Strict conformational demands of RNA cleavage in bulge-loops created by peptidyl-oligonucleotide conjugates.

Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. 'Smart' supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-ß anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an 'in-line' attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.

Process Design and Optimization in the Pharmaceutical Industry: A Suzuki-Miyaura Procedure for the Synthesis of Savolitinib.

A multidisciplinary approach covering synthetic, physical, and analytical chemistry, high-throughput experimentation and experimental design, process engineering, and solid-state chemistry is used to develop a large-scale (kilomole) Suzuki-Miyaura process. Working against clear criteria and targets, a full process investigation and optimization package is described highlighting how and why key decisions are made in the development of large-scale pharmaceutical processes.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution-induced fibrosis.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

Ultra-low voltage electrowetting using graphite surfaces.

The control of wetting behaviour underpins a variety of important applications from lubrication to microdroplet manipulation. Electrowetting is a powerful method to achieve external wetting control, by exploiting the potential-dependence of the liquid contact angle with respect to a solid substrate. Addition of a dielectric film to the surface of the substrate, which insulates the electrode from the liquid thereby suppressing electrolysis, has led to technological advances such as variable focal-length liquid lenses, electronic paper and the actuation of droplets in lab-on-a-chip devices. The presence of the dielectric, however, necessitates the use of large bias voltages (frequently in the 10-100 V range). Here we describe a simple, dielectric-free approach to electrowetting using the basal plane of graphite as the conducting substrate: unprecedented changes in contact angle for ultra-low voltages are seen below the electrolysis threshold (50° with 1 V for a droplet in air, and 100° with 1.5 V for a droplet immersed in hexadecane), which are shown to be reproducible, stable over 100 s of cycles and free of hysteresis. Our results dispel conventional wisdom that reversible, hysteresis-free electrowetting can only be achieved on solid substrates with the use of a dielectric. This work paves the way for the development of a new generation of efficient electrowetting devices using advanced materials such as graphene and monolayer MoS2.

The European New Car Assessment Programme: A historical review.

Established in 1997, the European New Car Assessment Programme (Euro NCAP) provides consumers with a safety performance assessment for the majority of the most popular cars in Europe. Thanks to its rigorous crash tests, Euro NCAP has rapidly become an important driver safety improvement to new cars. After ten years of rating vehicles, Euro NCAP felt that a change was necessary to stay in tune with rapidly emerging driver assistance and crash avoidance systems and to respond to shifting priorities in road safety. A new overall rating system was introduced that combines the most important aspects of vehicle safety under a single star rating. The overall rating system has allowed Euro NCAP to continue to push for better fitment and higher performance for vehicles sold on the European market. In the coming years, the safety rating is expected to play an important role in the support of the roll-out of highly automated vehicles.

Peptidyl-oligonucleotide conjugates demonstrate efficient cleavage of RNA in a sequence-specific manner.

Described here is a new class of peptidyl-oligonucleotide conjugates (POCs) which show efficient cleavage of a target RNA in a sequence-specific manner. Through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to amphiphilic peptide sequences containing leucine, arginine, and glycine, zero-linker conjugates are created which exhibit targeted phosphodiester cleavage under physiological conditions. tRNA(Phe) from brewer's yeast was used as a model target sequence in order to probe different structural variants of POCs in terms of selective TΨC-arm directed cleavage. Almost quantitative (97-100%) sequence-specific tRNA cleavage is observed for several POCs over a 24 h period with a reaction half-life of less than 1 h. Nontargeted cleavage of tRNA(Phe) or HIV-1 RNA is absent. Structure-activity relationships reveal that removal of the peptide's central glycine residue significantly decreases tRNA cleavage activity; however, this can be entirely restored through replacement of the peptide's C-terminal carboxylic acid group with the carboxamide functionality. Truncation of the catalytic peptide also has a detrimental effect on POC activity. Based on the encouraging results presented, POCs could be further developed with the aim of creating useful tools for molecular biology or novel therapeutics targeting specific messenger, miRNA, and genomic viral RNA sequences.

Enhancing the implementation of provider-to-provider telehealth in rural and remote areas: A mixed methods study protocol.

Background: Virtual healthcare solutions are proposed as a way to combat the inequity of access to healthcare in rural and remote areas, and to better support the front-line providers who work in these areas. Rural provider-to-provider telehealth (RPPT) connects rural and remote clinicians to a 'hub' of healthcare specialists who can increase access to emergency and specialised healthcare via an integrated model. Reported benefits for the place-based provider include enhanced knowledge, expanded professional development opportunities, improved scope of practice, and increased confidence in treating more complex cases. These reported benefits could have implications for supporting and futureproofing our health workforce in terms of productivity, burnout, recruitment, and retention. Methods: The research uses an explanatory sequential mixed methods approach across multiple phases to evaluate the current implementation of Western Australia Country Health Service's (WACHS) Command Centre (CC) services and explore factors associated with their differential use. The primary population of interest and participants in this study are the place-based providers in country Western Australia (WA). Patient data constitutes the secondary population, informing the access and reach of CC services into country WA. Data collection will include service data, an online survey, and semi-structured interviews with the primary population. The data will be interpreted to inform evidence-based strategies and recommendations to improve the implementation and sustainment of RPPT. Discussion: Innovative and sustained workforce models and solutions are needed globally. Virtual healthcare, including provider-to-provider models, demonstrate potential, especially in rural and remote areas, designed to increase access to specialised expertise for patients and to support the local workforce. This research will generate new data around behaviour, perceptions, and value from the WACHS rural and remote workforce about provider-to-provider telehealth, to explore the implementation and investigate strategies for the long-term sustainment of RPPT services.

A case of newly diagnosed primary hypothyroidism with deteriorating consciousness that did not respond to thyroxine--what else should be considered?

We present an unusual case of a 60 year old male who was noted to have primary hypothyroidism and decreased consciousness which did not respond to Levothyroxine. Further investigations revealed a diagnosis of Hashimoto's Encephalopathy which responded well to steroids. We describe the case in detail and discuss the available relevant literature on this topic.

Exploration of mentor and mentee perspectives of a mentored clinical practice programme to improve patient outcomes in musculoskeletal physiotherapy.

BACKGROUND: A recent randomised controlled trial has demonstrated the impact on practice of an educational programme for clinicians. Mentored clinical practice in musculoskeletal physiotherapy resulted in clinically significant improvements in both physiotherapist performance and patient outcomes. The objectives of this study were to explore mentor and mentee perceptions of a mentored clinical practice programme, in order to identify key factors in the process to improve patient outcomes. METHODS: Employing a case study design of a mentoring programme that led to improved patient outcomes, mentored clinical practice was explored from multiple perspectives using a grounded theory strategy of enquiry to derive a theory of mentored clinical practice grounded in the views of the participants. Semi-structured interviews with a purposive sample of mentors and mentees were employed along with qualitative observations of mentored clinical practice. Data analysis and collection were concurrent, with analysis an iterative process deriving inductive analytical categories from the data through constant comparison. FINDINGS: Highly informative themes of how the complex interaction between mentor, mentee, patient and environment worked successfully were identified from the data. The mentors' knowledge, additional perspectives, critical analysis and facilitatory style were enabling factors, as were mentees' motivation, openness to criticism and commitment to reflect on practice. Themes around potential threats to the mentees' development were also identified. Overloading or contradictory feedback and lack of relationship with mentees were barriers that mentors could bring; fear, defensiveness, routine working, people-pleasing and lack of experience were potential mentee barriers. A model emerges from the data demonstrating how these themes interact, providing guidance to mentors and mentees to optimise the effectiveness of mentored clinical practice. CONCLUSION: This study provides a sound basis for future mentored clinical practice, producing a model from key themes from a case study where impact on clinician performance and patient outcomes are established.

Adult day-case renal biopsy: a single-centre experience.

