Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.

Reducing opioid related deaths for individuals who are at high risk of death from overdose: a co-production study with people housed within prison and hostel accommodation during Covid-19.

Background: A record number of Opioid-related deaths occurred in Northern Ireland in 2021 and it is acknowledged that the Covid-19 pandemic compounded drugs related deaths crisis. This co-production study set out to refine the design of a wearable device for Opioid users to detect and subsequently prevent a potential overdose situation. Method: Purposive sampling was used to recruit people who had substance use disorders and were living in a hostel and prison during the Covid-19 pandemic. Principles of co-production influenced the study, which encompassed a focus group phase and a wearable phase. The initial phase included three focus groups with participants who inject Opioids and one focus group with workers from a street injector support service. During the wearable phase, the participant group tested the feasibility of the wearable technology in a controlled environment. This included testing the transferability of data from the device to a backend server on the cloud. Results: All focus group participants expressed an interest in the wearable technology when it was presented to them and agreed, that in principle, such a device would be extremely beneficial to help reduce the risk of overdose within the active drug using community. Participants outlined factors which would help or hinder the design of this proposed device and their decision to wear it, if it were readily available to them. Findings from wearable phase indicated that it was feasible to use a wearable device for monitoring Opioid users' biomarkers remotely. The provision of information regarding the specific functionality of the device was considered key and could be disseminated via front line services. The data acquisition and transfer process would not be a barrier for future research. Conclusion: Understanding the benefit and disadvantages of technologies such as a wearable device to prevent Opioid-related deaths will be critical for mitigating the risk of overdose for people who use Heroin. It was also clear that this would be particularly relevant during Covid-19 lock-down periods, when the effects of the pandemic further exacerbated the isolation and solitude experienced by people who use Heroin.

Impact of the COVID-19 pandemic on antimicrobial stewardship support for general practices in England: a qualitative interview study.

BACKGROUND: In England, clinical commissioning group (CCG; now replaced by Integrated Care Systems [ICSs]) and primary care network (PCN) professionals support primary care prescribers to optimise antimicrobial stewardship (AMS). AIM: To explore views and experiences of CCG and PCN staff in supporting AMS, and the impact of COVID-19 on this support. DESIGN & SETTING: Qualitative interview study in primary care in England. METHOD: Semi-structured interviews with staff from CCG and PCNs responsible for AMS were conducted at two timepoints via telephone. These were audio-recorded, transcribed, and analysed thematically. RESULTS: Twenty-seven interviews were conducted with 14 participants (nine CCG, five PCN) in December 2020-January 2021 and February-May 2021. The study found that AMS support was (1) deprioritised in order to keep general practice operational and deliver COVID-19 vaccines; (2) disrupted as social distancing made it harder to build relationships, conduct routine AMS activities, and challenge prescribing decisions; and (3) adapted, with opportunities identified for greater use of technology and changing patient and public perceptions of viruses and self-care. It was also found that resources to support AMS were valued if they were both novel, to counter AMS 'fatigue', and sufficiently familiar to fit with existing and/or future AMS. CONCLUSION: AMS needs to be reprioritised in general practice in the post-pandemic era and within the new ICSs in England. This should include interventions and strategies that combine novel elements with already familiar strategies to refresh prescribers' motivation and opportunities for AMS. Behaviour change interventions should be aimed at improving the culture and processes for how PCN pharmacists voice concerns about AMS to prescribers in general practice and take advantage of the changed patient and public perceptions of viruses and self-care.

Review: Inelastic Constitutive Modeling: Polycrystalline Materials.

This article provides a literature review that details the development of inelastic constitutive modeling as it relates to polycrystalline materials. This review distinguishes between inelastic constitutive models that account for nonlinear behavior at the microstructural level, time-independent classic plasticity models, and time-dependent unified models. Particular emphasis is placed on understanding the underlying theoretical framework for unified viscoplasticity models where creep and classical plasticity behavior are considered the result of applied boundary conditions instead of separable rates representing distinct physical mechanisms. This article establishes a clear understanding of the advantages of the unified approach to improve material modeling. This review also discusses recent topics in constitutive modeling that offer new techniques that bridge the gap between the microstructure and the continuum.

The impacts and implications of the community face mask use during the Covid-19 pandemic: A qualitative narrative interview study.

INTRODUCTION: A range of nonpharmaceutical public health interventions has been introduced in many countries following the rapid spread of Covid-19 since 2020, including recommendations or mandates for the use of face masks or coverings in the community. While the effectiveness of face masks in reducing Covid-19 transmission has been extensively discussed, scant attention has been paid to the lived experience of those wearing face masks. METHOD: Drawing on 40 narrative interviews with a purposive sample of people in the United Kingdom, with a particular focus on marginalised and minoritized groups, our paper explores experiences of face mask use during the pandemic. RESULTS: We find that face masks have a range of societal, health and safety impacts, and prompted positive and negative emotional responses for users. We map our findings onto Lorenc and Oliver's framework for intervention risks. We suggest that qualitative data offer particular insights into the experiences of public health interventions, allowing the potential downsides and risks of interventions to be more fully considered and informing public health policies that might avoid inadvertent harm, particularly towards marginalised groups. PATIENT OR PUBLIC CONTRIBUTION: The study primarily involved members of the public in the conduct of the research, namely through participation in interviews (email and telephone). The conception for the study involved extensive discussions on social media with a range of people, and we received input and ideas from presentations we delivered on the preliminary analysis.

Implementing antibiotic stewardship in high-prescribing English general practices: a mixed-methods study.

BACKGROUND: Trials have identified antimicrobial stewardship (AMS) strategies that effectively reduce antibiotic use in primary care. However, many are not commonly used in England. The authors co-developed an implementation intervention to improve use of three AMS strategies: enhanced communication strategies, delayed prescriptions, and point-of-care C-reactive protein tests (POC-CRPTs). AIM: To investigate the use of the intervention in high-prescribing practices and its effect on antibiotic prescribing. DESIGN AND SETTING: Nine high-prescribing practices had access to the intervention for 12 months from November 2019. This was primarily delivered remotely via a website with practices required to identify an 'antibiotic champion'. METHOD: Routinely collected prescribing data were compared between the intervention and the control practices. Intervention use was assessed through monitoring. Surveys and interviews were conducted with professionals to capture experiences of using the intervention. RESULTS: There was no evidence that the intervention affected prescribing. Engagement with intervention materials differed substantially between practices and depended on individual champions' preconceptions of strategies and the opportunity to conduct implementation tasks. Champions in five practices initiated changes to encourage use of at least one AMS strategy, mostly POC-CRPTs; one practice chose all three. POC-CRPTs was used more when allocated to one person. CONCLUSION: Clinicians need detailed information on exactly how to adopt AMS strategies. Remote, one-sided provision of AMS strategies is unlikely to change prescribing; initial clinician engagement and understanding needs to be monitored to avoid misunderstanding and suboptimal use.

Mental health service accessibility, development and research priority setting in Cambodia - a post-conflict nation.

BACKGROUND: The limited health and social care infrastructure that existed in the 1970s in Cambodia was destroyed due to the Khmer Rouge. Mental Health service infrastructures have developed in Cambodia in the last twenty five years, however, they have been shaped significantly by very limited funding being made available for human resources, support services and research. The lack of research on Cambodia's mental health systems and services is a significant barrier to the development of evidence-based mental health policies and practice. In order to address this barrier, effective research and development strategies are needed in Cambodia, which are based on locally well-informed research priorities. There are many possibilities for mental health research in LMIC countries such as Cambodia, therefore focused research priorities in these areas are needed to guide future research investment. This paper is the result of the development of international collaborative workshops, which focused on service mapping and research priority setting in the field of mental health in Cambodia. METHODS: A nominal group technique was used to gather ideas and insights from a range of key mental health service stakeholders in Cambodia. RESULTS: The key issues in service provisions for people with mental health issues and disorders, the interventions and programmes of support available, and currently needed, were identified. This paper also identifies five key mental health research priority areas which could form the basis for effective mental health research and development strategies in Cambodia. CONCLUSION: There is a clear need for the Cambodian government to devise a clear policy framework for health research. This framework could focus on the five research domains identified in this paper and could be incorporated within its National Health Strategic plans. The implementation of this approach would likely lead to the development of an evidence base which would allow the development of effective and sustainable strategies for mental health problem prevention and intervention. This would also contribute to promote the Cambodian government's capacity to take the deliberate, concrete, and targeted steps necessary to address the complex mental health needs of its population.

SEM and TEM data of nuclear graphite and glassy carbon microstructures.

Micrographs of multiple nuclear graphite grades were captured using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), complementing the data contained in the related manuscript, "A multi-technique image library of nuclear graphite microstructures of historical and modern grades." The SEM micrographs show the differences among filler particles, binder, and thermal cracks contained in nuclear graphite. This library of microstructures serves as a baseline of as-received material and enables understanding the phases and differences between nuclear grades. TEM micrographs included in this manuscript elucidate the content of a common material contained in the binder phase known as quinoline insoluble (QI) particles. These particles are a phase of graphite that can be used as a forensic fingerprint of the neutron irradiation effects in graphite. The manuscript also contains some data of glassy carbon, an allotrope of carbon that shares similarities with some of the chaotic structures in nuclear graphite. Combined, these micrographs provide a detailed overview of the microstructures of various graphite grades prior to neutron irradiation.

Experiences of face mask use during the COVID-19 pandemic: A qualitative study.

The use of face masks and coverings has been a central component of efforts to mitigate the impact of the COVID-19 pandemic and has been legally mandated in some countries. Most academic studies to date, however, have focussed primarily on its effectiveness in reducing SARS-CoV-2 transmission, largely neglecting the social dimensions of mask mandates. In this narrative interview-based study, we consider experiences of face masks, with a particular focus on groups considered to be at a potential disadvantage from compulsory masking. Drawing on 40 telephone, video-call and e-mail interviews, we highlight the impact of inconsistent communication and the notion of mask wearing as an act of altruism on participants' experiences. In particular, we show how intolerance towards individuals who did not wear masks could result in stigma and exclusion, regardless of the legitimacy of their reasons. We suggest that more is needed to mitigate the 'dark side' of discourses of collective effort and altruism at a time of societal stress and fracture, and to account for the needs and interests of groups for whom compulsory masking may result in further marginalisation.

'Getting involved in research': a co-created, co-delivered and co-analysed course for those with lived experience of health and social care services.

BACKGROUND: 'Getting Involved in Research' was co-created and delivered by a multi-organisational group to provide an accessible introduction to research for those with lived experience of health and social care services. METHOD: The evaluation of participants' perceptions adopted an exploratory mixed method research design and aimed to gather data to provide an in-depth understanding of the participants' experience of 'Getting Involved in Research' through the co-researchers' analysis of qualitative data using Participatory Theme Elicitation (PTE). PTE was used with the qualitative data to promote co-analysis by the course development group; analyses from an independent academic was also used to further validate the method of PTE. RESULTS: Thirty-five participants in total participated in 'Getting Involved in Research'. Age ranges varied from 19 to 73 years old. Participants were predominately female (n = 24), five males participated (n = 5) and there was one participant who identified as non-binary (n = 1). Six core themes were identified using the PTE approach: (1) A Meaningful Participatory Approach (2) Increasing the Confidence of Participants (3) Interactive Online Format (4) An Ambient Learning Environment (5) A Desire for Future Courses (6) A Balance of Course Content and Discussion. Participants in 'Getting Involved in Research' reported that the content of the training was applicable, relevant, fostered awareness of research methods and anticipated that it would support their involvement in research. CONCLUSION: 'Getting Involved in Research' has contributed innovatively to the evidence base for how to engage with and motivate those who have experience of health and social care to become actively involved in research. This study demonstrates that 'Getting Involved in Research' may be helpful to train those with lived experience and their care partners however, further research following up on the application of the course learning would be required to ascertain effectiveness. FUTURE DIRECTIONS: Future research should explore methods to apply research skills in practice to further develop participants' confidence in using the skills gained through 'Getting Involved in Research'.

The aim of this study was to gather information to help us understand the experience of participants' undertaking a research course called 'Getting Involved in Research'. A group of individuals including those with lived experience of health and social care services, academics, community and voluntary sector workers and a representative from the Department of Health in Northern Ireland worked together to develop a course to encourage and support people to engage with research. The 'Getting Involved in Research', course was designed to provide an understandable introduction to research for those people with lived experience of health and social care. We did this because research in health and social care sector should involve the patients and public who it is intended to help. The evaluation of the course had two distinct phases; we asked course participants to complete a survey before and after the course (pre- and post-course survey) and also asked them to complete a journal reflecting on their experiences after each lecture. This paper gives an overview of the profile of course participants and their responses to the survey questions. The survey answers were analysed using an approach to analysing information which encourages involvement from people with a range of experience of research methods, (known as Participatory Theme Elicitation). Thirty-five participants in total participated in 'Getting Involved in Research'. Age ranges varied from 19 to 73 years old. Participants were predominately female (n = 24), five males participated (n = 5) and there was one participant who identified as non-binary (n = 1). Participants in 'Getting Involved in Research' reported that the content of the course was relevant, encouraged awareness of research methods and encourage their future involvement in research.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein.

ß2-adrenoreceptor (ß2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of ß2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

What counts as a voiceable concern in decisions about speaking out in hospitals: A qualitative study.

OBJECTIVES: Those who work in health care organisations are a potentially valuable source of information about safety concerns, yet failures of voice are persistent. We propose the concept of 'voiceable concern' and offer an empirical exploration. METHODS: We conducted a qualitative study involving 165 semi-structured interviews with a range of staff (clinical, non-clinical and at different hierarchical levels) in three hospitals in two countries. Analysis was based on the constant comparative method. RESULTS: Our analysis shows that identifying what counts as a concern, and what counts as a occasion for voice by a given individual, is not a straightforward matter of applying objective criteria. It instead often involves discretionary judgement, exercised in highly specific organisational and cultural contexts. We identified four influences that shape whether incidents, events and patterns were classified as voiceable concerns: certainty that something is wrong and is an occasion for voice; system versus conduct concerns, forgivability and normalisation. Determining what counted as a voiceable concern is not a simple function of the features of the concern; also important is whether the person who noticed the concern felt it was voiceable by them. CONCLUSIONS: Understanding how those who work in health care organisations come to recognise what counts as a voiceable concern is critical to understanding decisions and actions about speaking out. The concept of a voiceable concern may help to explain aspects of voice behaviour in organisations as well as informing interventions to improve voice.

Freely Available Virtual Reality Experiences as Tools to Support Mental Health Therapy: a Systematic Scoping Review and Consensus Based Interdisciplinary Analysis.

The primary purpose of this article is to review the potential therapeutic value of freely available VR content as an addition to the practitioners 'toolkit'. Research has shown that virtual reality (VR) may be useful to extend existing guided imagery-based practices found in traditional mental health therapy. However, the use of VR technology within routine mental health practice remains low, despite recent reductions in equipment costs. A systematic scoping review and interdisciplinary analysis of freely available VR experiences was performed across two popular online databases (SteamVR and Oculus.com). A total of 1785 experiences were retrieved and screened for relevance with 46 meeting the inclusion criteria. VR content was then reviewed for potential therapeutic value by an interdisciplinary panel with experience across a number of therapeutic interventions including cognitive behavioural therapy, Rogerian counselling, mindfulness-based therapies. and family therapy. Eleven (22%) of the 50 freely available VR experiences were reported to have therapeutic potential as tools to support routine mental health therapy. These included support with the following mental health issues-low mood, social anxiety, stress reduction and fear of heights. Guidance of a qualified mental health practitioner was recommended in all cases to maximise the benefit of the VR experiences retrieved. While the quality is variable, freely available VR experiences may contain valuable content that could support mental health therapy. This includes as a homework activity or as an initial setting for case formulation and behavioural experiments.

Impact of the COVID-19 Pandemic on Community Antibiotic Prescribing and Stewardship: A Qualitative Interview Study with General Practitioners in England.

The COVID-19 pandemic has had a profound impact on the delivery of primary care services. We aimed to identify general practitioners' (GPs') perceptions and experiences of how the COVID-19 pandemic influenced antibiotic prescribing and antimicrobial stewardship (AMS) in general practice in England. Twenty-four semi-structured interviews were conducted with 18 GPs at two time-points: autumn 2020 (14 interviews) and spring 2021 (10 interviews). Interviews were audio-recorded, transcribed and analysed thematically, taking a longitudinal approach. Participants reported a lower threshold for antibiotic prescribing (and fewer consultations) for respiratory infections and COVID-19 symptoms early in the pandemic, then returning to more usual (pre-pandemic) prescribing. They perceived the pandemic as having had less impact on antibiotic prescribing for urinary and skin infections. Participants perceived the changing ways of working and consulting (e.g., proportions of remote and in-person consultations) in addition to changing patient presentations and GP workloads as influencing the fluctuations in antibiotic prescribing. This was compounded by decreased engagement with, and priority of, AMS due to COVID-19-related urgent priorities. Re-engagement with AMS is needed, e.g., through reviving antibiotic prescribing feedback and targets/incentives. The pandemic disrupted, and required adaptations in, the usual ways of working and AMS. It is now important to identify opportunities, e.g., for re-organising ways of managing infections and AMS in the future.

Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori VacA is a secreted toxin that assembles into water-soluble oligomeric structures and forms anion-selective membrane channels. Acidification of purified VacA enhances its activity in cell culture assays. Sites of protomer-protomer contact within VacA oligomers have been identified by cryoelectron microscopy, and in the current study, we validated several of these interactions by chemical cross-linking and mass spectrometry. We then mutated amino acids at these contact sites and analyzed the effects of the alterations on VacA oligomerization and activity. VacA proteins with amino acid charge reversals at interprotomer contact sites retained the capacity to assemble into water-soluble oligomers and retained cell-vacuolating activity. Introduction of paired cysteine substitutions at these sites resulted in formation of disulfide bonds between adjacent protomers. Negative-stain electron microscopy and single-particle two-dimensional class analysis revealed that wild-type VacA oligomers disassemble when exposed to acidic pH, whereas the mutant proteins with paired cysteine substitutions retain an oligomeric state at acidic pH. Acid-activated wild-type VacA caused vacuolation of cultured cells, whereas acid-activated mutant proteins with paired cysteine substitutions lacked cell-vacuolating activity. Treatment of these mutant proteins with both low pH and a reducing agent resulted in VacA binding to cells, VacA internalization, and cell vacuolation. Internalization of a nonoligomerizing mutant form of VacA by host cells was detected without a requirement for acid activation. Collectively, these results enhance our understanding of the molecular interactions required for VacA oligomerization and support a model in which toxin activity depends on interactions of monomeric VacA with host cells.

Development of an intervention to support the implementation of evidence-based strategies for optimising antibiotic prescribing in general practice.

BACKGROUND: Trials show that antimicrobial stewardship (AMS) strategies, including communication skills training, point-of-care C-reactive protein testing (POC-CRPT) and delayed prescriptions, help optimise antibiotic prescribing and use in primary care. However, the use of these strategies in general practice is limited and inconsistent. We aimed to develop an intervention to enhance uptake and implementation of these strategies in primary care. METHODS: We drew on the Person-Based Approach to develop an implementation intervention in two stages. (1) Planning and design: We defined the problem in behavioural terms drawing on existing literature and conducting primary qualitative research (nine focus groups) in high-prescribing general practices. We identified 'guiding principles' with intervention objectives and key features and developed logic models representing intended mechanisms of action. (2) Developing the intervention: We created prototype intervention materials and discussed and refined these with input from 13 health professionals and 14 citizens in two sets of design workshops. We further refined the intervention materials following think-aloud interviews with 22 health professionals. RESULTS: Focus groups highlighted uncertainties about how strategies could be used. Health professionals in the workshops suggested having practice champions, brief summaries of each AMS strategy and evidence supporting the AMS strategies, and they and citizens gave examples of helpful communication strategies/phrases. Think-aloud interviews helped clarify and shorten the text and user journey of the intervention materials. The intervention comprised components to support practice-level implementation: antibiotic champions, practice meetings with slides provided, and an 'implementation support' website section, and components to support individual-level uptake: website sections on each AMS strategy (with evidence, instructions, links to electronic resources) and material resources (patient leaflets, POC-CRPT equipment, clinician handouts). CONCLUSIONS: We used a systematic, user-focussed process of developing a behavioural intervention, illustrating how it can be used in an implementation context. This resulted in a multicomponent intervention to facilitate practice-wide implementation of evidence-based strategies which now requires implementing and evaluating. Focusing on supporting the uptake and implementation of evidence-based strategies to optimise antibiotic use in general practice is critical to further support appropriate antibiotic use and mitigate antimicrobial resistance.

The role of the informal and formal organisation in voice about concerns in healthcare: A qualitative interview study.

The importance of employee voice-speaking up and out about concerns-is widely recognised as fundamental to patient safety and quality of care. However, failures of voice continue to occur, often with disastrous consequences. In this article, we argue that the enduring sociological concepts of the informal organisation and formal organisation offer analytical purchase in understanding the causes of such problems and how they can be addressed. We report a qualitative study involving 165 interviews across three healthcare organisations in two high-income countries. Our analysis emphasises the interdependence of the formal and informal organisation. The formal organisation describes codified and formalised elements of structures, procedures and processes for the exercise of voice, but participants often found it frustrating, ambiguous, and poorly designed. The informal organisation-the informal practices, social connections, and methods for making decisions that are key to coordinating organisational activity-could facilitate voice through its capacity to help people to understand complex processes, make sense of their concerns, and frame them in ways likely to prompt an appropriate organisational response. Sometimes the informal organisation compensated for gaps, ambiguities and inconsistencies in formal policies and systems. At the same time, the informal organisation had a dark side, potentially subduing voice by creating informal hierarchies, prioritising social cohesion, and providing opportunities for retaliation. The formal and the informal organisation are not exclusive or independent: they interact with and mutually reinforce each other. Our findings have implications for efforts to improve culture and processes in relation to voice in healthcare organisations, pointing to the need to address deficits in the formal organisation, and to the potential of building on strengths in the informal organisation that are crucial in supporting voice.

Optimization of small-scale sample preparation for high-throughput OpenArray analysis.

OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.

Delineation of the pH-Responsive Regulon Controlled by the Helicobacter pylori ArsRS Two-Component System.

Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site-specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ΔarsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins (including sabA, alpA, alpB, hopD [labA], and horA). When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS-mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.