Daf-16 mediated repression of cytosolic ribosomal protein genes facilitates a hypoxia sensitive to hypoxia resistant transformation in long-lived germline mutants.

In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or "super-resistance", to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.

Functional analysis of a de novo variant in the neurodevelopment and generalized epilepsy disease gene NBEA.

Neurobeachin (NBEA) was initially identified as a candidate gene for autism. Recently, variants in NBEA have been associated with neurodevelopmental delay and childhood epilepsy. Here, we report on a novel NBEA missense variant (c.5899G > A, p.Gly1967Arg) in the Domain of Unknown Function 1088 (DUF1088) identified in a child enrolled in the Undiagnosed Diseases Network (UDN), who presented with neurodevelopmental delay and seizures. Modeling of this variant in the Caenorhabditis elegans NBEA ortholog, sel-2, indicated that the variant was damaging to in vivo function as evidenced by altered cell fate determination and trafficking of potassium channels in neurons. The variant effect was indistinguishable from that of the reference null mutation suggesting that the variant is a strong hypomorph or a complete loss-of-function. Our experimental data provide strong support for the molecular diagnosis and pathogenicity of the NBEA p.Gly1967Arg variant and the importance of the DUF1088 for NBEA function.

Light at the end of the tunnel: Visitors' virtual reality (versus in-person) attraction site tour-related behavioral intentions during and post-COVID-19.

Consumer behavior is changing as a result of the COVID-19 pandemic, thus compelling attraction sites to find new ways of offering safe tours to visitors. Based on protection motivation theory, we develop and test a model that examines key drivers of visitors' COVID-19-induced social distancing behavior and its effect on their intent to use virtual reality-based (vs. in-person) attraction site tours during and post-COVID-19. Our analyses demonstrate that visitor-perceived threat severity, response efficacy, and self-efficacy raise social distancing behavior. In turn, social distancing increases (decreases) visitors' intent to use virtual reality (in-person) tours during the pandemic. We find social distancing to boost visitors' demand for advanced virtual tours and to raise their advocacy intentions. Our results also reveal that social distancing has no effect on potential visitors' intent to use virtual reality vs. in-person tours post-the pandemic. We conclude by discussing vital implications that stem from our analyses.

Microbiological profile and antimicrobial resistance among diabetic foot infections in Lebanon.

Diabetic Foot Infection (DFI) is a challenging complication of diabetes mellitus with a high burden in the Middle East where there is a marked increase in diabetes prevalence and complications. Early detection of DFI and the infectious organisms could result in the early initiation of appropriate antibiotic therapy and improved outcomes. DFI microbiological profiles differ between countries. In our region, Western guidelines are used when initiating treatment for DFI in the absence of local guidance. The purpose of our study was to determine the microbiologic profile and antimicrobial susceptibility of the DFI admissions at a large tertiary referral centre in Beirut and review other reported series in Lebanon and our region. This is a retrospective observational study of patients with DFI admitted to the American University of Beirut Medical Centre from January 2008 to June 2017. The bacteriologic isolation and antimicrobial susceptibility tests were performed according to standard microbiological methods. Between 2008 and 2017, 319 diabetic patients with DFU were admitted to AUBMC, and deep-tissue cultures were taken for 179 patients. From 179 deep tissue cultures, 314 bacterial isolates were obtained. Fifty-four percent of patients had the polymicrobial infection. Aerobic gram-negative rods (GNR) were more prevalent than gram-positive cocci (GPC) (55%, 39%, respectively). The most common isolate was Escherichia coli (15%) followed by Enterococcus (14%) and Pseudomonas aeruginosa (11%). Staphylococcus aureus isolates accounted for 9% with 50% of them being methicillin-resistant (MRSA). Among Enterobacteriaceae, 37% of isolates were fluoroquinolone-resistant, 25% were ESBL producers, and 2% were carbapenem-resistant. Antibiotic resistance was significantly associated with prior usage of antibiotics. Anaerobes were isolated in 1% and Candida species in 5% of isolates. The sensitivity, specificity, PPV, and NPV of swab culture recovery of pathogens compared with deep tissue culture were (76%, 72%, 76%, 72%) and (94%, 81%, 91%, 86%) for gram-positive and gram-negative organisms, respectively. The microbiological profile of DFI in Lebanon is comparable to other countries in the MENA region with big differences compared with the West. Therefore, it is imperative to develop local guidelines for antimicrobial treatment. The high prevalence of GNR in DFI and the high fluoroquinolone resistance should be taken into consideration when choosing empiric antibiotics. Empiric treatment for MRSA or Pseudomonas does not appear necessary except for patients with specific risk factors.

