Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments

Activation of Akt at T308 and S473 in alcohol, tobacco and HPV-induced HNSCC:is there evidence to support a prognostic or diagnostic role?

BACKGROUND: Tobacco, alcohol and HPV infection are associated with increased risk of HNSCC. However, little is known about the underlying signaling events influencing risk. We aimed to investigate the relationship between these risk factors and Akt phosphorylation, to determine prognostic value. METHOD: VEGF-positive HNSCC biopsies, with known HPV status, were analyzed by immunohistochemistry (IHC) for Akt, phosphorylated at residues S473 and T308. Comparisons between the tissues were carried out using a Mann–Whitney U test. Associations between the variables and continuous immunohistochemical parameters were evaluated with general linear models. Patient characteristics and pAkt IHC score were analyzed for possible association with overall survival by Cox proportional hazard models. RESULTS: Immunohistochemistry revealed that cancer patients had significantly higher levels of pAkt T308 than S473 (P < 0.001). Smoking and alcohol were found to be independent risk factors for Akt phosphorylation at T308 (P = 0.022 and 0.027, respectively). Patients with tumors positive for HPV or pAkt S473 had a poorer prognosis (P = 0.005, and 0.004, respectively). Patients who were heavy drinkers were 49 times more likely to die than non-drinkers (P = 0.003). Patients with low pAkt T308 were more likely to be HPV positive (P = 0.028). Non-drinkers were also found to have lower levels of pAkt T308 and were more likely to have tumors positive for HPV than heavy drinkers (P = 0.044 and 0.007, respectively). CONCLUSION: This study suggests different mechanisms of carcinogenesis are initiated by smoking, alcohol and HPV. Our data propose higher phosphorylation of Akt at T308 as a reliable biomarker for smoking and alcohol induced HNSCC progression and higher phosphorylation of Akt at S473 as a prognostic factor for HNSCC

Losing It: Strategies for Reducing Archival Collection Backlogs

Archival backlogs have been discussed at length in professional literature and been the focus of many specially funded projects over the years. However, little is written about the successes and failures of these “elimination” projects, leaving institutions with minimal guidance for reducing their own un- or under-processed collection backlogs. This article will share details of a three-year archival backlog elimination project at the University of Nevada, Las Vegas and provide strategies for institutions planning to begin their own project. The authors will discuss project staffing models and accounting for turnover, establishing and re-evaluating project priorities, creating workflow and documentation strategies, and sharing communication and team building recommendations

RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1

Ribonucleases are critically important in many cellular and developmental processes and defects in their expression are associated with human disease. Pacman/XRN1 is a highly conserved cytoplasmic exoribonuclease which degrades RNAs in a 5' - 3' direction. In Drosophila, null mutations in pacman result in small imaginal discs, a delay in onset of pupariation and lethality during the early pupal stage. In this paper, we have used RNA-seq in a genome-wide search for mRNAs misregulated in pacman null wing imaginal discs. Only 4.2% of genes are misregulated ±>2-fold in pacman null mutants compared to controls, in line with previous work showing that Pacman has specificity for particular mRNAs. Further analysis of the most upregulated mRNAs showed that Pacman post-transcriptionally regulates the expression of the secreted insulin-like peptide Dilp8. Dilp8 is related to human IGF-1, and has been shown to co-ordinate tissue growth with developmental timing in Drosophila. The increased expression of Dilp8 is consistent with the developmental delay seen in pacman null mutants. Our analysis, together with our previous results, show that the normal role of this exoribonuclease in imaginal discs is to suppress the expression of transcripts that are crucial in apoptosis and growth control during normal development

Serious Game Evaluation as a Meta-game

Purpose – This paper aims to briefly outline the seamless evaluation approach and its application during an evaluation of ORIENT, a serious game aimed at young adults. Design/methodology/approach – In this paper, the authors detail a unobtrusive, embedded evaluation approach that occurs within the game context, adding value and entertainment to the player experience whilst accumulating useful data for the development team. Findings – The key result from this study was that during the “seamless evaluation” approach, users were unaware that they had been participating in an evaluation, with instruments enhancing rather than detracting from the in-role game experience. Practical implications – This approach, seamless evaluation, was devised in response to player expectations, perspectives and requirements, recognising that in the evaluation of games the whole process of interaction including its evaluation must be enjoyable and fun for the user. Originality/value – Through using seamless evaluation, the authors created an evaluation completely embedded within the “magic circle” of an in-game experience that added value to the user experience whilst also yielding relevant results for the development team

Regional Differences in Presence of Shiga toxin-producing Escherichia coli Virulence-Associated Genes in the Environment in the North West and East Anglian regions of England

Shiga toxin-producing Escherichia coli is carried in the intestine of ruminant animals, and outbreaks have occurred after contact with ruminant animals or their environment. The presence of STEC virulence genes in the environment was investigated along recreational walking paths in the North West and East Anglia regions of England. In all, 720 boot sock samples from walkers’ shoes were collected between April 2013 and July 2014. Multiplex PCR was used to detect E. coli based on the amplification of the uidA gene and investigate STEC-associated virulence genes eaeA, stx1 and stx2. The eaeA virulence gene was detected in 45·5% of the samples, where stx1 and/or stx2 was detected in 12·4% of samples. There was a difference between the two regions sampled, with the North West exhibiting a higher proportion of positive boot socks for stx compared to East Anglia. In univariate analysis, ground conditions, river flow and temperature were associated with positive boot socks. The detection of stx genes in the soil samples suggests that STEC is present in the English countryside and individuals may be at risk for infection after outdoor activities even if there is no direct contact with animals. Significance and Impact of the Study: Several outbreaks within the UK have highlighted the danger of contracting Shiga toxin-producing Escherichia coli from contact with areas recently vacated by livestock. This is more likely to occur for STEC infections compared to other zoonotic bacteria given the low infectious dose required. While studies have determined the prevalence of STEC within farms and petting zoos, determining the risk to individuals enjoying recreational outdoor activities that occur near where livestock may be present is less researched. This study describes the prevalence with which stx genes, indicative of STEC bacteria, were found in the environment in the English countryside

The alternate GNB3 splice variant, Gβ3s, exhibits an altered signalling response to EGF stimulation, which leads to enhanced cell migration

It has recently been reported that the duplication of the GNB3 gene has been shown to be directly linked to an obesity phenotype, both in humans and also in a humanised mouse model. Moreover, the common human GNB3 c.825C&gt;T polymorphism (rs5443) causes this ubiquitously expressed gene to be aberrantly spliced approximately 50% of the time leading to the production of both a normal Gβ3 protein and a truncated, possibly less stable subunit, known as Gβ3s. The presence of the GNB3 825T allele has previously been shown to be associated with predisposition to hypertension, obesity, various cancers, Alzheimers, age related cognitive function, erectile dysfunction as well as a marker for pharmacogenetic drug action. Great controversy, however, currently exists as to whether these phenotypes associated with the 825T allele are a) mainly due to the presence of the smaller, possibly more active, Gβ3s subunit or b) merely down to the haploinsufficiency of the normal GNB3 transcript, due to its frequent aberrant splicing. In order to try and address these two conflicting hypothesis, we report on the identification and characterisation of signalling alterations unique to the presence of Gβ3s protein subunit. Moreover we also show the physiological consequences associated with altered signalling, directly induced by the Gβ3s subunit. For this, we used both an EBV transformed lymphoblast cell line homozygote for GNB3 825T/825T (TT) and a stable Gβ3s expressing recombinant COS-7 clone. In both of these cell lines that express the Gβ3s subunit, we found enhanced cytosolic calcium influx upon stimulation with EGF, TGFα and VEGF ligands, as compared to “normal” GNB3 controls with the 825C/825C (CC) genotype. This aberrant calcium influx also led to an increase in ERK, but not AKT1, phosphorylation. Despite the lack of AKT1 activation, we paradoxically observed a significant increase in phosphorylation of its downstream substrates, namely mTOR and p70S6k (KS6B2). Moreover we observed a decrease in phospho FoxO3a only in Gβ3s expressing cells, but not in the “normal” GNB3 (CC) control cell line. The presence of the Gβ3s subunit also appeared to alter the distinct localisation patterns of both Foxo3a and AKT1, while also increasing the colocalisation of mTOR and p70S6K. Subsequent growth factor stimulation studies revealed that EGF treatment, of Gβ3s expressing cells, appeared to cause a significant decrease in cAMP levels, which, in turn resulted in both enhanced caveolin-1a phosphorylation, and an increase in actin stress fibre formation. The identification of these distinct Gβ3s specific signalling alterations were indicative of a more aggressive migratory phenotype. This led us to further investigate and confirm that the presence of the Gβ3s subunit also appears to cause significantly enhanced migration and robust scratch wound healing kinetics, as compared to cells harbouring only the normal copy of the gene. These data therefore present convincing evidence that the Gβ3s subunit is stable, functional and its presence can significantly alter signalling pathways, in different cell types

Balancing the Art and Science of Archival Processing Metrics and Assessment

At the University of Nevada, Las Vegas (UNLV) University Libraries, archival processing metrics are used to support value propositions, project proposals, project management, and strategic planning. When making data-driven decisions, UNLV Special Collections Technical Services staff strive to balance the art and science of archival processing metrics—to critically assess their data and look beyond the numbers for additional information that brings meaning to the metrics. In this research paper, the authors review processing metrics across the profession and place their own archival processing field data within the context of more than three decades of professional practice. They report and explore UNLV’s processing rates and the variables that potentially influence their rates. Learning from methods piloted during special projects, they reach toward a more streamlined, sustainable assessment practice. They also include a “work in progress”—an experimental framework that suggests core processing data points for UNLV with a second tier of optional data points that may add value to metrics in specific circumstances. The flexibility and extensibility of the framework give it the potential to serve as a model for other repositories

Perspectives and Practices: Archival Processing Metrics Survey Findings

Professional literature and case studies demonstrate that processing metrics are valuable in advocating for resources, informing priorities, supporting grant proposals, and predicting costs for collection storage and care. This article analyzes responses to an archival processing metrics survey that gathered perspectives and practices from archivists working in a variety of repository types. The findings describe how archivists collect processing metrics in different ways, what data points they view as essential, and how they use certain data points to serve specific purposes. The findings indicate that although most respondents acknowledge the value of processing metrics, the majority of them still do not employ metrics in daily operations. The findings note barriers to processing assessment and highlight disparities in resources, personnel, and practices across the profession. The findings reveal a lack of clarity about best practices for archival processing assessment and a need for profession-wide guidelines. The article suggests resources for those wishing to implement processing metrics

Calcium-Dependent But Action Potential-Independent BCM-Like Metaplasticity in the Hippocampus

The Bienenstock, Cooper and Munro (BCM) computational model, which incorporates a metaplastic sliding threshold for LTP induction, accounts well for experience-dependent changes in synaptic plasticity in the visual cortex. BCM-like metaplasticity over a shorter timescale has also been observed in the hippocampus, thus providing a tractable experimental preparation for testing specific predictions of the model. Here, using extracellular and intracellular electrophysiological recordings from acute rat hippocampal slices, we tested the critical BCM predictions (1) that high levels of synaptic activation will induce a metaplastic state that spreads across dendritic compartments, and (2) that postsynaptic cell-firing is the critical trigger for inducing that state. In support of the first premise, high-frequency priming stimulation inhibited subsequent long-term potentiation and facilitated subsequent long-term depression at synapses quiescent during priming, including those located in a dendritic compartment different to that of the primed pathway. These effects were not dependent on changes in synaptic inhibition or NMDA/ metabotropic glutamate receptor function. However, in contrast to the BCM prediction, somatic action potentials during priming were neither necessary nor sufficient to induce the metaplasticity effect. Instead, in broad agreement with derivatives of the BCM model, calcium as released from intracellular stores and triggered byM1 muscarinic acetylcholine receptor activation was critical for altering subsequent synapticplasticity. These results indicate that synaptic plasticity in stratum radiatum of CA1 can be homeostatically regulated by the cell-wide history of synaptic activity through a calcium-dependent but action potential-independent mechanis