The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle

For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.-Zolnerciks, J. K., Akkaya, B. G., Snippe, M., Chiba, P., Seelig, A., Linton, K. J. The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle

Food and nutrient intake in relation to mental wellbeing

BACKGROUND: We studied food consumption and nutrient intake in subjects with depressed mood, anxiety and insomnia as indices of compromised mental wellbeing. METHODS: The study population consisted of 29,133 male smokers aged 50 to 69 years who entered the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study in 1985–1988. This was a placebo-controlled trial to test whether supplementation with alpha-tocopherol or beta-carotene prevents lung cancer. At baseline 27,111 men completed a diet history questionnaire from which food and alcohol consumption and nutrient intake were calculated. The questionnaire on background and medical history included three symptoms on mental wellbeing, anxiety, depression and insomnia experienced in the past four months. RESULTS: Energy intake was higher in men who reported anxiety or depressed mood, and those reporting any such symptoms consumed more alcohol. Subjects reporting anxiety or depressed mood had higher intake of omega-3 fatty acids and omega-6 fatty acids. CONCLUSIONS: Our findings conflict with the previous reports of beneficial effects of omega-3 fatty acids on mood

Effects of acute tryptophan depletion on executive function in healthy male volunteers

BACKGROUND: Neurocognitive impairment is frequently described in a number of psychiatric disorders and may be a direct consequence of serotonergic dysfunction. As impairments in executive functions are some of the most frequently described, the purpose of this study was to examine the performance of normal volunteers on a range of executive tasks following a transient reduction of central serotonin (5-HT) levels using the method of acute tryptophan depletion (ATD). METHODS: Fifteen healthy male subjects participated in a within-subject, double-blind, counterbalanced crossover study. ATD was induced by ingestion of a 100 g amino-acid drink. Executive function was evaluated using the Wisconsin Card Sorting Test, Stroop, Verbal Fluency and Trail Making. Visual analogue scales were administered to assess mood. RESULTS: Plasma free and total tryptophan concentrations were significantly reduced by the depleting drink (P < 0.001). ATD selectively improved motor speed/ attention on the Trails A test (P = 0.027), with no effect on subjective ratings of mood. Interaction effects between drink and the order of drink administration were observed on most neurocognitive tests. CONCLUSIONS: The improvement in simple motor speed/ attention following ATD is in keeping with the ascribed role of 5-HT in the cortex, however performance on tests of executive function is not robustly altered. The presence of interaction effects on most tasks suggests that subtle changes may occur but are masked, possibly by simple learning effects, in the context of a crossover design. This has implications for the design of future studies, particularly those examining executive functions

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9 Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta

The potential for immunoglobulins and host defense peptides (HDPs) to reduce the use of antibiotics in animal production

Abstract Innate defense mechanisms are aimed at quickly containing and removing infectious microorganisms and involve local stromal and immune cell activation, neutrophil recruitment and activation and the induction of host defense peptides (defensins and cathelicidins), acute phase proteins and complement activation. As an alternative to antibiotics, innate immune mechanisms are highly relevant as they offer rapid general ways to, at least partially, protect against infections and enable the build-up of a sufficient adaptive immune response. This review describes two classes of promising alternatives to antibiotics based on components of the innate host defense. First we describe immunoglobulins applied to mimic the way in which they work in the newborn as locally acting broadly active defense molecules enforcing innate immunity barriers. Secondly, the potential of host defense peptides with different modes of action, used directly, induced in situ or used as vaccine adjuvants is described

Hypnosis Antenatal Training for Childbirth (HATCh): a randomised controlled trial [NCT00282204]

BACKGROUND: Although medical interventions play an important role in preserving lives and maternal comfort they have become increasingly routine in normal childbirth. This may increase the risk of associated complications and a less satisfactory birth experience. Antenatal hypnosis is associated with a reduced need for pharmacological interventions during childbirth. This trial seeks to determine the efficacy or otherwise of antenatal group hypnosis preparation for childbirth in late pregnancy. METHODS/DESIGN: A single centre, randomised controlled trial using a 3 arm parallel group design in the largest tertiary maternity unit in South Australia. Group 1 participants receive antenatal hypnosis training in preparation for childbirth administered by a qualified hypnotherapist with the use of an audio compact disc on hypnosis for re-enforcement; Group 2 consists of antenatal hypnosis training in preparation for childbirth using an audio compact disc on hypnosis administered by a nurse with no training in hypnotherapy; Group 3 participants continue with their usual preparation for childbirth with no additional intervention. Women > 34 and < 39 weeks gestation, planning a vaginal birth, not in active labour, with a singleton, viable fetus of vertex presentation, are eligible to participate. Allocation concealment is achieved using telephone randomisation. Participants assigned to hypnosis groups commence hypnosis training as near as possible to 37 weeks gestation. Treatment allocations are concealed from treating obstetricians, anaesthetists, midwives and those personnel collecting and analysing data. Our sample size of 135 women/group gives the study 80% power to detect a clinically relevant fall of 20% in the number of women requiring pharmacological analgesia – the primary endpoint. We estimate that approximately 5–10% of women will deliver prior to receiving their allocated intervention. We plan to recruit 150 women/group and perform sequential interim analyses when 150 and 300 participants have been recruited. All participant data will be analysed, by a researcher blinded to treatment allocation, according to the "Intention to treat" principle with comprehensive pre-planned cost- benefit and subgroup analyses. DISCUSSION: If effective, hypnosis would be a simple, inexpensive way to improve the childbirth experience, reduce complications associated with pharmacological interventions, yield cost savings in maternity care, and this trial will provide evidence to guide clinical practice

Distinct colonization patterns and cDNA-AFLP transcriptome profiles in compatible and incompatible interactions between melon and different races of Fusarium oxysporum f. sp. melonis

Background: Fusarium oxysporum f. sp. melonis Snyd. & Hans. (FOM) causes Fusarium wilt, the most important infectious disease of melon (Cucumis melo L.). The four known races of this pathogen can be distinguished only by infection on appropriate cultivars. No molecular tools are available that can discriminate among the races, and the molecular basis of compatibility and disease progression are poorly understood. Resistance to races 1 and 2 is controlled by a single dominant gene, whereas only partial polygenic resistance to race 1,2 has been described. We carried out a large-scale cDNA-AFLP analysis to identify host genes potentially related to resistance and susceptibility as well as fungal genes associated with the infection process. At the same time, a systematic reisolation procedure on infected stems allowed us to monitor fungal colonization in compatible and incompatible host-pathogen combinations. Results: Melon plants (cv. Charentais Fom-2), which are susceptible to race 1,2 and resistant to race 1, were artificially infected with a race 1 strain of FOM or one of two race 1,2 w strains. Host colonization of stems was assessed at 1, 2, 4, 8, 14, 16, 18 and 21 days post inoculation (dpi), and the fungus was reisolated from infected plants. Markedly different colonization patterns were observed in compatible and incompatible host-pathogen combinations. Five time points from the symptomless early stage (2 dpi) to obvious wilting symptoms (21 dpi) were considered for cDNA-AFLP analysis. After successful sequencing of 627 transcript-derived fragments (TDFs) differentially expressed in infected plants, homology searching retrieved 305 melon transcripts, 195 FOM transcripts expressed in planta and 127 orphan TDFs. RNA samples from FOM colonies of the three strains grown in vitro were also included in the analysis to facilitate the detection of in planta-specific transcripts and to identify TDFs differentially expressed among races/strains. Conclusion: Our data suggest that resistance against FOM in melon involves only limited transcriptional changes, and that wilting symptoms could derive, at least partially, from an active plant response. We discuss the pathogen-derived transcripts expressed in planta during the infection process and potentially related to virulence functions, as well as transcripts that are differentially expressed between the two FOM races grown in vitro. These transcripts provide candidate sequences that can be further tested for their ability to distinguish between races. Sequence data from this article have been deposited in GenBank, Accession Numbers: HO867279-HO867981

A single active catalytic site is sufficient to promote transport in P-glycoprotein

P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis

withdrawn 2017 hrs ehra ecas aphrs solaece expert consensus statement on catheter and surgical ablation of atrial fibrillation

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