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The development of many systemic autoimmune diseases, including systemic lupus erythematosus, is associated with overactivation of the type I interferon (IFN) pathway, lymphopenia and increased follicular helper T (Tfh)-cell differentiation. However, the cellular and molecular mechanisms underlying these immunological perturbations remain incompletely understood. Here, we show that the mechanistic target of rapamycin complex 2 (mTORC2) promotes Tfh differentiation and disrupts Treg homeostasis. Inactivation of mTORC2 in total T cells, but not in Tregs, greatly ameliorated the immunopathology in a systemic autoimmunity mouse model. This was associated with reduced Tfh differentiation, B-cell activation, and reduced T-cell glucose metabolism. Finally, we show that type I IFN can synergize with TCR ligation to activate mTORC2 in T cells, which partially contributes to T-cell lymphopenia. These data indicate that mTORC2 may act as downstream of type I IFN, TCR and costimulatory receptor ICOS, to promote glucose metabolism, Tfh differentiation, and T-cell lymphopenia, but not to suppress Treg function in systemic autoimmunity. Our results suggest that mTORC2 might be a rational target for systemic autoimmunity treatment.

Introduction: Uric acid (UA) level within carotid plaque specimens and its association with cerebrovascular events has not been detected or quantified. Systemically, serum uric acid (SUA) is a marker of inflammation and risk factor for atherosclerosis. However, its association with carotid plaque instability and subsequent cerebrovascular events remains unclear. Hypothesis: We hypothesized that UA may play a role in carotid artery atherosclerosis and stroke pathogenesis. In patients undergoing carotid endarterectomy (CEA), we aimed to determine whether UA is present differentially in carotid plaques of symptomatic versus asymptomatic patients and whether SUA is associated with cerebrovascular symptoms before surgery and major adverse cardiovascular events (MACE) in the follow-up to CEA. Methods: We prospectively collected 32 carotid plaques during CEA. We qualitatively assessed the presence of UA by using Gomori methenamine silver (GMS) staining and immunohistochemistry with anti-UA antibodies. We extracted and measured the quantity of UA in carotid plaques by using an enzymatic colorimetric assay. A retrospective analysis of a clinical database of 534 consecutive patients who underwent CEA was performed. We defined as symptomatic those patients with history of stroke, TIA and amaurosis fugax. Results: UA positive staining was detected in higher number of carotid plaques from symptomatic compared to asymptomatic patients on both staining, GMS [20 (86.9%) vs 2 (22.2%); p=0.001] and immunohistochemistry anti-UA [16 (69.5%) vs 1 (11.1%); p=0.004]. Moreover, we found a significantly higher concentration of UA in carotid plaque from symptomatic compared to asymptomatic patients [25.1 (9.5) μg/g vs 17.9 (3.8) μg/g; p=0.021]. SUA level in patients prior to CEA was significantly higher in the symptomatic compared to the asymptomatic group [6.0 (4.8-7.1) mg/dL vs 5.2 (4.4-6.3) mg/dL; p Conclusions: UA may provide a mechanistic explanation as systemic biomarker and potential tissue contributor for carotid plaque instability and subsequent cerebrovascular and cardiovascular symptoms.

Introduction Next‐generation sequencing (NGS) and digital polymerase chain reaction (PCR) based platforms have been used to detect EGFR mutations in plasma circulating tumor DNA (ctDNA) with high accuracy. Generally, molecular testing is performed after histopathological analysis. However, many patients with suspected advanced nonsmall cell lung cancer are unable to undergo biopsy thus forgoing potential treatment with highly effective tyrosine kinase inhibitors (TKIs) in patients with sensitizing EGFR mutations. We examined the utility of ctDNA testing to detect EGFR mutations in patients' plasma, where tissue biopsy is not feasible. Methods We conducted a single‐center, prospective study of 30 Chinese patients with suspected advanced lung cancer, who were unable to undergo a biopsy for initial diagnosis due to comorbidities or poor performance status. Patients with plasma EGFR sensitizing mutations were treated with first‐generation EGFR TKIs. Results Twenty of 30 patients enrolled had sensitizing EGFR mutations in ctDNA and were started on EGFR TKIs. After a median follow‐up of 12 months, median progression‐free survival (PFS) was 10 months and median overall survival (OS) was not reached. The median OS for the 10 untreated patients was 3 months. Conclusions In our study, patients with plasma EGFR mutations treated with TKIs showed disease control rate (DCR) and PFS similar to historical controls that were treated based on tissue testing. This is the first prospective study showing that ctDNA genotyping provides a feasible diagnostic approach for frail lung cancer patients who are unable to undergo biopsy, which subsequently leads to EGFR‐targeted therapy, and improved outcomes in this subgroup of patients., This study is the first to prospectively evaluate the utility and feasibility of plasma circulating tumor DNA to detect EGFR mutations in frail patients with lung cancer, when tissue biopsy was not feasible. Incorporating plasma molecular profiling with clinical data allowed patients with sensitizing EGFR mutations to undergo treatment with highly effective tyrosine kinase inhibitors in a timely manner and gain significant improvement in overall survival, disease control rate, and progression free survival.

