Radiopaque FeMnN-Mo composite drawn filled tubing wires for braided absorbable neurovascular devices.

Flow diverter devices are small stents used to divert blood flow away from aneurysms in the brain, stagnating flow and inducing intra-aneurysmal thrombosis which in time will prevent aneurysm rupture. Current devices are formed from thin (∼25 µm) wires which will remain in place long after the aneurysm has been mitigated. As their continued presence could lead to secondary complications, an absorbable flow diverter which dissolves into the body after aneurysm occlusion is desirable. The absorbable metals investigated to date struggle to achieve the necessary combination of strength, elasticity, corrosion rate, fragmentation resistance, radiopacity, and biocompatibility. This work proposes and investigates a new composite wire concept combining absorbable iron alloy (FeMnN) shells with one or more pure molybdenum (Mo) cores. Various wire configurations are produced and drawn to 25-250 µm wires. Tensile testing revealed high and tunable mechanical properties on par with existing flow diverter materials. In vitro degradation testing of 100 µm wire in DMEM to 7 days indicated progressive corrosion and cracking of the FeMnN shell but not of the Mo, confirming the cathodic protection of the Mo by the FeMnN and thus mitigation of premature fragmentation risk. In vivo implantation and subsequent µCT of the same wires in mouse aortas to 6 months showed meaningful corrosion had begun in the FeMnN shell but not yet in the Mo filament cores. In total, these results indicate that these composites may offer an ideal combination of properties for absorbable flow diverters.

Colectomy in patients with liver disease: albumin-bilirubin score accurately predicts outcomes.

BACKGROUND: Patients with liver disease undergoing colectomy have higher rates of complications and mortality. The Albumin-Bilirubin score is a recently developed system, established to predict outcomes after hepatectomy, that accounts for liver dysfunction. METHODS: All patients undergoing colectomy were identified in the 2015-2018 American College of Surgeons National Surgical Quality Improvement Program colectomy-targeted database. Demographics and outcomes were compared between patients with Albumin-Bilirubin Grade 1 vs. 2/3. Multivariable regression was performed for outcomes including colorectal-specific complications. Areas under the receiver operative characteristic curves were calculated to determine accuracy of the Albumin-Bilirubin score. RESULTS: Of 86,273 patients identified, 48% (N = 41,624) were Albumin-Bilirubin Grade 1, 45% (N = 38,370) Grade 2 and 7% (N = 6,279) Grade 3. Patents with Grade 2/3 compared to Grade 1 had significantly increased mortality (7.2% vs. 0.9%, p < 0.001) and serious morbidity (31% vs. 12%, p < 0.001). Colorectal-specific complications including anastomotic leak (3.7% vs. 2.8%, p < 0.001) and prolonged ileus (26% vs. 14%, p < 0.001) were higher in patients with Grade 2/3. Grade 2/3 had increased risk of mortality (odds ratio 3.07, p < 0.001) and serious morbidity (1.78, p < 0.001). Albumin-Bilirubin had excellent accuracy in predicting mortality (area under the curve 0.81, p < 0.001) and serious morbidity (0.70, p < 0.001). CONCLUSION: Albumin-Bilirubin is easily calculated using only serum albumin and total bilirubin values. Grade 2/3 is associated with increased rates of mortality and morbidity following colectomy. Albumin-Bilirubin can be applied to risk-stratify patients prior to colectomy.

Activation of intestinal immunity by pathogen effector-triggered aggregation of lysosomal TIR-1/SARM1.

TIR-domain proteins with enzymatic activity are essential for immunity in plants, animals, and bacteria. However, it is not known how these proteins function in pathogen sensing in animals. We discovered that a TIR-domain protein (TIR-1/SARM1) is strategically expressed on the membranes of a lysosomal sub-compartment, which enables intestinal epithelial cells in the nematode C. elegans to survey for pathogen effector-triggered host damage. We showed that a redox active virulence effector secreted by the bacterial pathogen Pseudomonas aeruginosa alkalinized and condensed a specific subset of lysosomes by inducing intracellular oxidative stress. Concentration of TIR-1/SARM1 on the surface of these organelles triggered its multimerization, which engages its intrinsic NADase activity, to activate the p38 innate immune pathway and protect the host against microbial intoxication. Thus, lysosomal TIR-1/SARM1 is a sensor for oxidative stress induced by pathogenic bacteria to activate metazoan intestinal immunity.

