Use of ctDNA in identifying an actionable BRAF mutation in stage 4 metastatic melanoma.

The identification of genetic variants in melanoma has enabled the development of targeted therapies. Under the National Institute for Health and Care Excellence (NICE) guidance, patients with BRAF V600E variant are eligible for BRAF and MEK inhibitor therapy. For those with advanced or highly symptomatic disease, a rapid response to treatment is often seen. Current practice relies on tissue biopsy to perform immunohistochemistry (IHC) or next generation sequencing (NGS) to identify these variants; however, this can take up to 2 weeks. In patients with widespread disease, rapid initiation of treatment can be lifesaving.We describe a case in which hotspot circulating tumour DNA (ctDNA) analysis confirmed BRAF variant 6 days prior to biopsy results. This was utilised to expedite treatment initiation and symptomatically, the patient had initial improvement within a few days.This article demonstrates the potential value of ctDNA analysis and the need for further research into this as an alternative to NGS for patients with rapidly progressive disease.

Establishing the selective phospholipid membrane coordination, permeation and lysis properties for a series of 'druggable' supramolecular self-associating antimicrobial amphiphiles.

The rise of antimicrobial resistance remains one of the greatest global health threats facing humanity. Furthermore, the development of novel antibiotics has all but ground to a halt due to a collision of intersectional pressures. Herein we determine the antimicrobial efficacy for 14 structurally related supramolecular self-associating amphiphiles against clinically relevant Gram-positive methicillin resistant Staphylococcus aureus and Gram-negative Escherichia coli. We establish the ability of these agents to selectively target phospholipid membranes of differing compositions, through a combination of computational host:guest complex formation simulations, synthetic vesicle lysis, adhesion and membrane fluidity experiments, alongside our novel 1H NMR CPMG nanodisc coordination assays, to verify a potential mode of action for this class of compounds and enable the production of evermore effective next-generation antimicrobial agents. Finally, we select a 7-compound subset, showing two lead compounds to exhibit 'druggable' profiles through completion of a variety of in vivo and in vitro DMPK studies.

Continuous enzymatic hydrolysis of sugar beet pectin and l-arabinose recovery within an integrated biorefinery.

Sugar beet pulp (SBP) fractionated by steam explosion, released sugar beet pectin (SB-pectin) which was selectively hydrolysed using a novel α-l-arabinofuranosidase (AF), yielding monomeric l-arabinose (Ara) and a galacturonic acid rich backbone (GABB). AF was immobilised on an epoxy-functionalised resin with 70% overall immobilisation yield. Pretreatment of SB-pectin, to remove coloured compounds, improved the stability of the immobilised AF, allowing its reutilisation for up to 10 reaction cycles in a stirred tank reactor. Continuous hydrolysis of SB-pectin was subsequently performed using a packed bed reactor (PBR) with immobilised AF. Reactor performance was evaluated using a Design of Experiment approach. Pretreated SB-pectin hydrolysis was run for 7 consecutive days maintaining 73% of PBR performance. Continuous separation of Ara from GABB was achieved by tangential flow ultrafiltration with 92% Ara recovery. These results demonstrate the feasibility of establishing a continuous bioprocess to obtain Ara from the inexpensive SBP biomass.

An integrated biorefinery concept for conversion of sugar beet pulp into value-added chemicals and pharmaceutical intermediates.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

Centrifugal partition chromatography in a biorefinery context: Optimisation and scale-up of monosaccharide fractionation from hydrolysed sugar beet pulp.

The isolation of component sugars from biomass represents an important step in the bioprocessing of sustainable feedstocks such as sugar beet pulp. Centrifugal partition chromatography (CPC) is used here, as an alternative to multiple resin chromatography steps, to fractionate component monosaccharides from crude hydrolysed sugar beet pulp pectin. CPC separation of samples, prepared in the stationary phase, was carried out using an ethanol: ammonium sulphate (300gL-1) phase system (0.8:1.8v:v) in ascending mode. This enabled removal of crude feedstream impurities and separation of monosaccharides into three fractions (l-rhamnose, l-arabinose and d-galactose, and d-galacturonic acid) in a single step. Throughput was improved three-fold by increasing sample injection volume, from 4 to 16% of column volume, with similar separation performance maintained in all cases. Extrusion of the final galacturonic acid fraction increased the eluted solute concentration, reduced the total separation time by 24% and removed the need for further column regeneration. Reproducibility of the separation after extrusion was validated by using multiple stacked injections. Scale-up was performed linearly from a semi-preparative 250mL column to a preparative 950mL column with a scale-up ratio of 3.8 applied to mobile phase flow rate and sample injection volume. Throughputs of 9.4gL-1h-1 of total dissolved solids were achieved at the preparative scale with a throughput of 1.9gL-1h-1 of component monosaccharides. These results demonstrate the potential of CPC for both impurity removal and target fractionation within biorefinery separations.

Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

BACKGROUND: Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process. RESULTS: Our technique incorporates counter-selection using ß-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation. CONCLUSION: The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

How to tackle bleeding and thrombosis in the newborn.

Bleeding and its management represent common clinical problems in neonatal intensive care, particularly in pre-term infants. Frank and severe single organ haemorrhage (e.g. pulmonary or gastrointestinal in association with necrotising enterocolitis) is less common, but may require urgent resuscitation and clinical stabilisation. Intracranial bleeding is always potentially of greatest concern because of the neurological consequences, but the pathophysiological mechanism of the most characteristic form, intraventricular haemorrhage, remains incomplete. Minor forms of bleeding are commonly seen in sicker neonates, ranging from blood stained endotracheal secretions to longer than expected oozing from phlebotomy sites, but may be manifestations of disseminated intravascular coagulation. The mainstay of treatment for bleeding in association with abnormalities of coagulation or thrombocytopenia remains blood products, although their role as prophylaxis to prevent bleeding in neonates without clinical signs of haemorrhage is less clear. The overwhelming majority of thromboembolic events in neonates occur in association with arterial or venous catheters, but the clinical features are very variable, including catheter dysfunction and local signs. The optimal treatment strategies including use of anticoagulants remain problematic in the absence of good clinical trials.

Characteristics of a Q-switched multicore photonic crystal fiber laser with a very large mode field area.

We model and characterize the behavior of a Q-switched fiber laser. The fiber is a doped multicore photonic crystal fiber having six cores in a ring-type geometry. The fiber laser is Q-switched using an intracavity acousto-optic modulator. Using a mode filtering technique in the far field, a mode very close to the fundamental in-phase supermode is obtained with a mode field area of 4200 microm(2) and a divergence of 9 mrad. Pulses with energies of up to 2.2 mJ and durations of 26 ns (limited by end facet damage) at a repetition rate of 10 kHz are obtained.

Cost-effectiveness of neonatal extracorporeal membrane oxygenation based on 7-year results from the United Kingdom Collaborative ECMO Trial.

OBJECTIVE: To assess the long-term cost-effectiveness of extracorporeal membrane oxygenation (ECMO) for mature newborn infants with severe respiratory failure. METHODS: A prospective economic evaluation was conducted alongside a pragmatic randomized, controlled trial in which 185 infants were randomly allocated to ECMO (n = 93) or conventional management (n = 92) and then followed up to 7 years of age. Information about their use of health services during the follow-up period was combined with unit costs (pound sterling, 2002-2003 prices) to obtain a net cost per child. The cost-effectiveness of neonatal ECMO was expressed in terms of incremental cost per additional life year gained and incremental cost per additional disability-free life year gained. The nonparametric bootstrap method was used to present cost-effectiveness acceptability curves and net benefit statistics at alternative willingness-to-pay thresholds held by decision-makers for an additional life year and for an additional disability-free life year. RESULTS: Over 7 years, neonatal ECMO was effective at reducing known death or severe disability. Mean health service costs during the first 7 years of life were 30,270 pound sterling in the ECMO group and 10,229 pound sterling in the conventional management group, generating a mean cost difference of 20,041 pound sterling that was statistically significant. The incremental cost per life year gained was estimated at 13,385 pound sterling. The incremental cost per disability-free life year gained was estimated at 23,566 pound sterling. At the notional willingness-to-pay threshold of 30,000 pound sterling for an additional life year, the probability that neonatal ECMO is cost-effective at 7 years was estimated at 0.98. This translated into a mean net benefit of 24,362 pound sterling for each adoption of neonatal ECMO rather than conventional management. CONCLUSIONS: This study provides rigorous evidence of the cost-effectiveness of neonatal ECMO during childhood.

United Kingdom collaborative randomized trial of neonatal extracorporeal membrane oxygenation: follow-up to age 7 years.

OBJECTIVE: The UK Collaborative ECMO trial provided an opportunity to describe mortality and morbidity associated with a neonatal ECMO policy compared with conventional management. The improved survival in the ECMO group was not offset by an increase in disability at 4 years, but the children were too young to assess educational and other longer-term impacts. The objective of this study was to assess the longer-term impact of these policies at age 7 years. METHODS: A psychologist assessed 90 of the 100 children available for follow-up without prior knowledge of treatment allocation. The assessments took place at the children's schools within 3 months of their 7th birthdays. RESULTS: Sixty-eight of 89 (76%) children recorded a cognitive level within the normal range. Learning problems were similar in the 2 groups, and there were notable difficulties with spatial and processing tasks. A higher respiratory morbidity and increased risk of behavioral problems among children treated conventionally persisted. Progressive sensorineural hearing loss was found in both groups. CONCLUSIONS: The underlying disease processes appear to be the major influence on morbidity at 7 years. The beneficial influence of an ECMO policy is still present at 7 years.

Maternal morbidity and pregnancy outcome in a cohort of mothers transferred out of perinatal centres during a national census.

OBJECTIVE: To record the maternal morbidity and pregnancy outcome in this cohort. DESIGN: Retrospective data collection from a prospectively defined cohort. SETTING: The 37 largest perinatal centres in the UK. POPULATION: 258 in utero transfers recorded during a three-month census (1/4/99-30/6/99). METHODS: A questionnaire regarding the outcome of each mother was sent to the perinatal centre and receiving hospital. RESULTS: Data were returned on 242/258 (94%) mothers. Fifty-eight percent were transferred out of their perinatal centre in preterm labour and 38% had coexisting disease necessitating early delivery. The median gestational age at transfer was 32 weeks (range 23-41). Sixty-one percent delivered at the receiving hospital; 12% were transferred on to a third hospital and 29% ultimately returned to deliver at the original perinatal centre. Fifty-two percent of mothers received postnatal care in hospitals other than those defined as a major perinatal centre. One mother delivered during transfer and a further nine within one hour of arrival. One mother received intensive care after delivery and later died, a further 7% required high dependency care postnatally. Data were available on 273/333 (82%) babies. The median gestational age at delivery was 34 weeks (range 24-41). Six infants were stillborn and 187/264 (71%) infants were admitted to a neonatal unit. CONCLUSIONS: This study has documented the maternal morbidity, potential risks and pregnancy outcome of a cohort of mothers transferred out of the largest perinatal centres in the UK because of a shortage of neonatal cots. A national standard for the delivery of high risk perinatal services is needed to uphold good clinical practice guidelines in the care of high risk mothers and their infants.

A comparison of clinical variables that predict adverse outcome in term infants with severe respiratory failure randomised to a policy of extracorporeal membrane oxygenation or to conventional neonatal intensive care.

OBJECTIVE: To identify clinical variables predicting adverse outcome in a group of infants with severe respiratory failure who were randomized either to referral for extra-corporeal membrane oxygenation (ECMO) or to conventional neonatal intensive care within the United Kingdom. METHODS: Adverse outcome was defined by death or disability by four years of age. Receiver operator characteristic (ROC) plots were constructed for variables with continuous data and relative risk (RR) with 95% confidence intervals (CI) calculated for binominal data. RESULTS: Of variables measurable at trial entry, congenital diaphragmatic hernia and lower birthweight was also associated with increased mortality and morbidity. Seizures or supplementary oxygen at discharge were markers of disease course, which predicted a poorer outcome amongst survivors. These variables behaved similarly in the two trial groups. Those infants in the ECMO group with an episode of sepsis, established full sucking feeds after 14 days of age or a hospital stay over 30 days were at increased risk of disability. CONCLUSIONS: This study has identified clinical variables that predict adverse outcome for term infants with severe respiratory failure. The results may assist clinicians caring for these babies, when counseling their families and in the development of guidelines for neonatal ECMO.