As a devastating complication of non-small cell lung cancer (NSCLC), the incidence of leptomeningeal metastasis (LM) is rising, largely due to overall longer survival of NSCLC, especially in patients with targetable molecular driver mutations. There is no clear consensus on the optimal management of LM. This review will cover recent advances in diagnosis, monitoring, and treatment of LM in NSCLC. In LM without oncogene drivers, systemic chemotherapy, intrathecal therapy, and radiation have modestly improved the clinical outcomes. Emerging data have also suggested encouraging activity of immunotherapy. At the same time, in LM with sensitizing EGFR mutations, osimertinib should be considered regardless of T790M status. Pulse erlotinib, afatinib, and newer agents with improved CNS penetration have also shown benefits. Moreover, accumulating evidences support potential benefits of molecularly targeted therapy in ALK-rearranged and other oncogene-driven NSCLC with LM. Future studies are warranted to better define the underlying mechanism, to optimize the clinical management, and to improve patient outcomes.

The past decade has seen a revolution of new advances in the management of non-small cell lung cancer (NSCLC) with remarkable progresses in screening, diagnosis, and treatment. The advances in systemic treatment have been driven primarily by the development of molecularly targeted therapeutics, immune checkpoint inhibitors, and anti-angiogenic agents, all of which have transformed this field with significantly improved patient outcomes. This review will address updates in lung cancer screening, liquid biopsy, and immunotherapy in the front-line setting. We discuss recent advances and highlight the plethora of new approvals of molecular-targeted therapy for subgroups of NSCLC patients with sensitizing EGFR, ALK, ROS1, RET, BRAF V600E, MET, and NTRK alterations.

Introduction: The immune checkpoint inhibitor, pembrolizumab, has revolutionized the treatment of non-small cell lung cancer (NSCLC). It is currently approved and widely used in patients with advanced NSCLC whose tumors have no EGFR or ALK genomic aberrations that express PD-L1 as single-agent treatment and irrespective of PD-L1 expression in combination with platinum-based doublet chemotherapy in the first-line setting.Areas covered: The authors have reviewed articles discussing pembrolizumab and NSCLC in MEDLINE between July 2013 to August 2019 and focus on recent advances in combining pembrolizumab with chemotherapy, radiotherapy and other novel agents in various stages of NSCLC.Expert opinion: Although pembrolizumab has revolutionized the treatment of advanced NSCLC, only a subset of patients benefit from single-agent therapy. Numerous trials combining pembrolizumab with chemotherapy and radiation have shown benefit and a large spectrum of novel combination strategies are being explored for improved synergies. In addition to PD-L1 tumor proportion score, validation of other biomarkers would be beneficial in stratifying patients and improving the predictive value of combining immune check point inhibitors and chemotherapy.

AimsTo evaluate if patients referred to Ealing Liaison Psychiatry Service (ELPS) with co-existing substance use are being appropriately identified as per NICE guidelines.Patients with co-existing substance misuse have greater morbidity and mortality and it is therefore important to identify these patients to optimise their management. NICE recommends that all patients are asked about their substance use.Anecdotally, our team felt we were doing a good job of identifying and managing such patients but we had no objective evidence of this.MethodCompleted a retrospective audit looking at a sample of patients referred to ELPS over two weeks in December 2019.A training session for ELPS was then held to highlight the initial audit results and NICE guideline recommendations.We then repeated the audit over two weeks in March 2020.ResultInitial audit (100 patients):Only 69% of patients asked about substance use. From those asked, 50-65.2% were using a substance, most commonly alcohol.None of the patients over the age of 80 were asked about substance use vs 79.5% of patients aged 20–40 years.55% of females vs 81% of males were asked about illicit substances.33.3% of ward referrals vs 74.2% of Emergency Department referrals asked about substance useRe-audit (53 patients):Significant improvement across all areas93% now asked about substance use60% of over 80s, 96% of females and 85% of ward referrals were now being correctly asked about substance useConclusionWe were surprised to find that we were initially not meeting NICE standards regarding asking patients about their substance use.Acknowledging this problem during our training session proved to be effective.This knowledge will help us develop our care pathways with our Acute colleagues and the Drug and Alcohol Liaison Service.

AimsTo host the first ELPS training day specifically for LAS staff to improve their knowledge and understanding about mental health issues and the role of ELPS.On average 13,000 calls are received by LAS relating to mental health issues every month. Many patients seen by ELPS will have multiple interactions with LAS. ELPS has previously held training for the Emergency Department team but this innovative day was designed to extend this training commitment to pre-hospital cliniciansMethodLAS training needs were initially assessed by a bespoke questionnaire and ELPS attending another LAS training event held by the new mental health joint response car team.We then developed a training programme to match the identified training needs and which utilised the specific expertise of individual ELPS staff.14 members of the local LAS stations attended including both Paramedics and Emergency Ambulance Clinicians. The presentations covered mental state examination, suicide, risk assessment, substance misuse, legal frameworks and then a ‘challenging cases’ session to bring it all together.Pre and post course questionnaires were completed by participants, exploring attitudes and knowledge.ResultThere was a statistically significant improvement in the average self-ratings for all of the categories assessed including attitudes to mental health, confidence in assessment and knowledge relating to the process the patient will experience in the emergency department.The knowledge about the pathway and role of liaison psychiatry showed the greatest improvement with an average 4.25 increase in pre and post course rating.Almost all participants (9.2/10) would recommend this training day to a colleagueConclusionWe met our objective of improving LAS staff knowledge and understanding about mental health issues and the role of ELPS. We plan to build on this successful pilot and expand our training programme for LAS with the ultimate aim of improving patient care.

189 Background: Data at our institution shows lung cancer is more prevalent and aggressive in HIV patients. A study of lung cancer patients revealed a mean age of 55.8 years in those with HIV vs. 68.0 in those without. Additionally, 67% of HIV patients had metastasis at time of diagnosis, compared to 49% in the overall population. One study found an 18.9% reduction in lung cancer mortality among HIV patients who receive NLST-recommended screening. Despite this, data from 2018 estimated only 13% of eligible HIV patients had completed screening at our institution. We pursued a quality improvement initiative to increase lung cancer screening in our HIV clinics. Methods: Our multi-disciplinary team studied charts of the 628 HIV clinic patients seen in a four-month span to identify those who had not received lung cancer screening and potential reasons why referrals were not made. We also spoke with clinic providers to identify improvement areas. Our intervention encompassed HIV patients that met CMS screening criteria (i.e. age 55-77, 30 pack-year smoking). Our process measure was new referrals to our dedicated screening coordinator, who contacts patients to arrange for CT scans. We plotted trends in appointment referrals on a run chart. Results: Areas for improvement included EMR documentation to assess screening eligibility and an occasional lack of awareness regarding criteria. Providers also cited time constraints may limit referrals. Our team identified patients that met screening criteria and generated EMR reminders for providers to refer patients to radiology. We also held sessions with providers and nursing staff to increase awareness of our screening program. Of 628 patients, 128 (20.4%) had sufficient documented smoking history to assess for screening eligibility. 81 patients (63.3%) met our criteria. Of these patients, 58 (71.6%) had not been screened or referred for screening. Through our most recent interventions, 16 (31.3%) patients have been referred to our screening coordinator, and 7 (12.1%) have received screening CT scans. Our interventions ultimately led to an increase from 23 of 81 (28.4%) patients with completed screening to a projected 46 of 81 (56.8%). Conclusions: Providing education and EMR alerts to raise awareness regarding eligibility, we substantially increased the screening rate in our clinics. Our interventions will be broadened as we return from COVID stoppages. Future interventions include increasing smoking history documentation in the EMR to allow for automated identification of screening eligibility. PDSA and interventions are ongoing with continued follow-up of efficacy.

