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.

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.

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.

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.

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.

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.

Therapeutic regulatory T-cell adoptive transfer ameliorates established murine chronic GVHD in a CXCR5-dependent manner.

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation. In cGVHD, alloreactive T cells and germinal center (GC) B cells often participate in GC reactions to produce pathogenic antibodies. Although regulatory T cells (Tregs) can inhibit GC reactions, Treg numbers are reduced in cGVHD, contributing to cGVHD pathogenesis. Here, we explored 2 means to increase Tregs in cGVHD: interleukin-2/monoclonal antibody (IL-2/mAb) complexes and donor Treg infusions. IL-2/mAb complexes given over 1 month were efficacious in expanding Tregs and treating established cGVHD in a multi-organ-system disease mouse model characterized by GC reactions, antibody deposition, and lung dysfunction. In an acute GVHD (aGVHD) model, IL-2/mAb complexes given for only 4 days resulted in rapid mortality, indicating IL-2/mAb complexes can drive conventional T-cell (Tcon)-mediated injury. In contrast, Treg infusions, which uniformly suppress aGVHD, increased Treg frequency and were effective in preventing the onset of, and treating, established cGVHD. Efficacy was dependent upon CXCR5-sufficient Tregs homing to, and inhibiting, GC reactions. These studies indicate that the infusion of Tregs, especially ones enriched for GC homing, may be desirable for cGVHD therapy. Although IL-2/mAb complexes can be efficacious in cGVHD, a cautious approach needs to be taken in settings in which aGVHD elements, and associated Tcon, are present.

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.

Depolarization of mitochondria in neurons promotes activation of nitric oxide synthase and generation of nitric oxide.

The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

An Unusual Ankle Injury: The Bosworth-Pilon Fracture.

The Bosworth injury occurs when the distal fibula becomes entrapped posterior to the posterior tibial tubercle, usually as a result of a supination external rotation injury. This uncommon occurrence is a recognized cause of an irreducible ankle dislocation. A pilon fracture is usually a high-energy injury caused by the talus being driven upward into the tibial plafond. The resulting bone and soft tissue injuries often require a staged approach to management. The present report is the first in the medical data to describe a Bosworth injury complicating a pilon fracture. We also discuss a management approach for this rare fracture.

Pantothenate and pantetheine antagonize the antitubercular activity of pyrazinamide.

Pyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth of Mycobacterium tuberculosis via an as yet undefined mechanism. An M. tuberculosis laboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains of M. tuberculosis. It was found that pantothenate and other ß-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that ß-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, including n-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates with panD-linked resistance.

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.

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.

Oncogenic Kras expression in postmitotic neurons leads to S100A8-S100A9 protein overexpression and gliosis.

Previous studies suggest that up-regulation of Ras signaling in neurons promotes gliosis and astrocytoma formation in a cell nonautonomous manner. However, the underlying mechanisms remain unknown. To address this question, we generated compound mice (LSL Kras G12D/+;CamKII-Cre) that express oncogenic Kras from its endogenous locus in postmitotic neurons after birth. These mice developed progressive gliosis, which is associated with hyperactivation of Ras signaling pathways. Microarray analysis identified S100A8 and S100A9 as two secreted molecules that are significantly overexpressed in mutant cortices. In contrast to their usual predominant expression in myeloid cells, we found that overexpression of S100A8 and S100A9 in the mutant cortex is primarily in neurons. This neuronal expression pattern is associated with increased infiltration of microglia in mutant cortex. Moreover, purified S100A8-S100A9 but not S100A8 or S100A9 alone promotes growth of primary astrocytes in vitro through both TLR4 and receptor of advanced glycation end product receptors. In summary, our results identify overexpression of S100A8-S100A9 in neurons as an early step in oncogenic Kras-induced gliosis. These molecules expressed in nonhematopoietic cells may be involved in tumorigenesis at a stage much earlier than what has been reported previously.

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.

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.

MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.

Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.

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.