Long-term voluntary running prevents the onset of symptomatic Friedreich's ataxia in mice.

The common clinical symptoms of Friedreich's ataxia (FRDA) include ataxia, muscle weakness, type 2 diabetes and heart failure, which are caused by impaired mitochondrial function due to the loss of frataxin (FXN) expression. Endurance exercise is the most powerful intervention for promoting mitochondrial function; however, its impact on FRDA has not been studied. Here we found that mice with genetic knockout and knock-in of the Fxn gene (KIKO mice) developed exercise intolerance, glucose intolerance and moderate cardiac dysfunction at 6 months of age. These abnormalities were associated with impaired mitochondrial respiratory function concurrent with reduced iron regulatory protein 1 (Irp1) expression as well as increased oxidative stress, which were not due to loss of mitochondrial content and antioxidant enzyme expression. Importantly, long-term (4 months) voluntary running in KIKO mice starting at a young age (2 months) completely prevented the functional abnormalities along with restored Irp1 expression, improved mitochondrial function and reduced oxidative stress in skeletal muscle without restoring Fxn expression. We conclude that endurance exercise training prevents symptomatic onset of FRDA in mice associated with improved mitochondrial function and reduced oxidative stress. These preclinical findings may pave the way for clinical studies of the impact of endurance exercise in FRDA patients.

Cross-sectional study of the prevalence, causes and management of hospital-onset diarrhoea.

BACKGROUND: The National Health Service in England advises hospitals collect data on hospital-onset diarrhoea (HOD). Contemporaneous data on HOD are lacking. AIM: To investigate prevalence, aetiology and management of HOD on medical, surgical and elderly-care wards. METHODS: A cross-sectional study in a volunteer sample of UK hospitals, which collected data on one winter and one summer day in 2016. Patients admitted ≥72 h were screened for HOD (definition: ≥2 episodes of Bristol Stool Type 5-7 the day before the study, with diarrhoea onset >48 h after admission). Data on HOD aetiology and management were collected prospectively. FINDINGS: Data were collected on 141 wards in 32 hospitals (16 acute, 16 teaching). Point-prevalence of HOD was 4.5% (230/5142 patients; 95% confidence interval (CI) 3.9-5.0%). Teaching hospital HOD prevalence (5.9%, 95% CI 5.1-6.9%) was twice that of acute hospitals (2.8%, 95% CI 2.1-3.5%; odds ratio 2.2, 95% CI 1.7-3.0). At least one potential cause was identified in 222/230 patients (97%): 107 (47%) had a relevant underlying condition, 125 (54%) were taking antimicrobials, and 195 (85%) other medication known to cause diarrhoea. Nine of 75 tested patients were Clostridium difficile toxin positive (4%). Eighty (35%) patients had a documented medical assessment of diarrhoea. Documentation of HOD in medical notes correlated with testing for C. difficile (78% of those tested vs 38% not tested, P<0.001). One-hundred and forty-four (63%) patients were not isolated following diarrhoea onset. CONCLUSION: HOD is a prevalent symptom affecting thousands of patients across the UK health system each day. Most patients had multiple potential causes of HOD, mainly iatrogenic, but only a third had medical assessment. Most were not tested for C. difficile and were not isolated.

Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle.

Deterioration of neuromuscular junction (NMJ) integrity and function is causal to muscle atrophy and frailty, ultimately hindering quality of life and increasing the risk of death. In particular, NMJ is vulnerable to ischemia reperfusion (IR) injury when blood flow is restricted followed by restoration. However, little is known about the underlying mechanism(s) and hence the lack of effective interventions. New evidence suggests that mitochondrial oxidative stress plays a causal role in IR injury, which can be precluded by enhancing mitochondrial protein S-nitrosation (SNO). To elucidate the role of IR and mitochondrial protein SNO in skeletal muscle, we utilized a clinically relevant model and showed that IR resulted in significant muscle and motor nerve injuries with evidence of elevated muscle creatine kinase in the serum, denervation at NMJ, myofiber degeneration and regeneration, as well as muscle atrophy. Interestingly, we observed that neuromuscular transmission improved prior to muscle recovery, suggesting the importance of the motor nerve in muscle functional recovery. Injection of a mitochondria-targeted S-nitrosation enhancing agent, MitoSNO, into ischemic muscle prior to reperfusion reduced mitochondrial oxidative stress in the motor nerve and NMJ, attenuated denervation at NMJ, and resulted in accelerated functional recovery of the muscle. These findings demonstrate that enhancing mitochondrial protein SNO protects against IR-induced denervation at NMJ in skeletal muscle and accelerates functional regeneration. This could be an efficacious intervention for protecting neuromuscular injury under the condition of IR and other related pathological conditions.