194 Background: Pneumococcal and influenza vaccinations are recommended for all patients with any malignancy in accordance with the Infectious Disease Society of America. Patients undergoing chemotherapy for solid tumors have a 40-50 fold higher risk for the development of invasive pneumococcal disease compared to healthy adults with a case fatality rate of 35% and should receive the sequential 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPSV23). In the outpatient setting, lack of provider knowledge, complexity of the pneumococcal vaccine regimen and disruption in work-flow of a busy clinic can lead to low rates of administration. Towards this end, we conducted a quality project to improve administration of both pneumococcal and influenza vaccines by at least 50% at one of our outpatient oncology clinics at Montefiore Medical Center in Bronx, NY during a 4 month period. Methods: We first provided provider and nursing education with regard to safety and efficacy of the vaccines in both the clinic as well as the infusion setting. Nurses were then prompted to screen patients and offer the vaccines during intake prior to all infusions and clinic visits. We created bulk orders which allowed nurses greater control of releasing vaccine orders previously entered by the director of the clinic. We also posted “cheat-sheets” on optimal timing, safety and sequence of administration of the vaccines in every patient room and nursing station. After our first cycle, we identified that there was a delay in work-flow in the outpatient clinic with delivery of the vaccines to the clinic from the pharmacy. We therefore obtained a secure vaccine fridge that was placed at the nursing station, which allowed nurses easy access to the vaccines. Results: When vaccine administration during the 2018-2019 influenza season was compared to the 2019-2020 influenza season, we found that these interventions improved the administration of the influenza vaccine by 70%. There was a dramatic increase in the number of PCV-13 vaccines administered by 350% (more than 5-fold) and increase in PPVS-23 by 12.5%. No immediate adverse reactions during this cycle were reported to our nurse manager. Conclusions: A simple intervention of improved work-flow of vaccine administration and increased education of providers and nurses translated to a dramatic improvement in the administration of influenza and PCV-13 vaccines in a busy outpatient oncology clinic.

MET exon 14 skipping mutations (METΔ14Ex) represent a new class of molecular alterations that occur in approximately 3%-5% of non-small cell lung cancer (NSCLC) cases, and recently emerged as an actionable oncogenic driver with significant responsiveness to MET tyrosine kinase inhibitors, including crizotinib and capmatinib. METΔ14Ex mutations are generally was found to be mutually exclusive with other recognized oncogenic drivers and higher frequency has been noted in a unique, highly aggressive subtype of lung cancer called pulmonary sarcomatoid carcinoma. In the current study, we sought to perform an array of experiments to determine the intrinsic tumorigenic role of METΔ14Ex in NSCLC. Using multiple isogenic METΔ14Ex cell models established with CRISPR, we first determined that METΔ14Ex increased HGF-dependent kinase activity and enhanced HGF/MET downstream signaling through impairing receptor endocytic degradation. In subsequent functional analyses, we identified that METΔ14Ex significantly increases HGF-dependent cell scatter, migration and invasion capacity in vitro as well as metastasis in vivo compared to wild-type MET (MET WT). To explore the intrinsic mechanism in regulation of aforementioned phenotypes, an RNA sequencing study was conducted to evaluate the global changes of dynamic gene expression profile and key functional pathways. Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) revealed a large cluster of cell movement-related signaling pathways that were remarkably activated in HGF-treated METΔ14Ex models (vs MET WT) in a time-dependent manner. In addition, a number of upregulated DEGs (Differently Expressed Genes) were enriched in functional categories of cell cytoskeleton reorganization and cell-matrix adhesion degradation in METΔ14Ex models (vs MET WT). Based on the information of GSEA, we further found that the activity of the small GTPase Rac1, a crucial regulator of cytoskeleton dynamics, was significantly increased by HGF treatment in METΔ14Ex cells, whereas inhibition of the PI3K/Akt pathway repressed HGF/METΔ14Ex-mediated Rac1 activity and cell movement. Moreover, a group of Matrix metallopeptidases (MMPs) were highly upregulated in our models. Notably, among these MMPs, HGF-dependent upregulation of MMP1 expression (>100 fold in both mRNA and protein level) was very prominent in METΔ14Ex compared to MET WT cells, and silencing MMP1 using siRNA dramatically repressed HGF-triggered METΔ14Ex cell invasion. We finally found that the MET inhibitors, MGCD516 and capmatinib remarkably inhibited HGF/METΔ14Ex signaling and repressed cell invasion in vitro and metastasis in vivo. In conclusion, these data demonstrate that METΔ14Ex plays a critical role in enhancing cell movement and metastasis, at least partially via upregulation of the PI3K/Akt-Rac1 pathway and expression of MMPs, and blockade of HGF/METΔ14Ex signaling using MET inhibitors might be potent therapeutic options for METΔ14Ex-mutated NSCLC patients and recognition of the unique biology of METΔ14Ex-mediated oncogenesis might yield additional synergistic treatment strategies. Citation Format: Feng Wang, Yang Liu, Elaine Shum, Mariam Alexander, Deyou Zheng, Roman Perez-Soler, Haiying Cheng, Balazs Halmos. A novel functional analysis of MET exon 14 skipping mutation in regulation of tumor cell invasion and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4907.