BACKGROUND: Despite improvements in safety seen over the last 20 years, percutaneous renal biopsy is still associated with haemorrhagic complications. Due to concerns over delayed bleeding, most nephrologists would advocate overnight observation. Recent evidence in both adult and paediatric populations suggest that in some groups, this is unnecessary. Since 1991, we have provided a day-case renal biopsy service performing 70 such procedures per year. In this study, we present a retrospective analysis of this practice. METHODS: A total of 192 patients over a consecutive 3-year period were analysed retrospectively. Patients were selected according to standardized criteria, and biopsy was performed using a modern technique (automated biopsy needles under ultrasound guidance). Complications were identified by examination of case notes and local hospital admission databases, and by telephone interview. Our pathology database was examined for sample adequacy and diagnosis. RESULTS: There were no delayed complications in the study group with 187 patients (97.4%) being discharged home on the same day. Major complications occurred in five patients (2.6%), all related to bleeding. Of these, two needed radiological intervention to achieve haemostasis. Sufficient tissue for diagnosis was achieved in 97% of cases, with a mean of 47 ± 23 glomeruli obtained per patient. Most biopsies were obtained with ≤ 2 passes (84%). CONCLUSIONS: Our findings show that in selected adult patients, renal biopsy can be performed as a day-case procedure. Given the benefits of day-case strategies in terms of patient and healthcare costs, we advocate increased utilization of this technique.

Omitting Cortical Renorrhaphy in Robot-Assisted Partial Nephrectomy: Is it Safe? A Single Center Large Case Series.

Introduction: Preserving renal function after robot-assisted partial nephrectomy (RAPN) is important and influenced by the technique of renal reconstruction among other parameters, including ischemia time and amount of healthy renal tissue resected. It is believed that reconstruction with a second layer of cortical renorrhaphy is necessary to prevent urinary leaks and postoperative bleeding, but this is associated with the potential loss of healthy renal parenchyma and may result in worse outcomes postoperatively. Purpose: To assess the safety of omitting cortical renorrhaphy during RAPN. Patients and Methods: A retrospective analysis of 146 consecutive patients undergoing a RAPN with single or double layer renorrhaphy at the Wirral University Teaching Hospital from 2014 to 2019. Data obtained included: Patient demographics, tumor RENAL nephrometry, Perioperative parameters; blood loss, duration, and warm ischemia time (WIT), Postop complications, change in estimated glomerular filtration rate (eGFR) (pre and 3 months postop), length of stay, and oncologic outcomes. Results: In total 146 patients were identified. One hundred-six had double renorrhaphy and 40 inner layer only renorrhaphy. No significant differences were seen between these two cohorts in terms of patient demographics, RENAL nephrometry score, tumor size, or location. Perioperative parameters showed a reduced duration of surgery in the single renorrhaphy group with a mean of 125 minutes compared to143 minutes in the double renorrhaphy (p = 0.006) and a tendency toward a shorter WIT of 12.9 minutes vs 14.0 minutes reaching borderline statistical significance (p = 0.05) but no difference in blood loss volume (p = 0.25). Postoperatively there was no statistical difference in the length of hospital stay (p = 0.85), loss in eGFR at 3 months (0.06), or complication (p = 0.56). After a median follow-up of 35 months no recurrences or deaths were observed in either group. Conclusions: Omission of cortical renorrhaphy appears feasible and safe with no urine leaks or excess complications observed.

Evaluation of the clinical effectiveness of a work-based mentoring programme to develop clinical reasoning on patient outcome: A stepped wedge cluster randomised controlled trial.

BACKGROUND: Despite persistent calls to measure the effectiveness of educational interventions on patient outcomes, few studies have been conducted. Within musculoskeletal physiotherapy, the effects of clinical mentoring on postgraduate physiotherapists have been explored, but its impact on patient outcomes is unknown. The objective of this trial was to assess the effectiveness of a work-based mentoring programme to facilitate physiotherapist clinical reasoning on patient outcomes. METHODS: In a stepped-wedge cluster RCT in the musculoskeletal physiotherapy outpatient departments of a large NHS organisation, 16 physiotherapists were randomised by cluster to receive the intervention-150 hours of mentored clinical practice-at one of 3 time periods; control was usual training. 441 patients submitted outcome measures: Patient-Specific Functional Scale (PSFS) (primary outcome measure), EQ-5D-5L, patient activation and patient satisfaction (secondary outcome measures). A further secondary outcome measure of physiotherapist performance was collected by an independent assessor observing the physiotherapists practice. RESULTS: 80.0% of intervention patients achieved clinically significant PSFS scores compared with 63.8% of control patients. Binary logistic regression analysis modelling for time, cluster and patient characteristics showed strong statistical evidence for this difference (p = 0.023; odds ratio 4.24, 95%CI 1.22, 14.79). Physiotherapist performance scores improved from a mean of 47.8% (SD 3.60) pre-intervention to a mean of 56.0% (SD 4.24) (p<0.001). There was no statistical evidence for differences between groups on other secondary outcomes. CONCLUSION: This is the first study that we aware of that provides patient outcomes measurement of an established educational intervention in physiotherapy, providing evidence that this type of intervention positively impacts patient outcomes and physiotherapist performance. This provides a basis for further research in education across other healthcare disciplines and outcome measures.