Ampk phosphorylation of Ulk1 is required for targeting of mitochondria to lysosomes in exercise-induced mitophagy.

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3-12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer, that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle.Exercise is associated with biogenesis and removal of dysfunctional mitochondria. Here the authors use a mitochondrial reporter gene to demonstrate the occurrence of mitophagy following exercise in mice, and show this is dependent on AMPK and ULK1 signaling.

Design and chemical evaluation of reduced machine-yield cigarettes.

Experimental cigarettes (ECs) were made by combining technological applications that individually reduce the machine measured yields of specific toxicants or groups of toxicants in mainstream smoke (MS). Two tobacco blends, featuring a tobacco substitute sheet or a tobacco blend treatment, were combined with filters containing an amine functionalised resin (CR20L) and/or a polymer-derived, high activity carbon adsorbent to generate three ECs with the potential for generating lower smoke toxicant yields than conventional cigarettes. MS yields of smoke constituents were determined under 4 different smoking machine conditions. Health Canada Intense (HCI) machine smoking conditions gave the highest MS yields for nicotine-free dry particulate matter and for most smoke constituents measured. Toxicant yields from the ECs were compared with those from two commercial comparator cigarettes, three scientific control cigarettes measured contemporaneously and with published data on 120 commercial cigarettes. The ECs were found to generate some of the lowest machine yields of toxicants from cigarettes for which published HCI smoke chemistry data are available; these comparisons therefore confirm that ECs with reduced MS machine toxicant yields compared to commercial cigarettes can be produced. The results encourage further work examining human exposure to toxicants from these cigarettes, including human biomarker studies.

Intrinsic dynamics and synaptic inputs control the activity patterns of subthalamic nucleus neurons in health and in Parkinson's disease.

Neurons in the subthalamic nucleus occupy a pivotal position in the circuitry of the basal ganglia. They receive direct excitatory input from the cerebral cortex and the intralaminar nuclei of the thalamus, and directly excite the inhibitory basal ganglia output neurons in the internal segment of the globus pallidus and the substantia nigra. They are also engaged in a reciprocal synaptic arrangement with inhibitory neurons in the external segment of the globus pallidus. Although once viewed as a simple relay of extrinsic input to the basal ganglia, physiological studies of subthalamic neurons have revealed that activity in these neurons does not directly reflect their pattern of extrinsic excitation. Subthalamic neurons are autonomously active at rates comparable to those observed in vivo, and they generate complex patterns of intrinsic activity arising from the interactions between voltage sensitive ion channels on the somatodendritic and axonal membranes. Extrinsic synaptic excitation does not create the firing pattern of the subthalamic neuron, but rather controls the timing of action potentials generated intrinsically. The dopaminergic innervation of the subthalamic nucleus, although moderate, can directly influence firing patterns by acting both on synaptic transmission and voltage-sensitive ion channels responsible for intrinsic properties. Furthermore, chronic dopamine depletion in Parkinson's disease may modify both synaptic transmission and integration in the subthalamic nucleus, in addition to its effects on other regions of the basal ganglia.

Vaccination against hepatitis B in an HIV outpatients' department: an audit against national vaccination guidelines.

Practice related to hepatitis B vaccination of HIV outpatients in a London teaching hospital was audited against the British HIV Association (BHIVA) immunization guidelines 2004 and 2008, both before and after the implementation of a vaccination record sheet in the patients' notes. Adherence to the guidelines in the original audit was poor - only 67% of patients requiring vaccination for hepatitis B received a full course of vaccination. Following the introduction of the vaccination record sheet, this vaccination completion rate increased to 79% (BHIVA target 95%). Overall the percentage of patients managed according to BHIVA guidelines, including those who did not require vaccination, improved from 33% in the original audit to 61% in the re-audit. Introduction of a simple hepatitis B vaccination record sheet improved the quality of care for our HIV outpatients. Further modification of this system is warranted, perhaps by the introduction of a computerized reminder system.

Therapeutic lung lavage in the piglet model of meconium aspiration syndrome.