Over the last 15 years, multiple generations of EGFR tyrosine kinase inhibitors (TKIs) have completely transformed the landscape of management of EGFR -mutated lung cancers but are rarely used in other settings. In the pre-molecular testing era, the 1 st generation reversible EGFR inhibitor, erlotinib, was shown to prolong survival in patients with stage IIIB/IV non-small cell lung cancer (NSCLC) after failure of first- or second-line chemotherapy compared to placebo [overall survival (OS) was 6.7 vs . 4.7 months, hazard ratio (HR) 0.70, P

Immunotherapy with immune-checkpoint inhibitors (ICIs) has shifted the therapeutic landscape in lung cancer. At the same time, the clinical benefits of ICIs in unselected patients remain modest.

Background: Over 200,000 people underwent weight loss surgery in the United States in 2017. The absorption of numerous drugs has been shown to be altered in patients after bariatric procedures, as gastrointestinal absorptive surface, food volume, and gastric pH all affect bioavailability. Dosing of warfarin, which is commonly used for the treatment of venous thromboembolism (VTE), often needs to be adjusted, based on INR, after bariatric surgery. Since direct oral anticoagulants (DOACs), which are rapidly replacing warfarin as standard anticoagulant therapy, are not monitored, there is concern regarding their efficacy and safety in patients who have had bariatric surgery, particularly rivaroxaban, which is absorbed primarily in the stomach and must be taken with food at therapeutic doses. One study found that 9 out of 9 apixaban patients (but only 2 of 7 rivaroxaban patients) had levels that fell within the expected range after bariatric surgery. Effects of bariatric surgery on DOACs may be further complicated by baseline obesity and subsequent weight loss of the patients. The goal of our current study is to determine whether DOACs are safe and effective in preventing recurrent VTE in patients who have undergone bariatric surgery. Methods: Using our institutional database, we identified all adult patients (age ≥18 years) with a history of bariatric surgery (gastric banding, sleeve gastrectomy, and Roux-en-Y gastric bypass) who were started on anticoagulation with apixaban or rivaroxaban for VTE, between July 1, 2013 and June 30, 2018. We performed retrospective chart review to obtain information on patient demographics, BMI and type of bariatric surgery. We documented clinical outcomes of recurrent VTE and bleeding from the first prescription date to the earliest of a thrombotic event, discontinuation of medication, death, or the end of study period, June 30, 2018. VTE events were confirmed by a review of imaging studies (compression ultrasonography, ventilation/perfusions scans, and CT scans). Bleeding events were included if they met criteria for clinically relevant non-major bleeding and/or major bleeding according to the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Safety outcomes included major bleeding (MB) and clinically relevant non-major bleeding (CRNMB). We also compared VTE recurrence and bleeding rates between the post-bariatric surgery patients and patients with BMI >40 from our prior study. Chi- squared tests were used to assess statistical significance of the differences in recurrent VTE and bleeding rates between anticoagulant cohorts. Results: Data on 102 patients were collected: 42 patients on apixaban and 60 patients on rivaroxaban. Our population was predominantly female (82.4%) with a mean age of 48.5 years and a median BMI of 35.7 at initiation of anticoagulation. Gastric bypass was the most common bariatric procedure (51%), followed by sleeve gastrectomy (37.3%), and gastric banding (11.8%). There were no recurrent VTE events in the apixaban cohort with a median follow-up duration of 137 days. Among patients on rivaroxaban, with a median follow-up of 232 days, there was one recurrent VTE (1.7%) in a patient with a BMI of 54 at the time of event. When we compared VTE recurrence rates of our combined DOAC cohort (apixaban + rivaroxaban) between our general morbidly obese population, from a prior study at our institution, and post-bariatric surgery patients, there was no statistically significant difference (2.0% vs. 1.0%, respectively, p=0.5). In bariatric surgery patients, one CRNMB event was recorded in apixaban group (2.4%) while 4 major bleeding events occurred on rivaroxaban (6.7%), p=0.3. There was no significant difference in the rate of composite MB and CRNMB between the general obesity and bariatric surgery patients (8.0% vs. 4.9%, respectively, p=0.3). Conclusions: In a review of post-bariatric surgery patients on anticoagulation for VTE, we found low rates of recurrent VTE for patients on DOACs. Although we had a relatively small sample size, the incidence of VTE recurrence was not higher in this cohort than was found in our previously published study of general obesity population. Prospective studies are needed to further investigate the efficacy and safety of direct oral anticoagulants in patients after bariatric surgery. Table. Disclosures Kushnir: Janssen Pharmaceuticals: Research Funding. Billett:Janssen: Research Funding.

Mutations in the human Mid1 gene cause Opitz G/BBB syndrome, which is characterized by various midline closure defects. The Caenorhabditis elegans homolog of Mid1, madd-2, positively regulates signaling by the unc-40 Netrin receptor during the extension of muscle arms to the midline and in axon guidance and branching. During uterine development, a specialized cell called anchor cell (AC) breaches the basal laminae separating the uterus from the epidermis and invades the underlying vulval tissue. AC invasion is guided by an UNC-6 Netrin signal from the ventral nerve cord and an unknown guidance signal from the vulval cells. Using genetic epistasis analysis, we show that madd-2 regulates AC invasion downstream of or in parallel with the Netrin signaling pathway. Measurements of AC shape, polarity and dynamics indicate that MADD-2 prevents the formation of ectopic AC protrusions in the absence of guidance signals. We propose that MADD-2 represses the intrinsic invasive capacity of the AC, while the Netrin and vulval guidance cues locally overcome this inhibitory activity of MADD-2 to guide the AC ventrally into the vulval tissue. Therefore, developmental cell invasion depends on a precise balance between pro- and anti-invasive factors.

A 37-year-old woman with known glioblastoma multiforme was admitted for treatment of new deep vein thrombosis. Anion gap and plasma lactate levels were found to be elevated. Magnetic resonance imaging of the brain showed a stable, advanced glioblastoma multiforme. All causes of lactic acidosis, including infections and medications, were ruled out. Aggressive tumors have been shown to produce lactate levels in minute quantities in their microenvironment, which helps them metastasize and evade immune response and even radiation.

This paper presents a visually servoed micropositioning system capable of automatically extracting locomotive features of Caenorhabditis elegans online at a full 30 Hz. The employment of Gaussian Pyramid Level-2 images significantly reduces the image size by 16-fold and permits real-time feature extraction, without sacrificing accuracy due to the cubic smoothing spline fitting. The automated micropositioning system is capable of revealing subtle differences in locomotive behavior across strains. A total of 128 worms of four C. elegans strains with different numbers of muscle arms were continuously tracked for 3 min per sample, and locomotive features were extracted online. Validated by experiments, the innovation in image analysis, or data reduction without sacrificing accuracy allows for rapid, online, and accurate analysis of streaming videos of C. elegans and other similar microorganisms.