Biofunctional few-layer metal dichalcogenides and related heterostructures produced by direct aqueous exfoliation using phospholipids.

We report a novel, inexpensive and green method for preparing aqueous dispersions of various biofunctional transition-metal dichalcogenides (MoS2, WS2, TiS2 and MoSe2) and their related heterostructures directly via ultrasonic exfoliation mediated by the presence of phospholipids. The dispersions predominantly consist of few-layer flakes coated with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), as confirmed by Raman, photoluminescence and X-ray photoelectron spectroscopies. The phospholipid coating renders the flakes biofunctional, which coupled with the unique properties of transition-metal dichalcogenides and their heterostructures, suggests this method will have great potential in biological applications.

Phospholipid-mediated exfoliation as a facile preparation method for graphene suspensions.

This paper deals with simple, inexpensive and 'green' methods of production for graphene in colloidal dispersion. Herein, we report on such a method by preparing aqueous graphene dispersions via ultrasonic exfoliation in the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The product predominantly consists of few-layer graphene flakes coated by DOPC with a lateral size of a few tens to hundreds of nm, as confirmed by Raman and X-ray photoelectron spectroscopies, thermogravimetric analysis (TGA), dynamic light scattering (DLS) and atomic force microscopy (AFM). The novelty of this method lies in its dependence on a typical soft matter property: the fluidity of the hydrophobic chains. Stiffer phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, which possesses two palmitoyl chains) or 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, one palmitoyl, one oleyl chain) are ineffective at dispersing graphene; however, in the presence of cholesterol these phospholipids also become effective mediators. The phospholipid coating renders the flakes compatible with biological environments.

'Dual' peptidyl-oligonucleotide conjugates: Role of conformational flexibility in catalytic cleavage of RNA.

Traditional therapeutic interventions against abnormal gene expression in disease states at the level of expressed proteins are becoming increasingly difficult due to poor selectivity, off-target effects and associated toxicity. Upstream catalytic targeting of specific RNA sequences offers an alternative platform for drug discovery to achieve more potent and selective treatment through antisense interference with disease-relevant RNAs. We report a novel class of catalytic biomaterials, comprising amphipathic RNA-cleaving peptides placed between two RNA recognition motifs, here demonstrated to target the TΨC loop and 3'- acceptor stem of tRNAPhe. These unique peptidyl-oligonucleotide 'dual' conjugates (DCs) were created by phosphoramidate or thiol-maleimide conjugation chemistry of a TΨC-targeting oligonucleotide to the N-terminus of the amphipathic peptide sequence, followed by amide coupling of a 3'-acceptor stem-targeting oligonucleotide to the free C-terminal carboxylic acid functionality of the same peptide. Hybridization of the DCs bearing two spatially-separated recognition motifs with the target tRNAPhe placed the peptide adjacent to a single-stranded RNA region and promoted cleavage within the 'action radius' of the catalytic peptide. Up to 100% cleavage of the target tRNAPhe was achieved by the best candidate (i.e. DC6) within 4 h, when conformational flexibility was introduced into the linker regions between the peptide and oligonucleotide components. This study provides the strong position for future development of highly selective RNA-targeting agents that can potentially be used for disease-selective treatment at the level of messenger, micro, and genomic viral RNA.

Impact of streaming "fast track" emergency department patients.

OBJECTIVE: Fast track systems to stream emergency department (ED) patients with low acuity conditions have been introduced widely, resulting in reduced waiting times and lengths of stay for these patients. We aimed to prospectively assess the impact on patient flows of a fast track system implemented in the emergency department of an Australian tertiary adult teaching hospital which deals with relatively few low acuity patients. METHODS: During the 12-week trial period, patients in Australasian Triage Scale (ATS) categories 3, 4 and 5 who were likely to be discharged were identified at triage and assessed and treated in a separate fast track area by ED medical and nursing staff rostered to work exclusively in the area. RESULTS: The fast track area managed 21.6% of all patients presenting during its hours of operation. There was a 20.3% (-18 min; 95%CI, -26 min to -10 min) relative reduction in the average waiting time and an 18.0% (-41 min; 95%CI, -52 min to -30 min) relative reduction in the average length of stay for all discharged patients compared with the same period the previous year. Compared with the 12-week period before the fast track trial, there was a 3.4% (-2.1 min; 95%CI, -8 min to 4 min) relative reduction in the average waiting time and a 9.7% (-20 min; 95%CI, -31 min to -9 min) relative reduction in the average length of stay for all discharged patients. There was no increase in the average waiting time for admitted patients. This was despite major increases in throughput and access block in the study period. CONCLUSION: Streaming fast track patients in the emergency department of an Australian tertiary adult teaching hospital can reduce waiting times and length of stay for discharged patients without increasing waiting times for admitted patients, even in an ED with few low acuity patients.

Regulation of synthesis and roles of hyaluronan in peritoneal dialysis.

Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating ß-1,4 and ß-1,3 glycosidic bonds. HA is synthesized in humans by HA synthase (HAS) enzymes 1, 2, and 3, which are encoded by the corresponding HAS genes. Previous in vitro studies have shown characteristic changes in HAS expression and increased HA synthesis in response to wounding and proinflammatory cytokines in human peritoneal mesothelial cells. In addition, in vivo models and human peritoneal biopsy samples have provided evidence of changes in HA metabolism in the fibrosis that at present accompanies peritoneal dialysis treatment. This review discusses these published observations and how they might contribute to improvement in peritoneal dialysis.

The effect of work-based mentoring on patient outcome in musculoskeletal physiotherapy: study protocol for a randomised controlled trial.

BACKGROUND: Despite persistent calls to measure the effectiveness of educational interventions on patient outcomes, few studies have been conducted. Within musculoskeletal physiotherapy, the effects of postgraduate clinical mentoring on physiotherapist performance have been assessed, but the impact of this mentoring on patient outcomes remains unknown. The objective of this trial is to assess the effectiveness of a work-based mentoring programme to facilitate physiotherapist clinical reasoning on patient outcomes in musculoskeletal physiotherapy. METHODS/DESIGN: A stepped wedge cluster randomised controlled trial (CRCT) has been designed to recruit a minimum of 12 senior physiotherapists who work in musculoskeletal outpatient departments of a large National Health Service (NHS) organization. Participating physiotherapists will be randomised by cluster to receive the intervention at three time periods. Patients will be blinded to whether their physiotherapist has received the intervention. The primary outcome measure will be the Patient-Specific Functional Scale; secondary outcome measures will include the EQ-5D, patient activation, patient satisfaction and physiotherapist performance. Sample size considerations used published methods describing stepped wedge designs, conventional values of 0.80 for statistical power and 0.05 for statistical significance, and pragmatic groupings of 12 participating physiotherapists in three clusters. Based on an intergroup difference of 1.0 on the PSFS with a standard deviation of 2.0, 10 patients are required to complete outcome measures per physiotherapist, at time period 1 (prior to intervention roll-out) and at each of time periods 2, 3 and 4, giving a sample size of 480 patients. To account for the potential loss to follow-up of 33%, 720 sets of patient outcomes will be collected.All physiotherapist participants will receive 150 hours of mentored clinical practice as the intervention and usual in-service training as control. Consecutive, consenting patients attending treatment by the participating physiotherapists during data collection periods will complete outcome measures at baseline, discharge and 12 months post-baseline. The lead researcher will be blinded to the allocation of the physiotherapist when analyzing outcome data; statistical analysis will involve classical linear models incorporating both an intervention effect and a random intercept term to reflect systematic differences among clusters. TRIAL REGISTRATION: Assigned 31 July 2012: ISRCTN79599220.