Therapeutic lung lavage is an emerging treatment for meconium aspiration syndrome. Our objective was to investigate the type of fluid and aliquot volume most appropriate for lung lavage in this condition. Meconium injury was induced in 2-week-old piglets, followed by a 30 ml/kg lavage in two aliquots 40 minutes later. Lavage with either dilute bovine surfactant (2.5 mg/ml) or a perfluorocarbon emulsion (20% wt/vol) improved oxygenation compared with a nonlavaged control group, but only with dilute surfactant was there a sustained improvement in oxygenation (alveolar-arterial oxygen difference at 5 hours: dilute surfactant 250 mm Hg; perfluorocarbon emulsion 460 mm Hg; controls 460 mm Hg; p = 0.0031). There was histologic and biochemical evidence of decreased lung injury in the dilute surfactant group. In a further study, 30 ml/kg dilute surfactant lavage was performed 40 minutes after meconium injury using either two aliquots of 15 ml/kg, or multiple 3-ml aliquots. Aliquot volume of 15 ml/kg was associated with increased meconium removal, better post-lavage lung function, and less lung injury. Dilute surfactant lavage using two 15-ml/kg aliquots is an effective therapy in the piglet model of meconium aspiration, and should be evaluated in human infants with this condition.

Regulation of the timing and pattern of action potential generation in rat subthalamic neurons in vitro by GABA-A IPSPs.

The regulation of activity in the subthalamic nucleus (STN) by GABAergic inhibition from the reciprocally connected globus pallidus (GP) plays an important role in normal movement and disorders of movement. To determine the precise manner in which GABAergic synaptic input, acting at A-type receptors, influences the firing of STN neurons, we recorded the response of STN neurons to GABA-A inhibitory postsynaptic potentials (IPSPs) that were evoked by supramaximal electrical stimulation of the internal capsule using the perforated-patch technique in slices at 37 degrees C. The mean equilibrium potential of the GABA-A IPSP (EGABA-A IPSP) was -79.4 +/- 7.0 mV. Single IPSPs disrupted the spontaneous oscillation that underlies rhythmic single-spike firing in STN neurons. As the magnitude of IPSPs increased, the effectiveness of prolonging the interspike interval was related more strongly to the phase of the oscillation at which the IPSP was evoked. Thus the largest IPSPs tended to reset the oscillatory cycle, whereas the smallest IPSPs tended to produce relatively phase-independent delays in firing. Multiple IPSPs were evoked at various frequencies and over different periods and their impact was studied on STN neurons held at different levels of polarization. Multiple IPSPs reduced and/or prevented action potential generation and/or produced sufficient hyperpolarization to activate a rebound depolarization, which generated a single spike or restored rhythmic spiking and/or generated a burst of activity. The pattern of IPSPs and the level of polarization of STN neurons were critical in determining the nature of the response. The duration of bursts varied from 20 ms to several hundred milliseconds, depending on the intrinsic rebound properties of the postsynaptic neuron. These data demonstrate that inhibitory input from the GP can produce a range of firing patterns in STN neurons, depending on the number and frequencies of IPSPs and the membrane properties and voltage of the postsynaptic neuron.

MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat.

We have undertaken a systematic reverse genetic approach to understand R2R3-MYB gene function in Arabidopsis. Here, we report the functional characterization of MYB61 based on the phenotype of three independent insertion alleles. Wide-ranging phenotype screens indicated that MYB61 mutants were deficient in seed mucilage extrusion upon imbibition. This phenotype was expressed in the sporophytic tissues of the seed. Deposition and extrusion of the principal component of the mucilage, a relatively unbranched rhamnogalacturonan, were reduced in the MYB61 mutant seed coats. Additional defects in the maturation of the testa epidermal cells suggested a potential deficiency in extracellular secretion in myb61 lines. Consistent with a proposed role in testa development, reverse transcription-polymerase chain reaction analysis showed the highest MYB61 expression in siliques, which was localized to the seed coat by a beta-glucuronidase (GUS) reporter gene fusion. Lower levels of GUS expression were detected in developing vascular tissue. Parallel analysis of the ttg1-1 mutant phenotype indicated that this mutant showed more severe developmental defects than myb61 and suggested that MYB61 may function in a genetic pathway distinct from that of TTG1. The transient nature of seed epidermal characteristics in the ttg1-1 mutant suggested that TTG1 was required for maintenance rather than initiation of testa epidermal differentiation. Germination and seedling establishment were compromised in the myb61 and ttg1-1 mutants under conditions of reduced water potential, suggesting a function for Arabidopsis seed mucilage during germination in dry conditions.