The postsynaptic membrane of the embryonic neuromuscular junction undergoes a dramatic expansion during later development to facilitate the depolarization of larger muscles. In C. elegans, the postsynaptic membrane resides at the termini of plasma membrane extensions called muscle arms. Membrane extension to the motor axons during larval development doubles the number of muscle arms, making them a tractable model to investigate both postsynaptic membrane expansion and guided membrane extension. To identify genes required for muscle arm extension, we performed a forward screen for mutants with fewer muscle arms. We isolated 23 mutations in 14 genes, including unc-40/Dcc, which encodes a transmembrane receptor that guides the migration of cells and extending axons in response to the secreted UNC-6/Netrin spatial cue. We discovered that UNC-40 is enriched at muscle arm termini and functions cell-autonomously to direct arm extension to the motor axons. Surprisingly, UNC-6 is dispensable for muscle arm extension, suggesting that UNC-40 relies on other spatial cues to direct arm extension. We provide the first evidence that the guanine-nucleotide exchange factor UNC-73/Trio,members of the WAVE actin-polymerization complex, and a homolog of the focal adhesion complex can function downstream of UNC-40 to direct membrane extension. Our work is the first to define a pathway for directed muscle membrane extension and illustrates that axon guidance components can play key roles in postsynaptic membrane expansion.

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction(NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs)also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.

SummaryThe body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

The body wall muscles (BWMs) of nematodes are connected to motor axons by muscle membrane extensions called muscle arms. To better understand how muscle arm extension is regulated, we screened conserved receptor tyrosine kinases for muscle arm defects in Caenorhabditis elegans. We discovered that mutations in daf-2, which encodes the only insulin-like receptor tyrosine kinase, confer a supernumerary muscle arm (Sna) phenotype. The Sna phenotype of daf-2 mutants is suppressed by loss-of-function in the canonical downstream FOXO-family transcription factor DAF-16 in either the muscles or the intestine, demonstrating that insulin-like signaling can regulate muscle arm extension non-autonomously. Furthermore, supernumerary arm extension requires the B isoform of the down-stream DAF-12 nuclear hormone receptor, which lacks the DNA-binding domain, but retains the ligand-binding domain. daf-2 regulates many processes in C. elegans including entry into dauer, which is a diapause-like state that facilitates survival of harsh environmental conditions. We found that wild-type dauers are also Sna. Unlike other changes associated with dauer, however, the Sna phenotype of dauers persists in recovered adults. Finally, disruption of a TGF-beta pathway that regulates dauer formation in parallel to the insulin-like pathway also confers the Sna phenotype. We conclude that supernumerary muscle arms are a novel dauer-specific modification that may facilitate some aspect of dauer behavior.

PURPOSE OF REVIEW The glomerular visceral epithelial cell plays a central role in ultrafiltration of the blood and in a wide variety of inherited and acquired diseases of the kidney. The discovery of nephrin and other slit diaphragm proteins has led to an explosion of knowledge in the biology of this cell type. The most significant recent discoveries are reviewed in this paper. RECENT FINDINGS Together with the glomerular endothelial cells and intervening glomerular basement membrane, the podocyte constitutes a major portion of the glomerular filtration barrier that separates blood from the urinary space. A number of proteins have been identified that are localized to the slit diaphragms that separate podocyte foot processes. Although it has been suggested that the slit diaphragm represents the ultimate filtration barrier, additional roles for this structure as a signaling centre and in endocytosis have been identified. Mutations in genes that reside in the slit pore or interact with the actin cytoskeleton have been linked to a variety of inherited diseases of the podocyte. Additional mutations in these genes have been linked to sporadic forms of nephrotic syndrome and proposed as modifiers of renal risk. The generation of podocyte-specific transgenic models and genomic tools for the murine podocyte provide important resources for the glomerular biologist. SUMMARY Over the past year, studies using human genetics, conditional gene targeting and cell biological approaches have led to a rapid increase in our understanding of podocyte and glomerular biology, which should lead to the development of novel therapies for individuals with glomerular disease.