Promoting anaesthetisia providers' non-technical skills through the Vital Anaesthesia Simulation Training (VAST) course in a low-resource setting.

BACKGROUND: Short educational programmes are important in building global anaesthesia workforce capacity. The Vital Anaesthesia Simulation Training (VAST) course is a 3-day immersive simulation-based programme concentrating on core clinical challenges and non-technical skills required by anaesthesia providers in low-resource settings. METHODS: This mixed methods study prospectively evaluated the impact of VAST in Rwanda. Anaesthetists' Non-Technical Skills (ANTS) scores were quantitatively assessed for 30 course participants at three time points (pre-, post-, and 4 months after VAST). Qualitative data were gathered during focus groups (4 months after VAST) to learn of participants' experiences implementing new knowledge into clinical practice. RESULTS: The ANTS total scores improved from pre- (11.0 [2.3]) (mean [standard deviation]) to post-test (14.0 [1.6]), and improvements were maintained at retention (14.2 [1.7]). A similar pattern was observed when data were analysed using the four ANTS categories (all P<0.001). The key theme that emerged during focus group discussions was that the use of cognitive aids and clinical algorithms, repeated and reinforced across simulated scenarios, encouraged a systematic approach to patient care. The participants attributed the systematic approach to improving their problem-solving skills and confidence, particularly during emergencies. They found value in well-functioning teams and shared decision-making. After VAST, the participants described empowerment to advocate for better patient care and system improvement. CONCLUSIONS: VAST offers a simulation-based training to anaesthesia providers working in low-resource settings. Skills retention and self-reported application of learning into the workplace reflect the scope of impact of this training.

Evaluating urban surface water quality in Luton.

Using a single numerical value to indicate the quality of water, a so-called Water Quality Index (WQI) is a well-established way of rating the overall water quality status of a given water body. During the last few years, researchers in the water sector have developed different such indices to address their specific needs. In this study, we attempt to obtain a WQI formula suited for evaluating the water quality of the River Lea. We have selected four different sites on the River Lea and explore the possibility of monitoring using a minimum number of parameters only. The results obtained are very encouraging and provide a strong indication that only three parameters are enough to indicate water quality of a water body.

Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in-vitro culture in cryopreservation studies.

Development of in vitro culture protocol for early stage ovarian follicles of zebrafish is important since cryopreserved early stage ovarian follicles would need to be matured in vitro following cryopreservation before they can be fertilised. Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in vitro culture of early stage zebrafish ovarian follicles in ovarian tissue fragments is reported here for the first time although some work has been reported for in vitro culture of isolated early stage zebrafish ovarian follicles. The main aim of the present study was to develop molecular markers in an optimised in vitro culture protocol for stage I and stage II zebrafish ovarian follicles in ovarian tissue fragments. The effect of concentration of the hormones human chorionic gonadotropin and follicle stimulating hormones, and additives such as Foetal Bovine Serum and Bovine Serum Albumin were studied. The results showed that early stage zebrafish ovarian fragments containing stage I and stage II follicles which are cultured in vitro for 24 h in 20% FBS and 100mIU/ml FSH in 90% L-15 medium at 28 °C can grow to the size of stage II and stage III ovarian follicles respectively. More importantly the follicle growth from stage I to stage II and from stage II to stage III were confirmed using molecular markers such as cyp19a1a (also known as P450aromA) and vtg1 genes respectively. However, no follicle growth was observed following cryopreservation and in vitro culture.

Highly potent and selective NaV1.7 inhibitors for use as intravenous agents and chemical probes.

The discovery and selection of a highly potent and selective NaV1.7 inhibitor PF-06456384, designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A proposed protein-ligand binding mode for this compound is also provided to rationalise the high levels of potency and selectivity over inhibition of related sodium channels. To further support the proposed binding mode, potent conjugates are described which illustrate the potential for development of chemical probes to enable further target evaluation.

The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics.

This paper describes our recent efforts to design and synthesise potent and selective PDE5 inhibitors and the use of in vitro predictors of clearance, absorption and permeability to maximise the potential for dose-proportional pharmacokinetics and good oral bioavailability in man. Optimisation of the preclinical profile resulted in the identification of UK-369003 (19a) and its nomination as a clinical candidate. The clinical pharmacokinetic and safety profile has enabled us to progress the compound to test its efficacy in patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) and a paper describing its efficacy has recently been published.

Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel.

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

Part 1: N-alkylated glycines as potent α2δ ligands.

A new series of glycine-derived ligands of the α(2)δ subunit of voltage gated calcium channels is described. Several novel compounds (7) based on (6) were prepared that possessed a potency <100 nM in the α(2)δ binding assay.

Part 3: Design and synthesis of proline-derived α2δ ligands.

A potent series of substituted (2S,4S)-benzylproline α(2)δ ligands have been designed from the readily available starting material (2S,4R)-hydroxy-L-proline. The ligands have improved pharmacokinetic profile over the (4S)-phenoxyproline derivatives described previously and have potential for development as oral agents for the treatment of neuropathic pain. Compound 16 has been progressed to clinical development.

Part 2: Design, synthesis and evaluation of hydroxyproline-derived α2δ ligands.

Conformational constraint has been used to design a potent series of α(2)δ ligands derived from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved physicochemistry and potency compared to their linear counterparts (described in our earlier publication) and the lead compound has been progressed to clinical development.

Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments.

Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution.

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes.

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs - these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science - and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.

Next-generation sequencing of non-small cell lung cancer using a customized, targeted sequencing panel: Emphasis on small biopsy and cytology.

BACKGROUND: Next-generation sequencing (NGS) with a multi-gene panel is now available for patients with lung adenocarcinoma, but the performance characteristics and clinical utility of this testing are not well-described. We present the results of an extended 467 gene panel in a series of advanced, highly selected nonsmall cell lung cancer (NSCLC) patients using a range of specimens, including predominantly small biopsy and cytology specimens. MATERIALS AND METHODS: A retrospective review of 22 NSCLC biopsies sent for NGS using an extended gene panel from January 2014 to July 2015. The customized NGS panel sequences 467 cancer-associated genes with exonic and intronic sequences obtained from purified tumor DNA. Genomic alterations, patient characteristics, and success of testing were determined. RESULTS: The majority of samples tested were metastatic lung adenocarcinoma on final pathology. Of the 22 specimens tested, 5 (22.7%) were surgical resections and 17 (77.3%) were small biopsy and cytology specimens. Twenty-one (95%) of the specimens were adequate for full sequencing and yielded a total of 204 genomic alterations (average 8.9 per tumor), of which 17 (average 0.81 per tumor) were actionable and/or clinically relevant. Genomic alterations were found most commonly in the TP53, EGFR, EPHB1, MLL3, APC, SETD2, KRAS, DNMT3A, RB1, CDKN2A, ARID1A, EP300, KDM6B, RAD50, STK11, and BRCA2 genes. CONCLUSIONS: NGS using a comprehensive gene panel was performed successfully in 95% of all NSCLC cases in this series, including 94% small biopsy and cytology specimens and 100% surgical resections. This custom assay was performed on a range of tumor specimens and demonstrates that small specimens are able to provide a similar depth of information as larger ones. As many patients present at an advanced stage and only small specimens are obtained, the information these provide has the potential for guiding treatment in highly selected patients with advanced lung adenocarcinoma.

Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

A novel series of potent and selective PDE5 inhibitors with potential for high and dose-independent oral bioavailability.

Sildenafil (5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one), a potent and selective phosphodiesterase type 5 (PDE5) inhibitor, provided the first oral treatment for male erectile dysfunction. The objective of the work reported in this paper was to combine high levels of PDE5 potency and selectivity with high and dose-independent oral bioavailability, to minimize the impact on the C(max) of any interactions with coadministered drugs in the clinic. This goal was achieved through identification of a lower clearance series with a high absorption profile, by replacing the 5'-piperazine sulfonamide in the sildenafil template with a 5'-methyl ketone. This novel series provided compounds with low metabolism in human hepatocytes, excellent caco-2 flux, and the potential for good aqueous solubility. The in vivo oral and iv pharmacokinetic profiles of example compounds confirmed the high oral bioavailability predicted from these in vitro screens. 5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (2) was selected for progression into the clinic.

Zebrafish embryos (Danio rerio) using microinjection.