Circular External Fixator Removal in the Outpatient Clinic Using Regional Anaesthesia: A Pilot Study of a Novel Approach.

Introduction: External fixator (EF) devices are commonly used in the management of complex skeletal trauma, as well as in elective limb reconstruction surgery for the management of congenital and acquired pathology. The subsequent removal of an EF is commonly performed under general anaesthesia in an operating theatre. This practice is resource-intensive and limits the amount of time available for other surgical cases in the operating theatre. We aimed to assess the use of regional anaesthesia as an alternative method of analgesia to facilitate the EF removal in an outpatient setting. Design and methods: This prospective case series evaluated the first 50 consecutive cases of EF removal in the outpatient clinic between 10/06/22 and 03/02/23. Regional anaesthesia using ultrasound-guided blockade of peripheral nerves was administered using 1% lidocaine due to its rapid onset and short half-life. Patients were assessed for additional analgesia requirements and then were asked to evaluate their experience and perceived pain using the visual analogue scale (VAS). Results: Fifty patients were included in the study. The mean age was 46.8 years (range 21-85 years). About 54% of the patients were male patients (N = 27). Post-procedure, all patients indicated positive satisfaction ratings, each participant responded as either 'satisfied' (N = 6), 'very satisfied' (N = 24) or 'highly satisfied' (N = 20). In addition, 90% of the participants reported that they would opt for this method of EF removal again in future. The VAS for pain immediately following completion of the procedure was low, with a mean score of 0.36 (range 0-4), where a score of 0 = 'No pain', and 10 = 'worst pain possible'. The median score was 0. Conclusion: We present the first description of outpatient EF removal using regional anaesthesia, with a prospective case series of 50 fully conscious patients from whom the EF was removed. This novel technique is likely to be cost-effective, reproducible, and safe. This technique reduces the burden of EF removal from an operating list and also improves the patient's experience when compared with other forms of conscious sedation. By eliminating the use of Entonox and methoxyflurane for sedation and analgesia, this technique also demonstrates a method of improving environmental sustainability. How to cite this article: Williams LM, Stamps G, Peak H, et al. Circular External Fixator Removal in the Outpatient Clinic Using Regional Anaesthesia: A Pilot Study of A Novel Approach. Strategies Trauma Limb Reconstr 2023;18(1):7-11.

Transcriptional suppression of sphingolipid catabolism controls pathogen resistance in C. elegans.

Sphingolipids are required for diverse biological functions and are degraded by specific catabolic enzymes. However, the mechanisms that regulate sphingolipid catabolism are not known. Here we characterize a transcriptional axis that regulates sphingolipid breakdown to control resistance against bacterial infection. From an RNAi screen for transcriptional regulators of pathogen resistance in the nematode C. elegans, we identified the nuclear hormone receptor nhr-66, a ligand-gated transcription factor homologous to human hepatocyte nuclear factor 4. Tandem chromatin immunoprecipitation-sequencing and RNA sequencing experiments revealed that NHR-66 is a transcriptional repressor, which directly targets sphingolipid catabolism genes. Transcriptional de-repression of two sphingolipid catabolic enzymes in nhr-66 loss-of-function mutants drives the breakdown of sphingolipids, which enhances host susceptibility to infection with the bacterial pathogen Pseudomonas aeruginosa. These data define transcriptional control of sphingolipid catabolism in the regulation of cellular sphingolipids, a process that is necessary for pathogen resistance.

Ether lipid biosynthesis promotes lifespan extension and enables diverse pro-longevity paradigms in Caenorhabditis elegans.

Biguanides, including the world's most prescribed drug for type 2 diabetes, metformin, not only lower blood sugar, but also promote longevity in preclinical models. Epidemiologic studies in humans parallel these findings, indicating favorable effects of metformin on longevity and on reducing the incidence and morbidity associated with aging-related diseases. Despite this promise, the full spectrum of molecular effectors responsible for these health benefits remains elusive. Through unbiased screening in Caenorhabditis elegans, we uncovered a role for genes necessary for ether lipid biosynthesis in the favorable effects of biguanides. We demonstrate that biguanides prompt lifespan extension by stimulating ether lipid biogenesis. Loss of the ether lipid biosynthetic machinery also mitigates lifespan extension attributable to dietary restriction, target of rapamycin (TOR) inhibition, and mitochondrial electron transport chain inhibition. A possible mechanistic explanation for this finding is that ether lipids are required for activation of longevity-promoting, metabolic stress defenses downstream of the conserved transcription factor skn-1/Nrf. In alignment with these findings, overexpression of a single, key, ether lipid biosynthetic enzyme, fard-1/FAR1, is sufficient to promote lifespan extension. These findings illuminate the ether lipid biosynthetic machinery as a novel therapeutic target to promote healthy aging.

Metformin is the drug most prescribed to treat type 2 diabetes around the world and has been in clinical use since 1950. The drug belongs to a family of compounds known as biguanides which reduce blood sugar, making them an effective treatment against type 2 diabetes. More recently, biguanides have been found to have other health benefits, including limiting the growth of various cancer cells and improving the lifespan and long-term health of several model organisms. Epidemiologic studies also suggest that metformin may increase the lifespan of humans and reduce the incidence of age-related illnesses such as cardiovascular disease, cancer and dementia. Given the safety and effectiveness of metformin, understanding how it exerts these desirable effects may allow scientists to discover new mechanisms to promote healthy aging. The roundworm Caenorhabditis elegans is an ideal organism for studying the lifespan-extending effects of metformin. It has an average lifespan of two weeks, a genome that is relatively easy to manipulate, and a transparent body that enables scientists to observe cellular and molecular events in living worms. To discover the genes that enable metformin's lifespan-extending properties, Cedillo, Ahsan et al. systematically switched off the expression of about 1,000 genes involved in C. elegans metabolism. They then screened for genes which impaired the action of biguanides when inactivated. This ultimately led to the identification of a set of genes involved in promoting a longer lifespan. Cedillo, Ahsan et al. then evaluated how these genes impacted other well-described pathways involved in longevity and stress responses. The analysis indicated that a biguanide drug called phenformin (which is similar to metformin) increases the synthesis of ether lipids, a class of fats that are critical components of cellular membranes. Indeed, genetically mutating the three major enzymes required for ether lipid production stopped the biguanide from extending the worms' lifespans. Critically, inactivating these genes also prevented lifespan extension through other known strategies, such as dietary restriction and inhibiting the cellular organelle responsible for producing energy. Cedillo, Ahsan et al. also showed that increasing ether lipid production alters the activity of a well-known longevity and stress response factor called SKN-1, and this change alone is enough to extend the lifespan of worms. These findings suggest that promoting the production of ether lipids could lead to healthier aging. However, further studies, including clinical trials, will be required to determine whether this is a viable approach to promote longevity and health in humans.

Non-canonical pattern recognition of a pathogen-derived metabolite by a nuclear hormone receptor identifies virulent bacteria in C. elegans.

Distinguishing infectious pathogens from harmless microorganisms is essential for animal health. The mechanisms used to identify infectious microbes are not fully understood, particularly in metazoan hosts that eat bacteria as their food source. Here, we characterized a non-canonical pattern-recognition system in Caenorhabditis elegans (C. elegans) that assesses the relative threat of virulent Pseudomonas aeruginosa (P. aeruginosa) to activate innate immunity. We discovered that the innate immune response in C. elegans was triggered by phenazine-1-carboxamide (PCN), a toxic metabolite produced by pathogenic strains of P. aeruginosa. We identified the nuclear hormone receptor NHR-86/HNF4 as the PCN sensor in C. elegans and validated that PCN bound to the ligand-binding domain of NHR-86/HNF4. Activation of NHR-86/HNF4 by PCN directly engaged a transcriptional program in intestinal epithelial cells that protected against P. aeruginosa. Thus, a bacterial metabolite is a pattern of pathogenesis surveilled by nematodes to identify a pathogen in its bacterial diet.

Routine Use of Closed Suction Drains Following Revision Arthroplasty May Not be Necessary.

BACKGROUND: There are numerous studies demonstrating that closed suction drainage (CSD) usage after primary total joint arthroplasty (TJA) has little to no benefit. There are little data on the role of CSDs after revision TJA. The purpose of our study was to evaluate whether there is any clinical advantage to CSD usage after revision TJA. METHODS: This retrospective study evaluated the clinical records of 2,030 patients undergoing revision TJA between 2007 and 2021. CSD was used in 472 patients and not used in 1,558 patients. Primary outcome was blood transfusion rate and secondary outcomes included total blood loss (TBL), as determined by Gross formula, wound complications (hematoma, infection, and dehiscence), and length of hospital stay. Patients undergoing revision TJA for oncologic reasons or those with incomplete datasets were excluded. RESULTS: There were no statistically significant differences in rates of allogeneic blood transfusion, TBL, and wound complications (hematoma, infection, and dehiscence) between the two groups (P = .159, .983, .192, .334, and .548, respectively). When adjusted for demographic and surgical confounders, there was no difference in transfusion and TBL rates between groups (Odds Ratio 1.04, 95% Confidence Interval 0.78-1.38, P = .780 and estimate -105.71 mL, 95% confidence interval -333.96 to 122.55, P = .364, respectively). CSD cohort had a shorter length of stay (4.30 versus 5.82 days, P < .001). CONCLUSION: We acknowledge that there is a role for CSD usage in a selected group of patients. Nevertheless, our study revealed that routine use of CSD after revision TJA does not provide an additional clinical benefit.

Low-Dose Aspirin for Venous Thromboembolism Prophylaxis is Associated With Lower Rates of Periprosthetic Joint Infection After Total Joint Arthroplasty.

BACKGROUND: Aspirin as a venous thromboembolism (VTE) prophylactic agent has been shown to have antistaphylococcal and antibiofilm roles. Optimal acetylsalicylic acid (ASA) dosage would facilitate antimicrobial effects while avoiding over-aggressive inhibition of platelet antimicrobial function. Our purpose was to determine the periprosthetic joint infection (PJI) rate after total joint arthroplasty in patients receiving low-dose ASA (81 mg twice a day), in comparison to high-dose ASA (325 mg twice a day). METHODS: We conducted a retrospective cohort study between 2008 and 2020. Eligible patients were older than 18 years, underwent primary total joint arthroplasty, both total knee arthroplasty and total hip arthroplasty, had a minimum 30-day follow-up, and received a full course ASA as VTE prophylaxis. Patients' records were reviewed for PJI, according to Musculoskeletal Infection Society criteria. Patients were excluded if they underwent revision arthroplasty, had a history of coagulopathy, or had an ASA regimen that was not completed. In total 15,825 patients were identified, 8,761 patients received low-dose ASA and 7,064 received high-dose ASA. RESULTS: The high-dose cohort had a higher PJI rate (0.35 versus 0.10%, P = .001). This relationship was maintained when comparing subgroups comprising total knee arthroplasty (0.32 versus 0.06%, P = .019) or total hip arthroplasty (0.38 versus 0.14%, P = .035) and accounting for potentially confounding demographic and surgical variables (odds ratio = 2.59, 95% CI = 1.15-6.40, P = .028). CONCLUSION: Comparing low-dose to high-dose ASA as a VTE prophylactic agent, low-dose ASA had a lower PJI rate. This may be attributable to a balance of anti-infective properties of ASA and antiplatelet effects.

Pathogen infection and cholesterol deficiency activate the C. elegans p38 immune pathway through a TIR-1/SARM1 phase transition.

Intracellular signaling regulators can be concentrated into membrane-free, higher ordered protein assemblies to initiate protective responses during stress - a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD+ glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD+ glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD+ glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that a loss-of-function mutation in nhr-8, which alters cholesterol metabolism and is used to study conditions of sterol deficiency, causes TIR-1/SARM1 to oligomerize into puncta in intestinal epithelial cells. Cholesterol scarcity increases p38 PMK-1 phosphorylation, primes immune effector induction in a manner that requires TIR-1/SARM1 oligomerization and its intrinsic NAD+ glycohydrolase activity, and reduces pathogen accumulation in the intestine during a subsequent infection. These data reveal a new adaptive response that allows a metazoan host to anticipate pathogen threats during cholesterol deprivation, a time of relative susceptibility to infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD+ glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.

From worms to humans, animals have developed various strategies ­ including immune defences ­ to shield themselves from disease-causing microbes. A type of roundworm, called C. elegans, lives in environments rich in microbes, so it needs effective immune defences to protect itself. The roundworms share a key regulatory pathway with mammals that helps to control their immune responses. This so-called p38 pathway relies on proteins that interact with each other to activate protective immune defences. Proteins contain different regions or domains that can give them a certain function. For example, proteins with a region called TIR play important roles in immune defences in both animals and plants. One such protein, called SARM1, is unique among animal and plant proteins in that it is an enzyme, which cleaves an important metabolite in the cell. In C. elegans, the SARM1 homolog, TIR-1, controls the p38 pathway during infection, but how TIR-1 activates it is unclear. To find out more, Peterson, Icso et al. modified C. elegans to generate a fluorescent form of TIR-1 and infected the worms with bacteria. Imaging techniques revealed that infection caused TIR-1 in gut cells to cluster into organized structures, which increases the enzymatic activity of the protein to activate the p38 immune pathway. Moreover, stress situations, such as cholesterol nutrient withdrawal, activated the p38 pathway in the same way. This adaptive stress response allows the animal to defend itself against pathogen threats during times, when they are most susceptible to infections. Cells in the gut provide a primary line of defence against infectious bacteria and are important for maintaining a healthy gut immune system. When the mechanisms for pathogen sensing and immune maintenance are disrupted, it can lead to inflammation and higher risk of infection. Peterson, Icso et al. show how a key regulator of gut immunity, TIR-1, provides protection in C. elegans, which may suggest that SARM1 could have a similar role in mammals.

Optimization of Spinal Cord Stimulation Using Bayesian Preference Learning and Its Validation.

Epidural spinal cord stimulation has been reported to partially restore volitional movement and autonomic functions after motor and sensory-complete spinal cord injury (SCI). Modern spinal cord stimulation platforms offer significant flexibility in spatial and temporal parameters of stimulation delivered. Heterogeneity in SCI and injury-related symptoms necessitate stimulation personalization to maximally restore functions. However, the large multi-dimensional stimulation space makes exhaustive tests impossible. In this paper, we present a Bayesian optimization strategy for identifying personalized optimal stimulation patterns based on the participant's expressed preference for stimulation settings. We present companion validation protocols for investigating the credibility of learned preference models. The results obtained for five participants in the E-STAND spinal cord stimulation clinical trial are reported. Personalized preference models produced by the proposed learning and optimization algorithm show that there is more similarity in optimal frequency than in pulse width across participants. Across five participants, the average model prediction accuracy is 71.5% in internal cross-validation and 65.6% in prospective validation. Statistical tests of both validation studies show that the ability of the preference models to correctly predict unseen preference data is significantly greater than chance. The personalized preference models are also shown to be significantly correlated with motor task performance across participants. We show that several aspects in participants' quality of life has been improved over the course of the trial. Overall, the results indicate that the Bayesian preference optimization algorithm could assist clinicians in the systematic programming of individualized therapeutic stimulation settings and improve the therapeutic outcomes.

Air pollution and retinal vessel diameter and blood pressure in school-aged children in a region impacted by residential biomass burning.

Little is known about the early-life cardiovascular health impacts of fine particulate air pollution (PM2.5) and oxidant gases. A repeated-measures panel study was used to evaluate associations between outdoor PM2.5 and the combined oxidant capacity of O3 and NO2 (using a redox-weighted average, Ox) and retinal vessel diameter and blood pressure in children living in a region impacted by residential biomass burning. A median of 6 retinal vessel and blood pressure measurements were collected from 64 children (ages 4-12 years), for a total of 344 retinal measurements and 432 blood pressure measurements. Linear mixed-effect models were used to estimate associations between PM2.5 or Ox (same-day, 3-day, 7-day, and 21-day means) and retinal vessel diameter and blood pressure. Interactions between PM2.5 and Ox were also examined. Ox was inversely associated with retinal arteriolar diameter; the strongest association was observed for 7-day mean exposures, where each 10 ppb increase in Ox was associated with a 2.63 µm (95% CI - 4.63, - 0.63) decrease in arteriolar diameter. Moreover, Ox modified associations between PM2.5 and arteriolar diameter, with weak inverse associations observed between PM2.5 and arteriolar diameter only at higher concentrations of Ox. Our results suggest that outdoor air pollution impacts the retinal microvasculature of children and interactions between PM2.5 and Ox may play an important role in determining the magnitude and direction of these associations.

Discovering Associations: Kawasaki Disease and COVID-19.

The COVID-19 pandemic has resulted in over 3.6 million confirmed cases and over 254,000 deaths worldwide. It has been theorized that children who are asymptomatic or who do not display significant respiratory symptoms are potential vectors for community transmission of the SARS-CoV-2 virus. This is incompletely understood due to the current lack of widespread testing in the pediatric population. We describe a case of a 2-year-old female who presented with symptoms of prolonged fever, conjunctivitis, extremity edema, rash, dry/cracked lips, fussiness and fatigue, and a notable absence of respiratory symptoms. She was diagnosed with and treated for Kawasaki disease. Due to her prolonged fever, she was tested for COVID-19 which was positive; however, she did not develop respiratory symptoms during her illness. At the time of manuscript submission, this is the second case report to our knowledge showing an association between Kawasaki Disease and SARS-CoV-2 virus, both of which are poorly understood diseases in the pediatric population. This case highlights the value of testing pediatric patients for COVID-19 who present with fever in the absence of other symptoms to improve epidemiologic measures during the ongoing pandemic, and it also adds to a foundation of cases for future research on the presence of a link between Kawasaki Disease and COVID-19.

Correction of the Neglected Clubfoot in the Adolescent and Adult Patient.

Clubfoot is a complex 4-dimensional deformity involving the hindfoot, midfoot, and forefoot. The fourth dimension is time. Treatment aims at achieving a pain-free, plantigrade, and mobile foot but, over time, flexible deformities become fixed and more difficult to manage. The Ponseti method of serial manipulation and casting can be used successfully in older children and may reduce the need for extensive open surgery. Alternatively, gradual correction of by an external device enables simultaneous correction of all components of the deformity without shortening the foot. Combining gradual soft tissue distraction with open releases and/or bony procedures may achieve a pain-free and plantigrade foot.

Innate Immunity in the C. elegans Intestine Is Programmed by a Neuronal Regulator of AWC Olfactory Neuron Development.

Olfactory neurons allow animals to discriminate nutritious food sources from potential pathogens. From a forward genetic screen, we uncovered a surprising requirement for the olfactory neuron gene olrn-1 in the regulation of intestinal epithelial immunity in Caenorhabditis elegans. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen, and resistance to bacterial infection. These data reveal an unexpected connection between olfactory receptor development and innate immunity and show that anti-pathogen defenses in the intestine are developmentally programmed.

The fatty acid oleate is required for innate immune activation and pathogen defense in Caenorhabditis elegans.

Fatty acids affect a number of physiological processes, in addition to forming the building blocks of membranes and body fat stores. In this study, we uncover a role for the monounsaturated fatty acid oleate in the innate immune response of the nematode Caenorhabditis elegans. From an RNAi screen for regulators of innate immune defense genes, we identified the two stearoyl-coenzyme A desaturases that synthesize oleate in C. elegans. We show that the synthesis of oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Together, these data add to the known health-promoting effects of monounsaturated fatty acids, and suggest an ancient link between nutrient stores, metabolism, and host susceptibility to bacterial infection.

Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

The nuclear hormone receptor NHR-86 controls anti-pathogen responses in C. elegans.

Nuclear hormone receptors (NHRs) are ligand-gated transcription factors that control adaptive host responses following recognition of specific endogenous or exogenous ligands. Although NHRs have expanded dramatically in C. elegans compared to other metazoans, the biological function of only a few of these genes has been characterized in detail. Here, we demonstrate that an NHR can activate an anti-pathogen transcriptional program. Using genetic epistasis experiments, transcriptome profiling analyses and chromatin immunoprecipitation-sequencing, we show that, in the presence of an immunostimulatory small molecule, NHR-86 binds to the promoters of immune effectors to activate their transcription. NHR-86 is not required for resistance to the bacterial pathogen Pseudomonas aeruginosa at baseline, but activation of NHR-86 by this compound drives a transcriptional program that provides protection against this pathogen. Interestingly, NHR-86 targets immune effectors whose basal regulation requires the canonical p38 MAPK PMK-1 immune pathway. However, NHR-86 functions independently of PMK-1 and modulates the transcription of these infection response genes directly. These findings characterize a new transcriptional regulator in C. elegans that can induce a protective host response towards a bacterial pathogen.

Caenorhabditis elegans in high-throughput screens for anti-infective compounds.

New classes of antimicrobials that are effective therapies for infections with multi-drug resistant pathogens are urgently needed. The nematode Caenorhabditis elegans has been incorporated into small molecule screening platforms to identify anti-infective compounds that provide protection of a host during infection. The use of a live animal in these screening systems offers several advantages, including the ability to identify molecules that boost innate immune responses in a manner advantageous to host survival and compounds that disrupt bacterial virulence mechanisms. In addition, new classes of antimicrobials that target the pathogen have been uncovered, as well as interesting chemical probes that can be used to dissect new mechanisms of host-pathogen interactions.