Comprehensive Biotransformation Analysis of Phenylalanine-Tyrosine Metabolism Reveals Alternative Routes of Metabolite Clearance in Nitisinone-Treated Alkaptonuria

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2–16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2–0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia

Durability of a primary care-led weight-management intervention for remission of type 2 diabetes: 2-year results of the DiRECT open-label, cluster-randomised trial

Background: The DiRECT trial assessed remission of type 2 diabetes during a primary care-led weight-management programme. At 1 year, 68 (46%) of 149 intervention participants were in remission and 36 (24%) had achieved at least 15 kg weight loss. The aim of this 2-year analysis is to assess the durability of the intervention effect. Methods: DiRECT is an open-label, cluster-randomised, controlled trial done at primary care practices in the UK. Practices were randomly assigned (1:1) via a computer-generated list to provide an integrated structured weight-management programme (intervention) or best-practice care in accordance with guidelines (control), with stratification for study site (Tyneside or Scotland) and practice list size (>5700 or ≤5700 people). Allocation was concealed from the study statisticians; participants, carers, and study research assistants were aware of allocation. We recruited individuals aged 20–65 years, with less than 6 years' duration of type 2 diabetes, BMI 27–45 kg/m2, and not receiving insulin between July 25, 2014, and Aug 5, 2016. The intervention consisted of withdrawal of antidiabetes and antihypertensive drugs, total diet replacement (825–853 kcal per day formula diet for 12–20 weeks), stepped food reintroduction (2–8 weeks), and then structured support for weight-loss maintenance. The coprimary outcomes, analysed hierarchically in the intention-to-treat population at 24 months, were weight loss of at least 15 kg, and remission of diabetes, defined as HbA1c less than 6·5% (48 mmol/mol) after withdrawal of antidiabetes drugs at baseline (remission was determined independently at 12 and 24 months). The trial is registered with the ISRCTN registry, number 03267836, and follow-up is ongoing. Findings: The intention-to-treat population consisted of 149 participants per group. At 24 months, 17 (11%) intervention participants and three (2%) control participants had weight loss of at least 15 kg (adjusted odds ratio [aOR] 7·49, 95% CI 2·05 to 27·32; p=0·0023) and 53 (36%) intervention participants and five (3%) control participants had remission of diabetes (aOR 25·82, 8·25 to 80·84; p<0·0001). The adjusted mean difference between the control and intervention groups in change in bodyweight was −5·4 kg (95% CI −6·9 to −4·0; p<0·0001) and in HbA1c was −4·8 mmol/mol (–8·3 to −1·4 [–0·44% (–0·76 to −0·13)]; p=0·0063), despite only 51 (40%) of 129 patients in the intervention group using anti-diabetes medication compared with 120 (84%) of 143 in the control group. In a post-hoc analysis of the whole study population, of those participants who maintained at least 10 kg weight loss (45 of 272 with data), 29 (64%) achieved remission; 36 (24%) of 149 participants in the intervention group maintained at least 10 kg weight loss. Serious adverse events were similar to those reported at 12 months, but were fewer in the intervention group than in the control group in the second year of the study (nine vs 22). Interpretation: The DiRECT programme sustained remissions at 24 months for more than a third of people with type 2 diabetes. Sustained remission was linked to the extent of sustained weight loss

Caribbean Corals in Crisis: Record Thermal Stress, Bleaching, and Mortality in 2005

BACKGROUND The rising temperature of the world's oceans has become a major threat to coral reefs globally as the severity and frequency of mass coral bleaching and mortality events increase. In 2005, high ocean temperatures in the tropical Atlantic and Caribbean resulted in the most severe bleaching event ever recorded in the basin. METHODOLOGY/PRINCIPAL FINDINGS Satellite-based tools provided warnings for coral reef managers and scientists, guiding both the timing and location of researchers' field observations as anomalously warm conditions developed and spread across the greater Caribbean region from June to October 2005. Field surveys of bleaching and mortality exceeded prior efforts in detail and extent, and provided a new standard for documenting the effects of bleaching and for testing nowcast and forecast products. Collaborators from 22 countries undertook the most comprehensive documentation of basin-scale bleaching to date and found that over 80% of corals bleached and over 40% died at many sites. The most severe bleaching coincided with waters nearest a western Atlantic warm pool that was centered off the northern end of the Lesser Antilles. CONCLUSIONS/SIGNIFICANCE Thermal stress during the 2005 event exceeded any observed from the Caribbean in the prior 20 years, and regionally-averaged temperatures were the warmest in over 150 years. Comparison of satellite data against field surveys demonstrated a significant predictive relationship between accumulated heat stress (measured using NOAA Coral Reef Watch's Degree Heating Weeks) and bleaching intensity. This severe, widespread bleaching and mortality will undoubtedly have long-term consequences for reef ecosystems and suggests a troubled future for tropical marine ecosystems under a warming climate.This work was partially supported by salaries from the NOAA Coral Reef Conservation Program to the NOAA Coral Reef Conservation Program authors. NOAA provided funding to Caribbean ReefCheck investigators to undertake surveys of bleaching and mortality. Otherwise, no funding from outside authors' institutions was necessary for the undertaking of this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Comprehensive LC-QTOF-MS Metabolic Phenotyping Strategy: Application to Alkaptonuria.

BACKGROUND:Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS:A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS:Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS:The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU

A Phase I study evaluating the safety and immunogenicity of MVA85A, a candidate TB vaccine, in HIV-infected adults

Objectives Control of the tuberculosis (TB) epidemic is a global health priority and one that is likely to be achieved only through vaccination. The critical overlap with the HIV epidemic requires any effective TB vaccine regimen to be safe in individuals who are infected with HIV. The objectives of this clinical trial were to evaluate the safety and immunogenicity of a leading candidate TB vaccine, MVA85A, in healthy, HIV-infected adults. Design This was an open-label Phase I trial, performed in 20 healthy HIV-infected, antiretroviral-naïve subjects. Two different doses of MVA85A were each evaluated as a single immunisation in 10 subjects, with 24 weeks of follow-up. The safety of MVA85A was assessed by clinical and laboratory markers, including regular CD4 counts and HIV RNA load measurements. Vaccine immunogenicity was assessed by ex vivo interferon γ (IFN-γ) ELISpot assays and flow-cytometric analysis. Results MVA85A was safe in subjects with HIV infection, with an adverse-event profile comparable with historical data from previous trials in HIV-uninfected subjects. There were no clinically significant vaccine-related changes in CD4 count or HIV RNA load in any subjects, and no evidence from qPCR analyses to indicate that MVA85A vaccination leads to widespread preferential infection of vaccine-induced CD4 T cell populations. Both doses of MVA85A induced an antigen-specific IFN-γ response that was durable for 24 weeks, although of a lesser magnitude compared with historical data from HIV-uninfected subjects. The functional quality of the vaccine-induced T cell response in HIV-infected subjects was remarkably comparable with that observed in healthy HIV-uninfected controls, but less durable. Conclusion MVA85A is safe and immunogenic in healthy adults infected with HIV. Further safety and efficacy evaluation of this candidate vaccine in TB- and HIV-endemic areas is merited

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle

Characterisation of proteins in excretory/secretory products collected from salmon lice, Lepeophtheirus salmonis

Background  The salmon louse, Lepeophtheirus salmonis, is an ectoparasitic copepod which feeds on the mucus, skin and blood of salmonid fish species. The parasite can persist on the surface of the fish without any effective control being exerted by the host immune system. Other ectoparasitic invertebrates produce compounds in their saliva, excretions and/or secretions which modulate the host immune responses allowing them to remain on or in the host during development. Similarly, compounds are produced in secretions of L. salmonis which are thought to be responsible for immunomodulation of the host responses as well as other aspects of crucial host-parasite interactions.  Methods  In this study we have identified and characterised the proteins in the excretory/secretory (E/S) products of L. salmonis using LC-ESI-MS/MS.  Results  In total 187 individual proteins were identified in the E/S collected from adult lice and pre-adult sea lice. Fifty-three proteins, including 13 serine-type endopeptidases, 1 peroxidase and 5 vitellogenin-like proteins were common to both adult and pre-adult E/S products. One hundred and seven proteins were identified in the adult E/S but not in the pre-adult E/S and these included serine and cysteine-type endopeptidases, vitellogenins, sphingomyelinase and calreticulin. A total of 27 proteins were identified in pre-adult E/S products but not in adult E/S.  Conclusions  The assigned functions of these E/S products and the potential roles they play in host-parasite interaction is discussed

Improving climate suitability for Bemisia tabaci in East Africa is correlated with increased prevalence of whiteflies and cassava diseases

Projected climate changes are thought to promote emerging infectious diseases, though to date, evidence linking climate changes and such diseases in plants has not been available. Cassava is perhaps the most important crop in Africa for smallholder farmers. Since the late 1990's there have been reports from East and Central Africa of pandemics of begomoviruses in cassava linked to high abundances of whitefly species within the Bemisia tabaci complex. We used CLIMEX, a process-oriented climatic niche model, to explore if this pandemic was linked to recent historical climatic changes. The climatic niche model was corroborated with independent observed field abundance of B. tabaci in Uganda over a 13-year time-series, and with the probability of occurrence of B. tabaci over 2 years across the African study area. Throughout a 39-year climate time-series spanning the period during which the pandemics emerged, the modelled climatic conditions for B. tabaci improved significantly in the areas where the pandemics had been reported and were constant or decreased elsewhere. This is the first reported case where observed historical climate changes have been attributed to the increase in abundance of an insect pest, contributing to a crop disease pandemic

Feasibility of Human Neural Stem Cell Transplantation for the Treatment of Acute Subdural Hematoma in a Rat Model: A Pilot Study

Human neural stem cells (hNSCs) transplantation in several brain injury models has established their therapeutic potential. However, the feasibility of hNSCs transplantation is still not clear for acute subdural hematoma (ASDH) brain injury that needs external decompression. Thus, the aim of this pilot study was to test feasibility using a rat ASDH decompression model with two clinically relevant transplantation methods. Two different methods, in situ stereotactic injection and hNSC-embedded matrix seating on the brain surface, were attempted. Athymic rats were randomized to uninjured or ASDH groups (F344/NJcl-rnu/rnu, n = 7–10/group). Animals in injury group were subjected to ASDH, and received decompressive craniectomy and 1-week after decompression surgery were transplanted with green fluorescent protein (GFP)-transduced hNSCs using one of two approaches. Histopathological examinations at 4 and 8 weeks showed that the GFP-positive hNSCs survived in injured brain tissue, extended neurite-like projections resembling neural dendrites. The in situ transplantation group had greater engraftment of hNSCs than matrix embedding approach. Immunohistochemistry with doublecortin, NeuN, and GFAP at 8 weeks after transplantation showed that transplanted hNSCs remained as immature neurons and did not differentiate toward to glial cell lines. Motor function was assessed with rotarod, compared to control group (n = 10). The latency to fall from the rotarod in hNSC in situ transplanted rats was significantly higher than in control rats (median, 113 s in hNSC vs. 69 s in control, P = 0.02). This study first demonstrates the robust engraftment of in situ transplanted hNSCs in a clinically-relevant ASDH decompression rat model. Further preclinical studies with longer study duration are warranted to verify the effectiveness of hNSC transplantation in amelioration of TBI induced deficits