Low membrane permeability is one of the major obstacles to the successful cryopreservation of zebrafish embryos. The aim of the present study was to explore if this could be overcome by yolk modification with different cryoprotectants by micro-injection. Initial investigation of two cryoprotectants, methanol and sucrose, was undertaken to determine their suitability for micro-injection supplementation of the yolk mass. Intact zebrafish embryos at 50% epiboly stage were injected with Hanks' solution, 5.2 M methanol or 1.3 M sucrose yielding approximate final concentrations of 2.0 and 0.5 M of the cryoprotectants within the yolk sac respectively. After micro-manipulation, the embryos were cultured at 28 degree C for three days and their survival assessed at the hatching stage. All micro-manipulations performed in the present study resulted in a significant decrease in embryo survival (P < 0.05). Embryos micro-injected with methanol or sucrose were also subjected to a cooling procedure. They were placed in 3M methanol + 0.5 M sucrose at room temperature for 30 min and then cooled from 20 degree C to 0 degree C at 2 degree/min, from 0 degree C to -7.5 degree/min at 1 degree/min, seeded at -7.5 degree C and held for 10 min, before cooling at 0.3 degree/min to - 20 degree C or until full crystallization in all embryos. The processes of extra- and intracellular crystallization were studied by cryomicroscopy. The temperature of intracellular crystallization did not differ significantly between control and injected embryos. However, it was found that intracellular crystallization did not always happen instantly after extracellular crystallization.

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.

The effect of external medium composition on membrane water permeability of zebrafish (Danio rerio) embryos.

The effect of external medium composition on chorion and plasma membrane permeability of zebrafish (Danio rerio) embryos was investigated in this study. Initially, survival of embryos spawned into varying strengths (10-40%) of Hank's solution (HBSS) was assessed. Development and hatching rates for embryos spawned into 30% and 40% HBSS were significantly lower than those obtained with embryos spawned into system water. The effect of embryo survival in 30% HBSS with different calcium levels was then investigated. Embryo survival in calcium free 30% HBSS or 30% HBSS with 10x the standard calcium concentration was similar to survival in standard 30% HBSS. Membrane water permeability was determined by measuring the floatation time of embryos in test solutions made up with heavy water (D2O) instead of deionized water. Intact embryos at early developmental stages were less permeable than later stages irrespective of the external medium that they were spawned into. In system water, the floatation time of embryos at one-cell and two-cell stages were 1323+/-83 and 1189+/-55 s, respectively, compared to 432+/-6 and 353+/-10 s at the high and 50% epiboly stages. Change of external medium composition had no effect on membrane permeability of intact embryos at early developmental stages. However, at later stages embryos spawned into 30% HBSS were less permeable than embryos spawned into system water, irrespective of calcium concentration. The flotation time of embryos at the high stage increased from 432+/-6s in system water to 468+/-10s in 30% HBSS. The study on dechorionated embryos showed that change of external medium composition had no effect on plasma membrane permeability.

Discovery and Optimization of Selective Nav1.8 Modulator Series That Demonstrate Efficacy in Preclinical Models of Pain.

Voltage-gated sodium channels, in particular Nav1.8, can be targeted for the treatment of neuropathic and inflammatory pain. Herein, we described the optimization of Nav1.8 modulator series to deliver subtype selective, state, and use-dependent chemical matter that is efficacious in preclinical models of neuropathic and inflammatory pain.

Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos.

Stage-dependent chilling sensitivity has been reported for many species of fish embryos. Most of these studies reveal that developmental stages beyond 50% epiboly are less sensitive to chilling, but the chilling sensitivity accelerates rapidly at subzero temperatures. In this study, the effects of methanol and developmental arrest on chilling injury were studied using zebrafish (Danio rerio) embryos at 64-cell, 50% epiboly, 6-somite, prim-6 and long-bud stages. Embryos were exposed to methanol or anoxic conditions before they were cooled to 0 or -5 degrees C with slow (1 degrees C/min), medium (30 degrees C/min) or fast ( approximately 300 degrees C/min) cooling rates and were held at these temperatures for different time periods. Embryo survival was evaluated in terms of the percentage of treated embryos with normal developmental appearance after 3-day culture. Experiments on the effect of methanol on chilling sensitivity of the embryos showed that the addition of methanol to embryo medium increased embryo survival significantly at all developmental stages and under all cooling conditions. Higher concentration of methanol treatment generally improved embryo survival when embryos were cooled at a fast cooling rate of 300 degrees C/min. Experiments on the effect of developmental arrest on chilling sensitivity of embryos showed that embryos at 50% epiboly and prim-6 stages underwent developmental arrest almost immediately after 15 min oxygen deprivation. After 4h in anoxia, the survival rates of the embryos were not significantly different from their respective aerobic controls. Anoxia and developmental arrest had no effect on the chilling sensitivity of zebrafish embryos.