Assessing mucociliary transport of single particles in vivo shows variable speed and preference for the ventral trachea in newborn pigs.

Mucociliary transport (MCT) is an innate defense mechanism that removes particulates, noxious material, and microorganisms from the lung. Several airway diseases exhibit abnormal MCT, including asthma, chronic bronchitis, and cystic fibrosis. However, it remains uncertain whether MCT abnormalities contribute to the genesis of disease or whether they are secondary manifestations that may fuel disease progression. Limitations of current MCT assays and of current animal models of human disease have hindered progress in addressing these questions. Therefore, we developed an in vivo assay of MCT, and here we describe its use in newborn wild-type pigs. We studied pigs because they share many physiological, biochemical, and anatomical features with humans and can model several human diseases. We used X-ray multidetector-row-computed tomography to track movement of individual particles in the large airways of newborn pigs. Multidetector-row-computed tomography imaging provided high spatial and temporal resolution and registration of particle position to airway anatomy. We discovered that cilia orientation directs particles to the ventral tracheal surface. We also observed substantial heterogeneity in the rate of individual particle movement, and we speculate that variations in mucus properties may be responsible. The increased granularity of MCT data provided by this assay may provide an opportunity to better understand host defense mechanisms and the pathogenesis of airway disease.

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.

Presentation of Gastrointestinal Stromal Tumor, Pancreatic Adenocarcinoma, and Gastric Adenocarcinoma in a Woman With No Identifiable Genetic Abnormalities.

This report details the first known case of co-occurrence of gastrointestinal stromal tumor (GIST), pancreatic adenocarcinoma, and gastric adenocarcinoma in an individual with no identifiable genetic abnormalities. The patient is a 57-year-old female who presented with abdominal pain. CT scan demonstrated a pancreatic mass, and endoscopic ultrasound demonstrated an additional gastric mass. Biopsy of both masses demonstrated adenocarcinoma; however, the masses were found to have different origins. The patient underwent neoadjuvant chemotherapy with excellent response. She then underwent surgical intervention, which demonstrated no ongoing adenocarcinoma in either location, and demonstrated a small focus of GIST. Genetic testing demonstrated no identifiable abnormalities. The presence of 3 primary neoplasms in an individual with no known genetic mutations represents a novel case study. These findings may suggest that screening for additional primary neoplasms may be indicated, even in patients for whom metastatic disease is initially suspected.

Distinct Mechanisms Underlie Electrical Coupling Resonance and Its Interaction with Membrane Potential Resonance.

Neurons in oscillatory networks often exhibit membrane potential resonance, a peak impedance at a non-zero input frequency. In electrically coupled oscillatory networks, the coupling coefficient (the ratio of post- and prejunctional voltage responses) could also show resonance. Such coupling resonance may emerge from the interaction between the coupling current and resonance properties of the coupled neurons, but this relationship has not been clearly described. Additionally, it is unknown if the gap-junction mediated electrical coupling conductance may have frequency dependence. We examined these questions by recording a pair of electrically coupled neurons in the oscillatory pyloric network of the crab Cancer borealis. We performed dual current- and voltage-clamp recordings and quantified the frequency preference of the coupled neurons, the coupling coefficient, the electrical conductance, and the postjunctional neuronal response. We found that all components exhibit frequency selectivity, but with distinct preferred frequencies. Mathematical and computational analysis showed that membrane potential resonance of the postjunctional neuron was sufficient to give rise to resonance properties of the coupling coefficient, but not the coupling conductance. A distinct coupling conductance resonance frequency therefore emerges either from other circuit components or from the gating properties of the gap junctions. Finally, to explore the functional effect of the resonance of the coupling conductance, we examined its role in synchronizing neuronal the activities of electrically coupled bursting model neurons. Together, our findings elucidate factors that produce electrical coupling resonance and the function of this resonance in oscillatory networks.

Comodulation reduces interindividual variability of circuit output.

Ionic current levels of identified neurons vary substantially across individual animals. Yet, under similar conditions, neural circuit output can be remarkably similar, as evidenced in many motor systems. All neural circuits are influenced by multiple neuromodulators which provide flexibility to their output. These neuromodulators often overlap in their actions by modulating the same channel type or synapse, yet have neuron-specific actions resulting from distinct receptor expression. Because of this different receptor expression pattern, in the presence of multiple convergent neuromodulators, a common downstream target would be activated more uniformly in circuit neurons across individuals. We therefore propose that a baseline tonic (non-saturating) level of comodulation by convergent neuromodulators can reduce interindividual variability of circuit output. We tested this hypothesis in the pyloric circuit of the crab, Cancer borealis. Multiple excitatory neuropeptides converge to activate the same voltage-gated current in this circuit, but different subsets of pyloric neurons have receptors for each peptide. We quantified the interindividual variability of the unmodulated pyloric circuit output by measuring the activity phases, cycle frequency and intraburst spike number and frequency. We then examined the variability in the presence of different combinations and concentrations of three neuropeptides. We found that at mid-level concentration (30 nM) but not at near-threshold (1 nM) or saturating (1 µM) concentrations, comodulation by multiple neuropeptides reduced the circuit output variability. Notably, the interindividual variability of response properties of an isolated neuron was not reduced by comodulation, suggesting that the reduction of output variability may emerge as a network effect.

The Antinarcolepsy Drug Modafinil Reverses Hypoglycemia Unawareness and Normalizes Glucose Sensing of Orexin Neurons in Male Mice.

Perifornical hypothalamus (PFH) orexin glucose-inhibited (GI) neurons that facilitate arousal have been implicated in hypoglycemia awareness. Mice lacking orexin exhibit narcolepsy, and orexin mediates the effect of the antinarcolepsy drug modafinil. Thus, hypoglycemia awareness may require a certain level of arousal for awareness of the sympathetic symptoms of hypoglycemia (e.g., tremors, anxiety). Recurrent hypoglycemia (RH) causes hypoglycemia unawareness. We hypothesize that RH impairs the glucose sensitivity of PFH orexin GI neurons and that modafinil normalizes glucose sensitivity of these neurons and restores hypoglycemia awareness after RH. Using patch-clamp recording, we found that RH enhanced glucose inhibition of PFH orexin GI neurons in male mice, thereby blunting activation of these neurons in low-glucose conditions. We then used a modified conditioned place preference behavioral test to demonstrate that modafinil reversed hypoglycemia unawareness in male mice after RH. Similarly, modafinil restored normal glucose sensitivity to PFH orexin GI neurons. We conclude that impaired glucose sensitivity of PFH orexin GI neurons plays a role in hypoglycemia unawareness and that normalizing their glucose sensitivity after RH is associated with restoration of hypoglycemia awareness. This suggests that the glucose sensitivity of PFH orexin GI neurons is a therapeutic target for preventing hypoglycemia unawareness.

Acute regulation of tight junction ion selectivity in human airway epithelia.

Electrolyte transport through and between airway epithelial cells controls the quantity and composition of the overlying liquid. Many studies have shown acute regulation of transcellular ion transport in airway epithelia. However, whether ion transport through tight junctions can also be acutely regulated is poorly understood both in airway and other epithelia. To investigate the paracellular pathway, we used primary cultures of differentiated human airway epithelia and assessed expression of claudins, the primary determinants of paracellular permeability, and measured transepithelial electrical properties, ion fluxes, and La(3+) movement. Like many other tissues, airway epithelia expressed multiple claudins. Moreover, different cell types in the epithelium expressed the same pattern of claudins. To evaluate tight junction regulation, we examined the response to histamine, an acute regulator of airway function. Histamine stimulated a rapid and transient increase in the paracellular Na(+) conductance, with a smaller increase in Cl(-) conductance. The increase was mediated by histamine H(1) receptors and depended on an increase in intracellular Ca(2+) concentration. These results suggest that ion flow through the paracellular pathway can be acutely regulated. Such regulation could facilitate coupling of the passive flow of counter ions to active transcellular transport, thereby controlling net transepithelial salt and water transport.

Neuromodulation reduces interindividual variability of neuronal output.

In similar states, neural circuits produce similar outputs across individuals despite substantial interindividual variability in neuronal ionic conductances and synapses. Circuit states are largely shaped by neuromodulators that tune ionic conductances. It is therefore possible that, in addition to producing flexible circuit output, neuromodulators also contribute to output similarity despite varying ion channel expression. We studied whether neuromodulation at saturating concentrations can increase the output similarity of a single identified neuron across individual animals. Using the LP neuron of the crab stomatogastric ganglion (STG), we compared the variability of f-I curves and rebound properties in the presence of neuropeptides. The two neuropeptides we used converge to activate the same target current, which increases neuronal excitability. Output variability was lower in the presence of the neuropeptides, regardless of whether the neuropeptides significantly changed the mean of the corresponding parameter or not. However, the addition of the second neuropeptide did not add further to the reduction of variability. With a family of computational LP-like models, we explored how increased excitability and target variability contribute to output similarity and found two mechanisms: Saturation of the responses and a differential increase in baseline activity. Saturation alone can reduce the interindividual variability only if the population shares a similar ceiling for the responses. In contrast, reduction of variability due to the increase in baseline activity is independent of ceiling effects.Significance StatementThe activity of single neurons and neural circuits can be very similar across individuals even though the ionic currents underlying activity are variable. The mechanisms that compensate for the underlying variability and promote output similarity are poorly understood but may involve neuromodulation. Using an identified neuron, we show that neuropeptide modulation of excitability can reduce interindividual variability of response properties at a single-neuron level in two ways. First, the neuropeptide increases baseline excitability in a differential manner, resulting in similar response thresholds. Second, the neuropeptide increases excitability towards a shared saturation level, promoting similar maximal firing rates across individuals. Such tuning of neuronal excitability could be an important mechanism compensating for interindividual variability of ion channel expression.

Frequency-Dependent Action of Neuromodulation.

In oscillatory circuits, some actions of neuromodulators depend on the oscillation frequency. However, the mechanisms are poorly understood. We explored this problem by characterizing neuromodulation of the lateral pyloric (LP) neuron of the crab stomatogastric ganglion (STG). Many peptide modulators, including proctolin, activate the same ionic current (IMI) in STG neurons. Because IMI is fast and non-inactivating, its peak level does not depend on the temporal properties of neuronal activity. We found, however, that the amplitude and peak time of the proctolin-activated current in LP is frequency dependent. Because frequency affects the rate of voltage change, we measured these currents with voltage ramps of different slopes and found that proctolin activated two kinetically distinct ionic currents: the known IMI, whose amplitude is independent of ramp slope or direction, and an inactivating current (IMI-T), which was only activated by positive ramps and whose amplitude increased with increasing ramp slope. Using a conductance-based model we found that IMI and IMI-T make distinct contributions to the bursting activity, with IMI increasing the excitability, and IMI-T regulating the burst onset by modifying the postinhibitory rebound in a frequency-dependent manner. The voltage dependence and partial calcium permeability of IMI-T is similar to other characterized neuromodulator-activated currents in this system, suggesting that these are isoforms of the same channel. Our computational model suggests that calcium permeability may allow this current to also activate the large calcium-dependent potassium current in LP, providing an additional mechanism to regulate burst termination. These results demonstrate a mechanism for frequency-dependent actions of neuromodulators.

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation.

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Perioperative statin therapy is associated with a significant and dose-dependent reduction of adverse cardiovascular outcomes after coronary artery bypass graft surgery.

OBJECTIVE: The aim of this study was to determine whether perioperative statin therapy was associated with a dose-dependent decrease in adverse cardiovascular events after coronary artery bypass graft (CABG) surgery. DESIGN: A prospective observational study. SETTING: A cardiovascular anesthesia unit in a university hospital. PARTICIPANTS: Four hundred eighteen consecutive patients undergoing CABG surgery between October 2004 and October 2005. INTERVENTIONS: Patients were divided in 2 groups depending on whether their preoperative treatment included statins or not. In patients receiving statins, high- and low-dose regimens were respectively defined as a regimen recognized to induce a theoretic reduction of low-density lipoprotein cholesterol level equal to 45% (n = 87) or <45% (n = 258). In treated patients, statin therapy was maintained until the day of surgery and was restarted soon thereafter. MEASUREMENTS AND MAIN RESULTS: The measured endpoint was adverse in-hospital cardiovascular outcomes including heart failure and/or malignant arrhythmia and/or cardiac death. Stepwise logistic regression and a multivariate analysis of propensity-matched cohort were used for analysis of the findings. After adjustment for propensity score, statin therapy was found to produce a significant reduction in cardiovascular outcomes (odds ratio = 0.56; 95% confidence interval [CI], 0.32-0.96, p < 0.05). By using multivariate analysis, the odds ratio for cardiovascular outcomes in patients receiving high-dose statins compared with those treated by low-dose statins was 0.62 (95% CI, 0.41-0.93; p < 0.05). CONCLUSION: Statin therapy is associated with a significant and dose-dependent reduction in adverse cardiovascular events after CABG surgery. However, further randomized trials still require confirming a causal association between statins and better postoperative outcomes and evaluating the tolerance of such perioperative therapy.

Requirement for translocon-associated protein (TRAP) α in insulin biogenesis.

The mechanistic basis for the biogenesis of peptide hormones and growth factors is poorly understood. Here, we show that the conserved endoplasmic reticulum membrane translocon-associated protein α (TRAPα), also known as signal sequence receptor 1, plays a critical role in the biosynthesis of insulin. Genetic analysis in the nematode Caenorhabditis elegans and biochemical studies in pancreatic ß cells reveal that TRAPα deletion impairs preproinsulin translocation while unexpectedly disrupting distal steps in insulin biogenesis including proinsulin processing and secretion. The association of common intronic single-nucleotide variants in the human TRAPα gene with susceptibility to type 2 diabetes and pancreatic ß cell dysfunction suggests that impairment of preproinsulin translocation and proinsulin trafficking may contribute to the pathogenesis of type 2 diabetes.

KCa3.1 potassium channels are critical for cAMP-dependent chloride secretion and cyst growth in autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by numerous fluid-filled kidney cysts. Net fluid secretion into renal cysts is caused by transepithelial transport mediated by the apical cystic fibrosis transmembrane conductance regulator chloride channel, which leads to cyst enlargement. Here we found that forskolin, a potent adenylyl cyclase agonist, stimulated anion secretion by monolayers of kidney cells derived from patients with ADPKD. TRAM-34, a specific KCa3.1 potassium channel blocker, inhibited this current, and in vitro cyst formation and enlargement by the cells cultured within a collagen gel. Net chloride secretion was enhanced by the KCa3.1 activator DCEBIO and both chloride secretion and in vitro cyst growth were inhibited by overexpression of myotubularin-related protein-6, a phosphatase that specifically inhibits KCa3.1 channel activity. Our study suggests that KCa3.1 channels play a critical role in transcellular chloride secretion and net fluid transport into the kidney cysts of patients with ADPKD by maintaining the electrochemical driving force for chloride efflux through apical chloride channels. Pharmacological inhibitors of KCa3.1 channels may provide a novel and effective therapy to delay progression to kidney failure in patients with ADPKD.

Parasacral sciatic nerve block: does the elicited motor response predict the success rate?

BACKGROUND: In this prospective, randomized, double-blind study, we compared the tibial and the peroneal evoked motor response with regard to efficacy of sciatic nerve block using the parasacral approach. METHODS: Twenty-six ASA I-III patients scheduled for elective lower limb surgery were randomized to receive a parasacral sciatic block, using a nerve stimulator technique seeking either a tibial (n = 14) or peroneal (n = 12) motor response. After the evoked motor response was obtained, a solution of 10 mL 2% lidocaine with epinephrine and 10 mL 0.75% ropivacaine (actual final concentration of epinephrine, 1/160,000) was slowly injected through the needle. Sensory and motor blocks were assessed every 5 min for 30 min by an anesthesiologist blinded to the elicited motor response. If the block was not complete 30 min after injection of the local anesthetics, it was considered as failed, and general anesthesia was supplemented. RESULTS: Time to perform the block and level of minimal and maximal stimulation were not different between groups. The success rate of complete block was significantly higher in the tibial compared to the peroneal group (11 of 14 vs 2 of 12; P = 0.002). CONCLUSIONS: Eliciting a tibial motor response predicts a higher success rate than eliciting a peroneal motor response with parasacral sciatic nerve block.

Ageing and hypoxia cause protein aggregation in mitochondria.

Aggregation of cytosolic proteins is a pathological finding in disease states, including ageing and neurodegenerative diseases. We have previously reported that hypoxia induces protein misfolding in Caenorhabditis elegans mitochondria, and electron micrographs suggested protein aggregates. Here, we seek to determine whether mitochondrial proteins actually aggregate after hypoxia and other cellular stresses. To enrich for mitochondrial proteins that might aggregate, we performed a proteomics analysis on purified C. elegans mitochondria to identify relatively insoluble proteins under normal conditions (110 proteins identified) or after sublethal hypoxia (65 proteins). A GFP-tagged mitochondrial protein (UCR-11 - a complex III electron transport chain protein) in the normally insoluble set was found to form widespread aggregates in mitochondria after hypoxia. Five other GFP-tagged mitochondrial proteins in the normally insoluble set similarly form hypoxia-induced aggregates. Two GFP-tagged mitochondrial proteins from the soluble set as well as a mitochondrial-targeted GFP did not form aggregates. Ageing also resulted in aggregates. The number of hypoxia-induced aggregates was regulated by the mitochondrial unfolded protein response (UPRmt) master transcriptional regulator ATFS-1, which has been shown to be hypoxia protective. An atfs-1(loss-of-function) mutant and RNAi construct reduced the number of aggregates while an atfs-1(gain-of-function) mutant increased aggregates. Our work demonstrates that mitochondrial protein aggregation occurs with hypoxic injury and ageing in C. elegans. The UPRmt regulates aggregation and may protect from hypoxia by promoting aggregation of misfolded proteins.