Paclitaxel inhibits the activity and membrane localization of PKCα and PKCβI/II to elicit a decrease in stimulated calcitonin gene-related peptide release from cultured sensory neurons

Peripheral neuropathy is a dose-limiting and debilitating side effect of the chemotherapeutic drug, paclitaxel. Consequently, elucidating the mechanisms by which this drug alters sensory neuronal function is essential for the development of successful therapeutics for peripheral neuropathy. We previously demonstrated that chronic treatment with paclitaxel (3–5 days) reduces neuropeptide release stimulated by agonists of TRPV1. Because the activity of TRPV1 channels is modulated by conventional and novel PKC isozymes (c/nPKC), we investigated whether c/nPKC mediate the loss of neuropeptide release following chronic treatment with paclitaxel (300 nM; 3 and 5 days). Release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Following paclitaxel treatment, cultured dorsal root ganglia sensory neurons were stimulated with a c/nPKC activator, phorbol 12,13-dibutyrate (PDBu), or a TRPV1 agonist, capsaicin, in the absence and presence of selective inhibitors of conventional PKCα and PKCβI/II isozymes (cPKC). Paclitaxel (300 nM; 3 days and 5 days) attenuated both PDBu- and capsaicin-stimulated release in a cPKC-dependent manner. Under basal conditions, there were no changes in the protein expression, phosphorylation or membrane localization of PKC α, βI or βII, however, paclitaxel decreased cPKC activity as indicated by a reduction in the phosphorylation of cPKC substrates. Under stimulatory conditions, paclitaxel attenuated the membrane translocation of phosphorylated PKC α, βI and βII, providing a rationale for the attenuation in PDBu- and capsaicin-stimulated release. Our findings suggest that a decrease in cPKC activity and membrane localization are responsible for the reduction in stimulated peptide release following chronic treatment with paclitaxel in sensory neurons

The role of Protein Kinase C in the modulation of neuronal sensitivity and neurite morphology following treatment with paclitaxel in cultured sensory neurons

Indiana University-Purdue University Indianapolis (IUPUI)Paclitaxel is a chemotherapeutic drug that is used in the treatment of solid tumors including breast and ovarian cancer. However, a debilitating and severe side effect associated with paclitaxel treatment is peripheral neuropathy. Clinically, peripheral neuropathy is characterized by a gain and loss of sensory neuronal function with symptoms including burning pain, tingling and numbness, respectively. In addition, paclitaxel also elicits a reduction in the length of intraepidermal nerve fibers. Currently, there are no effective therapies to prevent or alleviate the symptoms of peripheral neuropathy. Further studies are needed to elucidate the mechanisms underlying the changes in neuronal function and neurite morphology induced by paclitaxel in order to develop therapeutics to address the symptoms of peripheral neuropathy. Our laboratory previously demonstrated that paclitaxel altered capsaicin stimulated release of calcitonin gene-related peptide (CGRP) from cultured sensory neurons, indicating that paclitaxel altered the function of the transient receptor potential vanilloid I (TRPV1) channels. Because protein kinase C (PKC) modulates the function of TRPV1, we questioned whether PKC mediated changes in neuronal sensitivity induced by chronic treatment with paclitaxel. We used the release of CGRP as an index of neuronal sensitivity. Our data show that paclitaxel decreased the activity and membrane localization of the conventional PKC isozymes, PKCα and PKCβI/II, to elicit a reduction in the release of CGRP from cultured sensory neurons. For our neurite morphology studies, we focused on the importance of the novel PKC isozyme, PKCε, in mediating the changes in neurite morphology induced by treatment with paclitaxel because studies have demonstrated that PKCε is important for enhancing neurite outgrowth. Since our preliminary data showed a correlative reduction in PKCε protein expression and neurite length and branching following treatment with paclitaxel, we questioned whether loss of PKCε mediated altered neurite morphology induced by paclitaxel. Unexpectedly, we found that downregulation of PKCε did not exacerbate the reduction in neurite length and branching induced by paclitaxel. Our work highlights the significance of PKCα and PKCβI/II as critical mediators of changes in neuronal sensitivity induced by paclitaxel and illuminates our understanding of the mechanisms underlying the neurotoxic effects of paclitaxel on sensory neuronal function

The Missing Piece: Drought Impacts Monitoring Report from a Workshop in Tucson, AZ MARCH 5-6, 2013

Based on a shared interest to better understand the impacts of drought and the potential utility of using drought impacts reporting as a tool for monitoring conditions, researchers from the Carolinas RISA (Dow, Lackstrom, and Brennan), the Climate Assessment for the Southwest (Crimmins and Ferguson), and the Southwest Climate Science Center (Meadow) decided to convene a workshop in Tucson in March 2013. The primary goal was to assemble a small group of university and agency scientists involved with drought impacts monitoring to discuss opportunities and barriers associated with drought impacts reporting, recommend best practices for implementing a drought impacts reporting system, and develop a path forward for addressing or overcoming barriers. The longer-term objective of the initial meeting was to explore the feasibility of creating a community of practice that could share information and integrate activities related to drought impacts research and reporting

Intervening to eliminate the centre-effect variation in home dialysis use: protocol for Inter-CEPt - a sequential mixed-methods study designing an intervention bundle

Introduction: Use of home dialysis by centres in the UK varies considerably and is decreasing despite attempts to encourage greater use. Knowing what drives this unwarranted variation requires in-depth understanding of centre cultural and organisational factors and how these relate to quantifiable centre performance, accounting for competing treatment options. This knowledge will be used to identify components of a practical and feasible intervention bundle ensuring this is realistic and cost-effective. Methods and analysis: Underpinned by the non-adoption, abandonment, scale-up, spread and sustainability framework, our research will use an exploratory sequential mixed-methods approach. Insights from multisited focused team ethnographic and qualitative research at four case study sites will inform development of a national survey of 52 centres. Survey results, linked to patient-level data from the UK Renal Registry, will populate a causal graph describing patient and centre-level factors, leading to uptake of home dialysis and multistate models incorporating patient-level treatment modality history and mortality. This will inform a contemporary economic evaluation of modality cost-effectiveness that will quantify how modification of factors facilitating home dialysis, identified from the ethnography and survey, might yield the greatest improvements in costs, quality of life and numbers on home therapies. Selected from these factors, using the capability, opportunity and motivation for behaviour change framework (COM-B) for intervention design, the optimal intervention bundle will be developed through workshops with patients and healthcare professionals to ensure acceptability and feasibility. Patient and public engagement and involvement is embedded throughout the project. Ethics and dissemination: Ethics approval has been granted by the Health Research Authority reference 20-WA-0249. The intervention bundle will comprise components for all stake holder groups: commissioners, provider units, recipients of dialysis, their caregivers and families. To reache all these groups, a variety of knowledge exchange methods will be used: short guides, infographics, case studies, National Institute for Health and Care Excellence guidelines, patient conferences, ‘Getting it Right First Time’ initiative, Clinical Reference Group (dialysis)

Solution structure of the SGTA dimerisation domain and investigation of its interactions with the ubiquitin-like domains of BAG6 and UBL4A

BACKGROUND: The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. METHODOLOGY AND PRINCIPAL FINDINGS: SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. SIGNIFICANCE: This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex

A Bad Air Day in Houston

© Copyright 2005 American Meteorological Society (AMS). Permission to use figures, tables, and brief excerpts from this work in scientific and educational works is hereby granted provided that the source is acknowledged. Any use of material in this work that is determined to be “fair use” under Section 107 of the U.S. Copyright Act September 2010 Page 2 or that satisfies the conditions specified in Section 108 of the U.S. Copyright Act (17 USC §108, as revised by P.L. 94-553) does not require the AMS’s permission. Republication, systematic reproduction, posting in electronic form, such as on a web site or in a searchable database, or other uses of this material, except as exempted by the above statement, requires written permission or a license from the AMS. Additional details are provided in the AMS Copyright Policy, available on the AMS Web site located at (https://www.ametsoc.org/) or from the AMS at 617-227-2425 or [email protected] case study from the Texas Air Quality Study 2000 field campaign illustrates the complex interaction of meteorological and chemical processes that produced a high-pollution event in the Houston area on 30 August 2000. High 1-h ozone concentrations of nearly 200 ppb were measured near the surface, and vertical profile data from an airborne differential-absorption lidar (DIAL) system showed that these high-ozone concentrations penetrated to heights approaching 2 km into the atmospheric boundary layer. This deep layer of pollution was transported over the surrounding countryside at night, where it then mixed out the next day to become part of the rural background levels. These background levels thus increased during the course of the multiday pollution episode. The case study illustrates many processes that numerical forecast models must faithfully represent to produce accurate quantitative predictions of peak pollutant concentrations in coastal locations such as Houston. Such accurate predictions will be required for useful air-quality forecasts for urban areas.Southern Oxidant Study Texas Commission on Environmental Qualit

Rehabilitation aimed at improving outdoor mobility for people after stroke: a multicentre randomised controlled study (the Getting out of the House Study)

Background: One-third of stroke patients are dependent on others to get outside their homes. This can cause people to become housebound, leading to increased immobility, poor health, isolation and misery. There is some evidence that outdoor mobility rehabilitation can reduce these limitations. Objective: To test the clinical effectiveness and cost-effectiveness of an outdoor mobility rehabilitation intervention for stroke patients. Design: Multicentre, parallel-group randomised controlled trial, with two groups allocated at a 1 : 1 ratio plus qualitative participant interviews. Setting: Fifteen UK NHS stroke services throughout England, Scotland and Wales. Participants: A total of 568 stroke patients who wished to get out of the house more often, mean age of 71 years: 508 reached the 6-month follow-up and 10 were interviewed. Intervention: Control was delivered prior to randomisation to all participants, and consisted of verbal advice and transport and outdoor mobility leaflets. Intervention was a targeted outdoor mobility rehabilitation programme delivered by 29 NHS therapists to 287 randomly chosen participants for up to 12 sessions over 4 months. Main outcome measures: Primary outcome was participant health-related quality of life, measured by the Short Form questionnaire-36 items, version 2 (Social Function domain), 6 months after baseline. Secondary outcomes were functional ability, mobility, number of journeys (from monthly travel diaries), satisfaction with outdoor mobility (SWOM), psychological well-being and resource use [health care and Personal Social Services (PSS)] 6 months after baseline. Carer well-being was recorded. All outcome measures were collected by post and repeated 12 months after baseline. Outcomes for the groups were compared using statistical significance testing and adjusted for multiple membership to account for the effect of multiple therapists at different sites. Interviews were analysed using interpretive phenomenology to explore confidence. Results: A median of seven intervention sessions [interquartile range (IQR) 3–7 sessions], median duration of 369 minutes (IQR 170–691.5 minutes) per participant was delivered. There was no significant difference between the groups on health-related quality of life (social function). There were no significant differences between groups in functional ability, psychological well-being or SWOM at 6- or 12-month follow-ups. There was a significant difference observed for travel journeys with the intervention group being 42% more likely to make a journey compared with the control group [rate ratio 1.42, 95% confidence interval (95% CI) 1.14 to 1.67] at 6 months and 76% more likely (rate ratio 1.76, 95% CI 1.36 to 1.95) at 12 months. The number of journeys was affected by the therapist effect. The mean incremental cost (total NHS and PSS cost) of the intervention was £3413.75 (95% CI –£448.43 to £7121.00), with an incremental quality-adjusted life-year gain of –0.027 (95% CI –0.060 to 0.007) according to the European Quality of Life-5 Dimensions and –0.003 (95% CI –0.016 to 0.006) according to the Short Form questionnaire-6 Dimensions. At baseline, 259 out of 281 (92.2%) participants in the control group were dissatisfied with outdoor mobility but at the 6-month assessment this had reduced to 77.7% (181/233), a 15% reduction. The corresponding reduction in the intervention group was slightly greater (21%) than 268 out of 287 (93.4%) participants dissatisfied with outdoor mobility at baseline to 189 out of 261 (72.4%) at 6 months. Participants described losing confidence after stroke as being detrimental to outdoor mobility. Recruitment and retention rates were high. The intervention was deliverable by the NHS but had a neutral effect in all areas apart from potentially increasing the number of journeys. This was dependent on the therapist effect, meaning that some therapists were more successful than others. The control appeared to affect change. Conclusions: The outdoor mobility intervention provided in this study to these stroke patients was not clinically effective or cost-effective. However, the provision of personalised information and monthly diaries should be considered for all people who wish to get out more

BUILDING A SUSTAINABLE NETWORK OF DROUGHT COMMUNITIES

The first step in managing large-scale (national) collaborations and networks is to consider and address how a group and a potential partnership may match up (Luther, 2005). To explore this concept and many other collaborative concepts, the National Integrated Drought Information System (NIDIS) hosted a workshop, “Building a Sustainable Network of Drought Communities,” which was facilitated by the National Drought Mitigation Center (NDMC) in Chicago, IL, June 8-9, 2011. The workshop explored current examples of good communication and lessons learned within the realm of drought planning in order to address a future NIDIS Engaging Preparedness Communities (EPC) working group that is solution-focused and collaborative. With the diversity and experience of the participants at this meeting, a wealth of good practices or lessons learned in drought planning, preparedness, and general stakeholder engagement set the pathway for building a sustainable community of drought practitioners. In his opening remarks, NIDIS Director Roger Pulwarty noted that adaptive institutions can show robustness in the following ways: Levels of alertness—monitoring the external world for early warning signs that key assumptions are likely to verify/fail and a commitment to rigorous monitoring of performance; Agility—the ability to react to early warning signs of problems or opportunities; flow of knowledge across components, and to adjust strategies and tactics rapidly to meet changes in the environment; and Alignment—the ability to align the whole organization (and partners) to its mission-policies and practices that give rise to failures/successes. Through an interactive workshop format that used Appreciative Inquiry (framing breakout sessions on success), the group was able to effectively discuss topics such as: • Integrating Planning Efforts • Planning Under Uncertainty • Evaluating, Assessing, and Updating Drought Plans • Leveraging Resources for Risk Management • Implementing Plans and Planning Information • Synthesizing Success Stories and Lessons Learned • Creating a Sustainable Network of Drought Professionals The most common themes resulting from the workshop included: • Importance of networking and collaboration—this is a necessity. Figuring out how to make it seamless is the main goal that the NIDIS EPC Community should foster. Good communication is the key among the drought practitioners and their stakeholders. • Celebrate success—in this future drought network, successes related to drought efforts should be highlighted within the community and to the public. This will help drive future positive interactions and collaborations. It also gives the community a sense of pride. • “Stakeholder Buy-In”—why should stakeholders stay engaged in an ongoing drought community? Especially when there is no drought? Again, good communication and collaborations with other multi-hazard, sustainability, and natural resources planning efforts will help keep drought a priority. • Economic, environmental, and social aspects of planning for drought—these should always be considered. This was a recurrent theme in the workshop. • Planners should not “reinvent the wheel”—planners involved in climate adaptation work can and should reference the best drought planning resources and case studies to help them incorporate drought in their overall planning efforts. • “Have a plan for the plan”—how and who will make it happen? What kind of leadership is needed within the NIDIS EPC community to track its progress and success? • Sharing of resources—as budgets become slimmer, a central location of available resources and the sharing of resources in the area of drought preparedness and mitigation is necessary. Communication regarding these potential resources should also be integrated into this NIDIS EPC community. Since the occurrence of the workshop, several EPC-related activities have taken place, including a webinar in December 2011. This workshop report and additional EPC updates will be placed on the U.S. Drought portal (www.drought.gov). Currently, the American Planning Association (APA), NIDIS and the NDMC are collaborating to produce a Planning Advisory Service (PAS) Report to connect drought mitigation resources with the planning practices of local, regional, tribal and state governments. This builds on the work of the APA’s Hazard Planning Center, which produced a similar PAS Report (sponsored by FEMA) on how to integrate multi-hazard planning into planning practices. In May 2012: The APA’s drought planning project webpage went live and can be found at: http://www.planning.org/research/drought/index.ht

Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmi

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions