicit tacit knowledge in each layer, the ability to infer tacit knowledge in other layers from tacit knowledge gained in another layer and the exploration of the dynamics of tacit knowledge among the various layers presented in the model. Conceptual and practical advantages compared to prior works on tacit knowledge are discussed.

Design/methodology/approach

The main theoretical and methodological dilemmas discussed in the literature regarding tacit knowledge are reviewed. The “onion model” presented in this paper suggests an approach and methodologies that address the issues raised in the literature. The different layers of the model are demonstrated by prior research studies.

Findings

The “onion model” discussed in this study points to various layers of tacit knowledge and the links among them, allowing a research-based approach, as well as various practices.

Research limitations/implications

This paper discusses different layers of tacit knowledge relying on previous works that have dealt with these layers independently. The model as a whole and the dynamics among the layers are yet to be empirically investigated.

Practical implications

The “onion model” provides a conceptual framework that can be used for research and diagnosis aimed at exploring tacit knowledge that can serve individual and organizational development.

Originality/value

The approach discussed in this paper addresses some major problems discussed in the literature on tacit knowledge.
Investigating the role of UTAUT and e-service quality in internet banking adoption setting
Authors: Rahi Samar , Abd.Ghani Mazuri
Published: April 2019

Purpose

Technology adoption is a great challenge in the banking sector of Pakistan. A recent report issued by state bank of Pakistan revealed that there is a squeak growth, only 3 percent, in internet banking adoption. In order to effectively delve into the issue of internet banking adoption, the purpose of this paper is to use unified theory of acceptance and use of technology factors, namely performance expectancy and effort expectancy and e-service quality (E-SQ), as theoretical lens for this study.

Design/methodology/approach

The research model was empirically tested using 398 valid responses from customers of commercial banks in Pakistan. The theoretical model was tested using structural equation modeling.

Findings

Findings indicate that performance expectancy, effort expectancy, website design, customer service, assurance and reliability have direct influence on user intention to adopt internet banking. Results revealed that approximately 79 percent of variance in user intention to adopt internet banking was explained by predictors. In addition, the mediating role of performance expectancy and effort expectancy among website design, customer service and user intention was also confirmed.

Practical implications

For researchers, this study provides a base of integrated technology model and it suggests using this model in other online domains such as mobile payment and online web-shopping for further refinement. For policymakers, understanding the key constructs is important to design, refine and implement new internet banking website that, in turn, will boost internet banking adoption trend among users of commercial banks.

Originality/value

This paper makes a unique contribution toward information system and services marketing literature. The study schematized that website design, customer service, assurance and reliability are the key dimensions of E-SQ and significantly influence the user intention to adopt internet banking.
Co‐authoring better futures for children and families – Anticipation Dialogue in children’s social care networks
Authors: Huhnen Mark
Published: April 2019

Anticipation Dialogue (AD), in comparison to Open Dialogue, the lesser known form of dialogical network practices introduced by Seikkula and Arnkil, seems not to be used in children’s social care in the UK, and there is little literature about it. In this article I will introduce and explain Anticipation Dialogue and make links to other systemic ideas including Signs of Safety. I will review the literature and give a case example from my children’s social care practice.