Using Exploratory Data Analytics to Identify Deficiencies in mCherry Red Fluorescent Protein and Suggest Improvements

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Regulation of β cell glucokinase by S-nitrosylation and association with nitric oxide synthase

Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic β cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured β cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic β cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS

Structural Studies of Computationally Designed Inhibitors of HIV Gp41

Human immunodeficiency virus (HIV) infection is a major public health concern. New strategies that target viral entry hold promise for preventing infection or for enhancing the efficacy of existing anti-retroviral therapy. We have targeted the HIV protein gp41, required for initial membrane fusion and viral entry, for computational lead compound development and structural characterization. We have successfully purified an engineered version of this protein (gp41-5) that has an exposed patch that resembles the pre-fusion complex that is presumed to be an important intermediate in fusion. We crystallized this protein and collected a complete data set, however the crystals suffer from an unusual order-disorder pathology. Current work is identifying improved crystallization conditions that will allow determination of the crystal structure of gp41-5 with computationally identified compounds that inhibit viral entry

Structural Studies of Computationally Designed Inhibitors of HIV Gp41

Human immunodeficiency virus (HIV) infection is a major public health concern. New strategies that target viral entry hold promise for preventing infection or for enhancing the efficacy of existing anti-retroviral therapy. We have targeted the HIV protein gp41, required for initial membrane fusion and viral entry, for computational lead compound development and structural characterization. We have successfully purified an engineered version of this protein (gp41-5) that has an exposed patch that resembles the pre-fusion complex that is presumed to be an important intermediate in fusion. We crystallized this protein and collected a complete data set, however the crystals suffer from an unusual order-disorder pathology. Current work is identifying improved crystallization conditions that will allow determination of the crystal structure of gp41-5 with computationally identified compounds that inhibit viral entry

Marginal zone lymphomas in children and the young adult population; characterization of genetic aberrations by FISH and RT-PCR

Marginal zone lymphomas present rarely in children and young adults as either primary nodal or extranodal disease and have an excellent prognosis. To date, chromosomal aberrations have not been analyzed in the pediatric and young adult population. We undertook a study to analyze genetic alterations in nodal and extranodal marginal zone lymphomas in children and young adults using fluorescence in situ hybridization (FISH) and RT-PCR. These findings were correlated with clinical features at presentation and immunophenotype. Forty-one cases were identified meeting these criteria. The age range was 1.5-29 years old with 49% of the cases <18 years of age. 73% of the marginal zone lymphoma cases showed evidence of light chain restriction by immunohistochemistry or flow cytometry. CD43 was coexpressed in 83%. 85% of the marginal zone lymphoma cases tested showed evidence of immunoglobulin heavy chain gene rearrangement. Fifty-nine percent of the cases were nodal marginal zone lymphomas with a median age at presentation of 16 years and an M/F ratio of 7:1. Twenty-one percent of the nodal marginal zone lymphoma cases contained genetic aberrations. Seventeen percent contained trisomy 18 with one case containing an additional trisomy 3. A translocation of the immunoglobulin heavy chain gene to an unknown partner gene was present in one case. Forty-one percent of the cases were extranodal marginal zone lymphomas with a median age of 24 years and a M/F ratio of 1.4:1. Eighteen percent of the extranodal marginal zone lymphoma cases contained genetic aberrations. The t(14;18) involving the IGH and MALT1 genes was present in one case, tetraploidy was present in one case, and another case contained trisomy 3. Overall the incidence of genetic aberrations in marginal zone lymphomas in the pediatric and young adult population is low, but the aberrations seen are similar to those seen in the adult population

A search for heavy Kaluza-Klein electroweak gauge bosons at the LHC

The feasibility for the observation of a certain leptonic Kaluza-Klein (KK) hard process in {\em pp} interactions at the LHC is presented. Within the $S^1/Z_2$ TeV$^{-1}$ extra dimensional theoretical framework with the focus on the KK excitations of the Standard Model $\gamma$ and $Z^0$ gauge bosons, the hard-process, $f\bar f \to \sum_n\left(\gamma^*/Z^*\right)_n \to F \bar F$, has been used where $f$ is the initial state parton, $F$ the final state lepton and $\left(\gamma^*/Z^*\right)_{n}$ is the $n^{\rm th}$ KK excitation of the $\gamma/Z^0$ boson. For this study the analytic form for the hard process cross section has been independently calculated by the authors and has been implemented using the {\sc Moses} framework. The Moses framework itself, that has been written by the authors, was used as an external process within the {\sc Pythia} Monte Carlo generator which provides the phase space generation for the final state leptons and partons from the initial state hadrons, and the simulation of initial and final state radiation and hadronization. A brief discussion of the possibility for observing and identifying the unique signature of the KK signal given the current LHC program is also presented.Comment: 16 pages 10 figures, MCnet number: MCnet/10/06, Accepted by JHE

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

BACKGROUND: Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. RESULTS: Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. CONCLUSIONS: Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor

Considering patient safety in autonomous e-mental health systems - detecting risk situations and referring patients back to human care

Background: Digital health interventions can fill gaps in mental healthcare provision. However, autonomous e-mental health (AEMH) systems also present challenges for effective risk management. To balance autonomy and safety, AEMH systems need to detect risk situations and act on these appropriately. One option is sending automatic alerts to carers, but such 'auto-referral' could lead to missed cases or false alerts. Requiring users to actively self-refer offers an alternative, but this can also be risky as it relies on their motivation to do so. This study set out with two objectives. Firstly, to develop guidelines for risk detection and auto-referral systems. Secondly, to understand how persuasive techniques, mediated by a virtual agent, can facilitate self-referral. Methods: In a formative phase, interviews with experts, alongside a literature review, were used to develop a risk detection protocol. Two referral protocols were developed - one involving auto-referral, the other motivating users to self-refer. This latter was tested via crowd-sourcing (n = 160). Participants were asked to imagine they had sleeping problems with differing severity and user stance on seeking help. They then chatted with a virtual agent, who either directly facilitated referral, tried to persuade the user, or accepted that they did not want help. After the conversation, participants rated their intention to self-refer, to chat with the agent again, and their feeling of being heard by the agent. Results: Whether the virtual agent facilitated, persuaded or accepted, influenced all of these measures. Users who were initially negative or doubtful about self-referral could be persuaded. For users who were initially positive about seeking human care, this persuasion did not affect their intentions, indicating that a simply facilitating referral without persuasion was sufficient. Conclusion: This paper presents a protocol that elucidates the steps and decisions involved in risk detection, something that is relevant for all types of AEMH systems. In the case of self-referral, our study shows that a virtual agent can increase users' intention to self-refer. Moreover, the strategy of the agent influenced the intentions of the user afterwards. This highlights the importance of a personalised approach to promote the user's access to appropriate care.Interactive Intelligenc

Trends for Influenza-related Deaths during Pandemic and Epidemic Seasons, Italy, 1969–2001

During epidemics, excess deaths were similar in amplitude and time across 3 regions