The identification and characterisation of novel antimicrobial resistance genes from human and animal metagenomes

Antimicrobial resistance genes are harboured by bacteria in the human oral cavity and ruminant faeces and they are shed in particularly high abundances in calf faeces. Furthermore, bacteriocin (antimicrobial peptide) producing bacteria have been isolated from these environments. In recent times bacteriocins have received much attention as potential alternatives to antibiotics. Human saliva and calf faeces harbour ‘yet-to-be cultured bacteria’ that can only be studied by analysing their DNA. To this end, two metagenomic libraries were created from human saliva and calf faeces metagenomic DNA with the aim of identifying novel antimicrobial resistance and bacteriocin genes. Screening these libraries for tetracycline resistance identified two tetracycline resistant clones. Clone PS9 was also tigecycline resistant and contained a 7,765 bp insert that encoded two half-ABC transporter genes; subcloning of these genes showed that they were responsible for the observed resistance phenotype. As the ABC transporter conferred resistance only to tetracyclines and its putative amino acid sequence showed <80 % identity to known tetracycline resistance proteins, it was named TetAB(60). Clone TT31 contained a 14,226 bp insert. 7, 216 bp of the insert had 97 % nucleotide identity to Tn916 and contained part of tet(M) and a full length tet(L) gene. This gene organisation has not been described in Tn916-like elements and it may represent a novel Tn916-like element. The human saliva library was also screened for antiseptic resistance revealing a CTAB resistant clone. Random transposon mutagenesis of the 19.1 Kb insert and subcloning of a UDP-glucose 4-epimerase revealed it to be solely required for the observed resistance. This study identified novel tetracycline, tigecycline and CTAB resistance genes from the human saliva metagenome, demonstrating the importance of this environment as a source of resistance genes that may compromise the effectiveness of these antibiotics and antimicrobials. Additionally, this work highlights the relevance of house-keeping genes to the development of antimicrobial resistance

Patterns of regional variation of opioid prescribing in primary care in England: a retrospective observational study

BACKGROUND: Opioids are a widely prescribed class of drug with potentially harmful short-term and long-term side effects. There are concerns about the amounts of these drugs being prescribed in England given that they are increasingly considered ineffective in the context of long-term non-cancer pain, which is one of the major reasons for their prescription. AIM: To assess the amount and type of opioids prescribed in primary care in England, and patterns of regional variation in prescribing. DESIGN AND SETTING: Retrospective observational study using publicly available government data from various sources pertaining to opioids prescribed in primary practice in England and Indices of Social Deprivation. METHOD: Official government data were analysed for opioid prescriptions from August 2010 to February 2014. The total amount of opioid prescribed was calculated and standardised to allow for geographical comparisons. RESULTS: The total amount of opioid prescribed, in equivalent milligrams of morphine, increased (r = 0.48) over the study period. More opioids were prescribed in the north than in the south of England (r = 0.66, P<0.0001), and more opioids were prescribed in areas of greater social deprivation (r = 0.56, P<0.0001). CONCLUSION: Long-term opioid prescribing is increasing despite poor efficacy for non-cancer pain, potential harm, and incompatibility with best practice. Questions of equality of care arise from higher prescription rates in the north of England and in areas of greater social deprivation. A national registry of patients with high opioid use would improve patient safety for this high-risk demographic, as well as provide more focused epidemiological data regarding patterns of prescribing

Toward a conceptual framework of the acceptability of tuberculosis treatment in children using a theory generative approach

To describe an early-stage holistic framework towards evaluating factors that impact the overall acceptability of TB treatment along the TB care cascade in children. We developed a conceptual framework utilising a theory generative approach. Domains were developed through review of existing definitions and analysis of existing qualitative data undertaken in acceptability studies of TB treatment in children. Clarity of domain definitions was achieved through iterative refinement among the research team. Three domains, each comprising several dimensions, were identified to holistically evaluate treatment acceptability: (1) usability, which involves the alignment between the requirements of treatment use and caregivers’ and children’s ability to integrate TB treatment into their everyday routines, (2) receptivity, which describes the end-user’s perception and expectations of treatment and its actual use, and (3) integration, which describes the relationship between available health services and caregivers/children’s capacity to make use of those services. Our framework addresses the gaps in current research which do not account for the influence of caregivers’ and children’s contexts on TB treatment uptake and overall acceptability. This approach may support the development of more standard, holistic measures to improve TB treatment delivery and experiences and future research in children

Parental social contact in the work place and the risk of childhood acute lymphoblastic leukaemia

To study the possible relation between parental social contact through occupation, a marker for a child's risk of infection, and childhood acute lymphoblastic leukaemia (ALL), the parents of 294 children with ALL aged 0–14.9 years and 376 matched controls were interviewed about their jobs after their child's birth up to the age of 3 years. Job titles were assigned to a level of social contact, and an index of occupational social contact months was created using the level and the job duration. Positive interactions between this index and rural residence associated with an increased risk of childhood ALL and common ALL (c-ALL) were observed (interaction P-value=0.02 for both, using tertiles of contact months; interaction P-value=0.05 and 0.02 for ALL and c-ALL, respectively, using continuous contact months); such findings were not observed when job durations were ignored. Our data suggest that duration of parental occupation may be important when examining the association between parental social contact in the workplace and childhood leukaemia

Critical evaluation of current data analysis strategies for psychophysiological measures of fear conditioning and extinction in humans

Fear conditioning and extinction is a construct integral to understanding trauma-, stress- and anxiety-related disorders. In the laboratory, associative learning paradigms that pair aversive with neutral stimuli are used as analogues to real-life fear learning. These studies use physiological indices, such as skin conductance, to sensitively measure rates and intensity of learning and extinction. In this review, we discuss some of the potential limitations in interpreting and analysing physiological data during the acquisition or extinction of conditioned fear. We argue that the utmost attention should be paid to the development of modelling approaches of physiological data in associative learning paradigms, by illustrating the lack of replicability and interpretability of results in current methods. We also show that statistical significance may be easily achieved in this paradigm without more stringent data and data analysis reporting requirements, leaving this particular field vulnerable to misleading conclusions. This review is written so that issues and potential solutions are accessible to researchers without mathematical training. We conclude the review with some suggestions that all laboratories should be able to implement, including visualising the full data set in publications and adopting modelling, or at least regression-based, approaches

Mapped aboveground carbon stocks to advance forest conservation and recovery in Malaysian Borneo

Forest carbon stocks in rapidly developing tropical regions are highly heterogeneous, which challenges efforts to develop spatially-explicit conservation actions. In addition to field-based biodiversity information, mapping of carbon stocks can greatly accelerate the identification, protection and recovery of forests deemed to be of high conservation value (HCV). We combined airborne Light Detection and Ranging (LiDAR) with satellite imaging and other geospatial data to map forest aboveground carbon density at 30m (0.09ha) resolution throughout the Malaysian state of Sabah on the island of Borneo. We used the mapping results to assess how carbon stocks vary spatially based on forest use, deforestation, regrowth, and current forest protections. We found that unlogged, intact forests contain aboveground carbon densities averaging over 200MgCha−1, with peaks of 500MgCha−1. Critically, more than 40% of the highest carbon stock forests were discovered outside of areas designated for maximum protection. Previously logged forests have suppressed, but still high, carbon densities of 60–140MgCha−1. Our mapped distributions of forest carbon stock suggest that the state of Sabah could double its total aboveground carbon storage if previously logged forests are allowed to recover in the future. Our results guide ongoing efforts to identify HCV forests and to determine new areas for forest protection in Borneo

The human liver microenvironment shapes the homing and function of CD4+ T-cell populations.

OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance

Deletion of the GABAA α2-subunit does not alter self dministration of cocaine or reinstatement of cocaine seeking

Rationale GABAA receptors containing α2-subunits are highly represented in brain areas that are involved in motivation and reward, and have been associated with addiction to several drugs, including cocaine. We have shown previously that a deletion of the α2-subunit results in an absence of sensitisation to cocaine. Objective We investigated the reinforcing properties of cocaine in GABAA α2-subunit knockout (KO) mice using an intravenous self-administration procedure. Methods α2-subunit wildtype (WT), heterozygous (HT) and KO mice were trained to lever press for a 30 % condensed milk solution. After implantation with a jugular catheter, mice were trained to lever press for cocaine (0.5 mg/kg/infusion) during ten daily sessions. Responding was extinguished and the mice tested for cue- and cocaine-primed reinstatement. Separate groups of mice were trained to respond for decreasing doses of cocaine (0.25, 0.125, 0.06 and 0.03 mg/kg). Results No differences were found in acquisition of lever pressing for milk. All genotypes acquired self-administration of cocaine and did not differ in rates of self-administration, dose dependency or reinstatement. However, whilst WT and HT mice showed a dose-dependent increase in lever pressing during the cue presentation, KO mice did not. Conclusions Despite a reported absence of sensitisation, motivation to obtain cocaine remains unchanged in KO and HT mice. Reinstatement of cocaine seeking by cocaine and cocaine-paired cues is also unaffected. We postulate that whilst not directly involved in reward perception, the α2-subunit may be involved in modulating the “energising” aspect of cocaine’s effects on reward-seeking