Indirect analysis of oligonucleotides using cleavable small molecule mass tags with detection by mass spectrometry

In the 1990s, siRNAs and microRNAs were discovered to be naturally occurring genetic regulators. This provided a new potential mechanism of action for drugs with applicability to a wide range of therapeutic areas. Consequently, a substantial increase into oligonucleotide research has occurred, leading to the need for improved and novel techniques for their analysis. Standard methods of oligonucleotide analysis are based on hybridisation assays with analysis via detection probes labelled with fluorescent tags. However, multiplexing potential is limited due to the broad, and thus often overlapping, signals emitted.An alternative to labelling detection probes with fluorescent tags is to use cleavable small molecule mass tags with detection by mass spectrometry. Herein, a self-reporting detection probe was designed for use in a hybridisation assay for indirect oligonucleotide detection via cleavable small molecule mass tags. The self-reporting detection probe contains an analyte complementary region and a reporter region. The reporter region is a custom designed DNA/RNA chimeric nucleotide sequence. The ribose-phosphate backbone is used as a built-in enzyme cleavable linker, generating small nucleotide products upon cleavage by RNase A. These nucleotides can then serve as mass tags for indirect detection of oligonucleotides. This system avoids the need to design or synthesise a cleavable linker by exploiting the properties of the RNA molecule. This approach was used for the successful detection of a synthetic microRNA and the multiplexing potential was demonstrated by the simultaneous detection of two RNAs

Contributing and constraining factors to collaborative land use planning: Consequences of proposed housing development in and around the New River Gorge National River

In recent years, population increases in gateway communities has led to developers wanting to development adjacent to public lands. Although gateway community developments provides economic benefits such as increases in jobs and tax revenues, the population increase can cause a negative change in the landscape of the public land. As a result, public land managers are approaching more collaborative ways to engage in land use planning.;The purpose of this study was to assess the level of support for collaborative land use planning in and around the New River Gorge National River in Fayetteville, WV, particularly focused on the context of rapid population growth in the Fayetteville area. This qualitative analysis used a triangulation method consisting of newspaper articles, documents, and 17 semi-structured interviews of individuals most directly involved in the New River Gorge housing development issue. From that information, we were able to identify an increased need to participate in collaborative land use planning both in the National Park Service (NPS) and stakeholders. We noticed several constraints to collaborative land use planning such as human and financial capacity and community resistance as well as how groups such as the Transition Team and the New River Working Group can better enhance collaborative land use planning efforts. Finally, we identified several keys to successful collaborative land use planning such as building relationships and better NPS engagement. For that reason, public land use managers and planners will be able to work with community members and with their help, make more effective policies and planning decisions

The physiological expression of inducible nitric oxide synthase (iNOS) in the human colon

Inducible nitric oxide synthase (iNOS) is expressed in the colonic epithelium in both inflammatory bowel disease and colorectal cancer. Nitric oxide (NO), the product of this enzyme, has been implicated in the pathogenesis of both conditions. However, there are conflicting data on whether iNOS is expressed in the normal, uninflamed human colon. To evaluate the expression of iNOS in histologically normal, non-inflamed human colonic mucosa. Reverse transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to investigate the expression of iNOS in 17 histologically normal specimens obtained at colectomy performed for colorectal neoplasia. In addition, 16 endoscopic mucosal biopsies, taken from normal individuals, were also evaluated. Eleven surgical specimens and 16 endoscopic biopsies from patients with refractory ulcerative colitis were used as inflammatory controls. All types of specimens expressed iNOS mRNA. Immunoblotting revealed a protein of approximately 130 kDa consistent with iNOS in mucosal extracts of 77% of normal individuals, and 85% of diseased controls. Immunolabelling localised this protein to the surface epithelium in most of the normal specimens and also to the crypt epithelium and inflammatory cells in the diseased controls. These findings provide evidence that iNOS is often expressed in the surface epithelium of non-inflamed human colon, suggesting that it is induced by local luminal factors, such as bacterial lipopolysaccharide (endotoxin). The resultant NO produced at this site might act as an oxidative barrier, reducing bacterial translocation and providing a means of defence against pathogenic microorganisms

Medication decision making and patient outcomes in GP, nurse and pharmacist prescriber consultations

UNLABELLED: Aim The aims of this study were twofold: (a) to explore whether specific components of shared decision making were present in consultations involving nurse prescribers (NPs), pharmacist prescribers (PPs) and general practitioners (GPs) and (b) to relate these to self-reported patient outcomes including satisfaction, adherence and patient perceptions of practitioner empathy.BACKGROUND: There are a range of ways for defining and measuring the process of concordance, or shared decision making as it relates to decisions about medicines. As a result, demonstrating a convincing link between shared decision making and patient benefit is challenging. In the United Kingdom, nurses and pharmacists can now take on a prescribing role, engaging in shared decision making. Given the different professional backgrounds of GPs, NPs and PPs, this study sought to explore the process of shared decision making across these three prescriber groups.METHODS: Analysis of audio-recordings of consultations in primary care in South England between patients and GPs, NPs and PPs. Analysis of patient questionnaires completed post consultation. Findings A total of 532 consultations were audio-recorded with 20 GPs, 19 NPs and 12 PPs. Prescribing decisions occurred in 421 (79%). Patients were given treatment options in 21% (102/482) of decisions, the prescriber elicited the patient's treatment preference in 18% (88/482) and the patient expressed a treatment preference in 24% (118/482) of decisions. PPs were more likely to ask for the patient's preference about their treatment regimen (χ 2=6.6, P=0.036, Cramer's V=0.12) than either NPs or GPs. Of the 275 patient questionnaires, 192(70%) could be matched with a prescribing decision. NP patients had higher satisfaction levels than patients of GPs or PPs. More time describing treatment options was associated with increased satisfaction, adherence and greater perceived practitioner empathy. While defining, measuring and enabling the process of shared decision making remains challenging, it may have patient benefit

Community participation for malaria elimination in Tafea Province, Vanuatu: part I. Maintaining motivation for prevention practices in the context of disappearing disease

Background: In the 1990s, the experience of eliminating malaria from Aneityum Island, Vanuatu is often given as evidence for the potential to eliminate malaria in the south-west Pacific. This experience, however, cannot provide a blueprint for larger islands that represent more complex social and environmental contexts. Community support was a key contributor to success in Aneityum. In the context of disappearing disease, obtaining and maintaining community participation in strategies to eliminate malaria in the rest of Tafea Province, Vanuatu will be significantly more challenging. Method: Nine focus group discussions (FGDs), 12 key informant interviews (KIIs), three transect walks and seven participatory workshops were carried out in three villages across Tanna Island to investigate community perceptions and practices relating to malaria prevention (particularly relating to bed nets); influences on these practices including how malaria is contextualized within community health and disease priorities; and effective avenues for channelling health information. Results: The primary protection method identified by participants was the use of bed nets, however, the frequency and motivation for their use differed between study villages on the basis of the perceived presence of malaria. Village, household and personal cleanliness were identified by participants as important for protection against malaria. Barriers and influences on bed net use included cultural beliefs and practices, travel, gender roles, seasonality of mosquito nuisance and risk perception. Health care workers and church leaders were reported to have greatest influence on malaria prevention practices. Participants preferred receiving health information through visiting community health promotion teams, health workers, church leaders and village chiefs. Conclusion: In low malaria transmission settings, a package for augmenting social capital and sustaining community participation for elimination will be essential and includes: 'sentinel sites' for qualitative monitoring of evolving local socio-cultural, behavioural and practical issues that impact malaria prevention and treatment; mobilizing social networks; intersectoral collaboration; integration of malaria interventions with activities addressing other community health and disease priorities; and targeted implementation of locally appropriate, multi-level, media campaigns that sustain motivation for community participation in malaria elimination

Novel methods for estimating the instantaneous and overall COVID-19 case fatality risk among care home residents in England

The COVID-19 pandemic has had high mortality rates in the elderly and frail worldwide, particularly in care homes. This is driven by the difficulty of isolating care homes from the wider community, the large population sizes within care facilities (relative to typical households), and the age/frailty of the residents. To quantify the mortality risk posed by disease, the case fatality risk (CFR) is an important tool. This quantifies the proportion of cases that result in death. Throughout the pandemic, CFR amongst care home residents in England has been monitored closely. To estimate CFR, we apply both novel and existing methods to data on deaths in care homes, collected by Public Health England and the Care Quality Commission. We compare these different methods, evaluating their relative strengths and weaknesses. Using these methods, we estimate temporal trends in the instantaneous CFR (at both daily and weekly resolutions) and the overall CFR across the whole of England, and dis-aggregated at regional level. We also investigate how the CFR varies based on age and on the type of care required, dis-aggregating by whether care homes include nursing staff and by age of residents. This work has contributed to the summary of measures used for monitoring the UK epidemic

Self reporting RNA probes as an alternative to cleavable small molecule mass tags

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker

Antilymphoid antibody preconditioning and tacrolimus monotherapy for pediatric kidney transplantation

Objective: Heavy post-transplant immunosuppression may contribute to long-term immunosuppression dependence by subverting tolerogenic mechanisms; thus, we sought to determine if this undesirable consequence could be mitigated by pretransplant lymphoid depletion and minimalistic post-transplant monotherapy. Study design: Lymphoid depletion in 17 unselected pediatric recipients of live (n = 14) or deceased donor kidneys (n = 3) was accomplished with antithymocyte globulin (ATG) (n = 8) or alemtuzumab (n = 9). Tacrolimus was begun post-transplantation with subsequent lengthening of intervals between doses (spaced weaning). Maintenance immunosuppression, morbidity, graft function, and patient/graft survival were collated. Results: Steroids were added temporarily to treat rejection in two patients (both ATG subgroup) or to treat hemolytic anemia in two others. After 16 to 31 months (mean 22), patient and graft survival was 100% and 94%, respectively. The only graft loss was in a nonweaned noncompliant recipient. In the other 16, serum creatinine was 0.85 ± 0.35 mg/dL and creatinine clearance was 90.8 ± 22.1 mL/1.73 m2. All 16 patients are on monotherapy (15 tacrolimus, one sirolimus), and 14 receive every other day or 3 times per week doses. There were no wound or other infections. Two patients developed insulin-dependent diabetes. Conclusion: The strategy of lymphoid depletion and minimum post-transplant immunosuppression appears safe and effective for pediatric kidney recipients. © 2006 Elsevier Inc. All rights reserved

Staffing in postnatal units: is it adequate for the provision of quality care? Staff perspectives from a state-wide review of postnatal care in Victoria, Australia

BACKGROUND: State-wide surveys of recent mothers conducted over the past decade in Victoria, one state of Australia, have identified that women are consistently less satisfied with the care they received in hospital following birth compared with other aspects of maternity care. Little is known of caregivers' perspectives on the provision ofhospital postnatal care: how care is organised and provided in different hospitals; what constrains the provision of postnatal care (apart from funding) and what initiatives are being undertaken to improve service delivery. A state-widereview of organisational structures and processes in relation to the provision of hospital postnatal care in Victoria was undertaken. This paper focuses on the impact of staffing issues on the provision of quality postnatal care from the perspective of care providers. METHODS: A study of care providers from Victorian public hospitals that provide maternity services was undertaken. Datawere collected in two stages. Stage one: a structured questionnaire was sent to all public hospitals in Victoria that provided postnatal care (n = 73), exploring the structure and organisation of care (e.g. staffing, routine observations, policy framework and discharge planning). Stage two: 14 maternity units were selected and invited to participate in a more in-depth exploration of postnatal care. Thirty-eight key informant interviews were undertaken with midwives (including unit managers, associate unit managers and clinical midwives) and a medical practitioner from eachselected hospital. RESULTS: Staffing was highlighted as a major factor impacting on the provision of quality postnatal care. There were significant issues associated with inadequate staff/patient ratios; staffing mix; patient mix; prioritisation of birth suites over postnatal units; and the use of non-permanent staff. Forty-three percent of hospitals reported having only midwives (i.e. no non-midwives) providing postnatal care. Staffing issues impact on hospitals' ability to provide continuity of care. Recruitment and retention of midwives are significant issues, particularly in rural areas. CONCLUSION: Staffing in postnatal wards is a challenging issue, and varies with hospital locality and model of care. Staff/patient ratios and recruitment of midwives in rural areas are the two areas that appear to have the greatest negative impact on staffing adequacy and provision of quality care. Future research on postnatal care provision should include consideration of any impact on staff and staffing