Identification and characterization of thymus LIM protein: targeted disruption reduces thymus cellularity.

We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag(-/-) OT-1) compared to its expression in a thymus in which selection is blocked at the CD4+ CD8+ stage of T-cell development (Rag(-/-) Tap(-/-) OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation.

Dopamine regulates the impact of the cerebral cortex on the subthalamic nucleus-globus pallidus network.

The subthalamic nucleus-globus pallidus network plays a central role in basal ganglia function and dysfunction. To determine whether the relationship between activity in this network and the principal afferent of the basal ganglia, the cortex, is altered in a model of Parkinson's disease, we recorded unit activity in the subthalamic nucleus-globus pallidus network together with cortical electroencephalogram in control and 6-hydroxydopamine-lesioned rats under urethane anaesthesia. Subthalamic nucleus neurones in control and 6-hydroxydopamine-lesioned animals exhibited low-frequency oscillatory activity, which was tightly correlated with cortical slow-wave activity (approximately 1 Hz). The principal effect of dopamine depletion was that subthalamic nucleus neurones discharged more intensely (233% of control) and globus pallidus neurones developed low-frequency oscillatory firing patterns, without changes in mean firing rate. Ipsilateral cortical ablation largely abolished low-frequency oscillatory activity in the subthalamic nucleus and globus pallidus. These data suggest that abnormal low-frequency oscillatory activity in the subthalamic nucleus-globus pallidus network in the dopamine-depleted state is generated by the inappropriate processing of rhythmic cortical input. A component (15-20%) of the network still oscillated following cortical ablation in 6-hydroxydopamine-lesioned animals, implying that intrinsic properties may also pattern activity when dopamine levels are reduced. The response of the network to global activation was altered by 6-hydroxydopamine lesions. Subthalamic nucleus neurones were excited to a greater extent than in control animals and the majority of globus pallidus neurones were inhibited, in contrast to the excitation elicited in control animals. Inhibitory responses of globus pallidus neurones were abolished by cortical ablation, suggesting that the indirect pathway is augmented abnormally during activation of the dopamine-depleted brain. Taken together, these results demonstrate that both the rate and pattern of activity of subthalamic nucleus and globus pallidus neurones are altered profoundly by chronic dopamine depletion. Furthermore, the relative contribution of rate and pattern to aberrant information coding is intimately related to the state of activation of the cerebral cortex.

Antigen presentation to CD8+ T cells: cross-priming in infectious diseases.

Recent studies indicate that, in most types of infections, antigen presentation by 'professional' bone-marrow-derived cells is essential for priming pathogen-specific CD8+ T cells. This is true even in the absence of direct infection of these cells, which indicates that cross-priming is an essential component of the immune response against pathogens.

Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signalling.

Plants both produce and utilize carbohydrates and have developed mechanisms to regulate their sugar status and co-ordinate carbohydrate partitioning. High sugar levels result in a feedback inhibition of photosynthesis and an induction of storage processes. We used a genetic approach to isolate components of the signalling pathway regulating the induction of starch biosynthesis. The regulatory sequences of the sugar inducible ADP-glucose pyrophosphorylase subunit ApL3 were fused to a negative selection marker. Of the four impaired sucrose induction (isi) mutants described here, two (isi1 and isi2) were specific to this screen. The other two mutants (isi3 and isi4) showed additional phenotypes associated with sugar-sensing screens that select for seedling establishment on high-sugar media. The isi3 and isi4 mutants were found to be involved in the abscisic acid signalling pathway. isi3 is allelic to abscisic acid insensitive4 (abi4), a gene encoding an Apetala2-type transcription factor; isi4 was found to be allelic to glucose insensitive1 (gin1) previously reported to reveal cross-talk between ethylene and glucose signalling. Here we present an alternative interpretation of gin1 as an allele of the ABA-deficient mutant aba2. Expression analysis showed that ABA is unable to induce ApL3 gene expression by itself, but greatly enhances ApL3 induction by sugar. Our data suggest a major role for ABA in relation to sugar-signalling pathways, in that it enhances the ability of tissues to respond to subsequent sugar signals.

Conservation of microstructure between a sequenced region of the genome of rice and multiple segments of the genome of Arabidopsis thaliana.

The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that the Arabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes.