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Golden Gate cloning allows rapid and reliable assembly of multiple DNA fragments in a defined orientation. Golden Gate cloning requires careful design of the restriction fragment overhangs to minimize undesired products and to generate the desired junctions. The ApE (A plasmid Editor) software package can assist in silico design of input fragments or to generate expected assembly products., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Calcium channels at synaptic boutons are critical for synaptic function, but their number and distribution are poorly understood. This gap in knowledge is primarily due to the resolution limits of fluorescence microscopy. In the last decade, the diffraction limit of light was surpassed, and fluorescent molecules can now be localized with nanometer precision. Concurrently, new gene editing strategies allowed direct tagging of the endogenous calcium channel genes-expressed in the correct cells and at physiological levels. Further, the repurposing of self-labeling enzymes to attach fluorescent dyes to proteins improved photon yields enabling efficient localization of single molecules. Here, we describe tagging strategies, localization microscopy, and data analysis for calcium channel localization. In this case, we are imaging calcium channels fused with SNAP or HALO tags in live anesthetized C. elegans nematodes, but the analysis is relevant for any super-resolution preparations. We describe how to process images into localizations and protein clusters into confined nanodomains. Finally, we discuss strategies for estimating the number of calcium channels present at synaptic boutons. Key features • Super-resolution imaging of live anesthetized C. elegans. • Three-color super-resolution reconstruction of synapses. • Nanodomains and the distribution of proteins. • Quantification of the number of proteins at synapses from single-molecule localization data., Competing Interests: Competing interestsE.M.J holds a patent on optical biplanes, which are integral to the design of the microscope discussed in this study. However, E.M.J does not have any financial stake in Bruker., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

Amyotrophic lateral sclerosis can be caused by abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping and subsequent dissection of the neuromuscular circuit, we show that TDP-43 worms have profound defects in GABA neurons. Moreover, acetylcholine neurons appear functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of, among others, G-protein-coupled receptors restores neurotransmission, but inefficiently rescues locomotion. Rebalancing the excitatory-to-inhibitory ratio in the neuromuscular system by simultaneous stimulation of the affected GABA- and acetylcholine neurons, however, not only synergizes the effects of boosting individual neurotransmitter systems, but instantaneously improves movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations., Competing Interests: Declaration of interests L.J. is currently employed by AstraZeneca as a medical advisor and M.K. is employed by the health insurance company Zilveren Kruis as a data scientist; both positions are unrelated to the current article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks.

Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release., Competing Interests: BM, SM, SW, PL, LN, AS, PM, AC, MR, MS, AM, ZW, EJ No competing interests declared, (© 2023, Mueller, Merrill et al.)

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle. Unc13 mediates synaptic vesicle docking and converts syntaxin into the permissive "open" configuration. The SM protein, Unc18, is required to initiate and proofread SNARE assembly. The SNAREs are then held in a half-zippered state by synaptotagmin and complexin. Calcium removes the synaptotagmin and complexin block, and the SNAREs drive vesicle fusion. After fusion, NSF and alpha-SNAP unwind the SNAREs and thereby recharge the system for further rounds of fusion. In this chapter, we will describe the discovery of the SNAREs, their relevant structural features, models for their function, and the central role of Unc18. In addition, we will touch upon the regulation of SNARE complex formation by Unc13, complexin, and synaptotagmin., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: To describe the sonographic findings of endometrial intraepithelial neoplasia (EIN), a precursor of endometrial cancer., Methods: Cases were found by word search of pathology database 1/2013 to 6/2019. One hundred and seventy-eight patients with ultrasound <1 year prior to biopsy were included. Medical records were searched for patient data. Two radiologists blindly classified images. Differences of opinion were decided by clinical report. Univariate and multivariate analyses were performed., Results: Median time between ultrasound and first sampling procedure was 49 days. Median age was 55 (range 28-85) years. Endometrial thickness ranged from 2 to 90 mm. Mean endometrial thickness was 13 ± 6 mm in the noncancer group and 16 ± 11 mm in the cancer group (P = .02). The endometrium was almost always heterogeneous 175/178 (98%). Cysts were almost always multiple (89/109, 82%) and >1 mm (72/109, 66%). Masses were most often >5 mm (56/105, 55%) and ill-defined (41/105, 39%). Vascularity was present in 93/178 examinations (52%) and always associated with cysts and/or mass. There were 92 cancers, 25 with invasion (including 4 with tumor extension into adenomyosis). In 47 cases, the endometrial-myometrial interface was graded as ill-defined, 39 of whom had hysterectomy. There was macroscopic cancer in 11, microscopic cancer in 4, and invasive carcinoma in 12 patients (P for invasive cancer versus other outcomes = .02). Depth of invasion was 5- >95%, with 6 cancers >50%. Multivariate analysis showed thickness, polyps, and type of bleeding as the best set of independent variables for cancer (area under the receiver operating characteristic (ROC) curve [AUC] = .75). Replacing type of bleeding with age or menopausal status had AUC of .73 and .74, respectively., Conclusions: EIN has a variety of sonographic appearances with thickened endometrium with cysts and masses being common. Ill-definition of the endometrial-myometrial interface is a poor prognostic finding when seen in the absence of adenomyosis., (© 2021 American Institute of Ultrasound in Medicine.)

Unc18 and SNARE proteins form the core of the membrane fusion complex at synapses. To understand the functional interactions within the core machinery, we adopted an "interspecies complementation" approach in Caenorhabditis elegans . Substitutions of individual SNAREs and Unc18 proteins with those from yeast fail to rescue fusion. However, synaptic transmission could be restored in worm-yeast chimeras when two key interfaces were present: an Habc-Unc18 contact site and an Unc18-SNARE motif contact site. A constitutively open form of Unc18 bypasses the requirement for the Habc-Unc18 interface. These data suggest that the Habc domain of syntaxin is required for Unc18 to adopt an open conformation; open Unc18 then templates SNARE complex formation. Finally, we demonstrate that the SNARE and Unc18 machinery in the nematode C. elegans can be replaced by yeast proteins and still carry out synaptic transmission, pointing to the deep evolutionary conservation of these two interfaces., Competing Interests: The authors declare no competing interests. L.A.P. is currently employed in the Department of Pathology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA.

Endometriosis, a painful gynecological condition accompanied by inflammation in women of reproductive age, is associated with an increased risk of ovarian cancer. We evaluated the role of peritoneal heme accumulated during menstrual cycling, as well as peritoneal and lesional macrophage phenotype, in promoting an oncogenic microenvironment. We quantified the heme-degrading enzyme, heme oxygenase-1 (HO-1, encoded by Hmox 1) in normal peritoneum, endometriotic lesions and endometriosis-associated ovarian cancer (EAOC) of clear cell type (OCCC). HO-1 was expressed primarily in macrophages and increased in endometrioma and OCCC tissues relative to endometriosis and controls. Further, we compared cytokine expression profiles in peritoneal macrophages (PM) and peripheral blood mononuclear cells (PBMC) in women with endometriosis versus controls as a measure of a tumor-promoting environment in the peritoneum. We found elevated levels of HO-1 along with IL-10 and the pro-inflammatory cytokines (IL-1β, IL-16, IFNγ) in PM but not in PBMC from endometriosis patients. Using LysM-Cre:Hmox1 flfl conditional knockout mice, we show that a deficiency of HO-1 in macrophages led to the suppression of growth of ID8 ovarian tumors implanted into the peritoneum. The restriction of ID8 ovarian tumor growth was associated with an increased number of Mac3 + macrophage and B cells in LysM-Cre:Hmox1 flfl mice compared to controls. Functional experiments in ovarian cancer cell lines show that HO-1 is induced by heme. Low levels of exogenous heme promoted ovarian cancer colony growth in soft agar. Higher doses of heme led to slower cancer cell colony growth in soft agar and the induction of HO-1. These data suggest that perturbation of heme metabolism within the endometriotic niche and in cancer cells themselves may be an important factor that influences tumor initiation and growth.

A plasmid Editor (ApE) is a free, multi-platform application for visualizing, designing, and presenting biologically relevant DNA sequences. ApE provides a flexible framework for annotating a sequence manually or using a user-defined library of features. ApE can be used in designing plasmids and other constructs via in silico simulation of cloning methods such as PCR, Gibson assembly, restriction-ligation assembly and Golden Gate assembly. In addition, ApE provides a platform for creating visually appealing linear and circular plasmid maps. It is available for Mac, PC, and Linux-based platforms and can be downloaded at https://jorgensen.biology.utah.edu/wayned/ape/., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Davis and Jorgensen.)

Deep-brain microscopy is strongly limited by the size of the imaging probe, both in terms of achievable resolution and potential trauma due to surgery. Here, we show that a segment of an ultra-thin multi-mode fiber (cannula) can replace the bulky microscope objective inside the brain. By creating a self-consistent deep neural network that is trained to reconstruct anthropocentric images from the raw signal transported by the cannula, we demonstrate a single-cell resolution (< 10μm), depth sectioning resolution of 40 μm, and field of view of 200 μm, all with green-fluorescent-protein labelled neurons imaged at depths as large as 1.4 mm from the brain surface. Since ground-truth images at these depths are challenging to obtain in vivo, we propose a novel ensemble method that averages the reconstructed images from disparate deep-neural-network architectures. Finally, we demonstrate dynamic imaging of moving GCaMp-labelled C. elegans worms. Our approach dramatically simplifies deep-brain microscopy.

We mapped rol-9 to the mlt-11 locus (encoded by the gene W01F3.3) on the far-right end of chromosome V. The canonical allele of rol-9 , sc148 , is an in-frame deletion in a conserved exon of the protein that creates a gain-of-function roller phenotype. sc148 deletes a short peptide of unknown function conserved in nematodes., (Copyright: © 2022 by the authors.)

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50-100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits., Competing Interests: The authors have declared that no competing interests exist.

Study Objective: To compare the Fundamentals of Laparoscopic Surgery (FLS) exam scores between obstetrics and gynecology (OBGYN) and general surgery (GS) providers., Design: This is a retrospective cohort study at a single institution from July 2007 to May 2018. Categorical and continuous variables were analyzed with χ2 test, t test, and Wilcoxon rank sum test., Setting: Beth Israel Deaconess Medical Center (BIDMC), Boston, MA, a tertiary care academic medical center., Patients: All providers who took the FLS exam at the Carl J. Shapiro Simulation and Skills Center at BIDMC., Interventions: FLS certification., Measurements and Main Results: A total of 205 BIDMC trainees and faculty took the FLS exam between July 2007 and May 2018, of which 176 were identified to be OBGYN or GS providers. The FLS certification pass rate was high for both specialties (97.0% OBGYN vs 96.1% sGS, p = .76). When comparing all providers, no significant difference was found in the mean manual skill test scores between surgical specialties (594.9 OBGYN vs 601.0 GS, p = .59); whereas, a significant difference was noted in the mean cognitive scores, with GS providers scoring higher than OBGYN providers (533.8 OBGYN vs 583.4 GS, p <.001). However, when adjusting for several variables in a multivariate linear regression model, surgical specialty was not a predictor for cognitive scores. In the multivariate analysis, age, sex, and test year were predictors for cognitive scores, with higher scores associated with younger age, male sex, and advancing calendar year. None of the variables were significant predictors of manual scores., Conclusion: Both OBGYN and GS providers had extremely high FLS pass rates. In the multivariate analysis, surgical specialty was not a predictor for higher FLS test scores for either manual or cognitive test scores. Although OBGYN residency programs offer fewer years of training, OBGYN trainees demonstrate the capacity to perform well on the FLS exam., (Published by Elsevier Inc.)

The structural features of a synapse help determine its function. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified, a process called morphometry. Three parameters are typically assessed from each specimen: (1) the sizes of individual vesicles and organelles; (2) the absolute number and densities of organelles; and (3) distances between organelles and key features at synapses, such as active zone membranes and dense projections. For data to be meaningful, the analysis must be repeated from hundreds to thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze key structural features of synapses: SynapsEM. In short, we developed ImageJ/Fiji macros to record x,y-coordinates of segmented structures. The coordinates are then exported as text files. Independent investigators can reload the images and text files to reexamine the segmentation using ImageJ. The Matlab program then calculates and reports key synaptic parameters from the coordinates. Since the values are calculated from coordinates, rather than measured from each micrograph, other parameters such as locations of docked vesicles relative to the center of an active zone can be extracted in Matlab by additional scripting. Thus, this program can accelerate the morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Watanabe, Davis, Kusick, Iwasa and Jorgensen.)

The high-gamma-amino butyric acid (GABA)-producing bacterium Levilactobacillus brevis strain NPS-QW 145, along with Streptococcus thermophilus (one of the two starter bacteria used to make yogurt for its proteolytic activity), enhances GABA production in milk. However, a mechanistic understanding of how Levilactobacillus brevis cooperates with S. thermophilus to stimulate GABA production has been lacking. Comparative peptidomic and metatranscriptomic analyses were carried out to unravel the casein and lactose utilization patterns during milk fermentation with the coculture. We found that particular peptides hydrolyzed by S. thermophilus ASCC1275 were transported and biodegraded with peptidase in Lb. brevis 145 to meet the growth needs of the latter. In addition, amino acid synthesis and metabolism in Lb. brevis 145 were activated to further support its growth. Glucose, as a result of lactose hydrolysis by S. thermophilus 1275, but not available lactose in milk, was metabolized as the main carbon source by Lb. brevis 145 for ATP production. In the stationary phase, under acidic conditions due to the accumulation of lactic acid produced by S. thermophilus 1275, the expression of genes involved in pyridoxal phosphate (coenzyme of glutamic acid decarboxylase) metabolism and glutamic acid decarboxylase (Gad) in Lb. brevis 145 was induced for GABA production. SIGNIFICANCE A huge market for GABA-rich milk as a dietary therapy for the management of hypertension is anticipated. The novelty of this work lies in applying peptide profiles supported by metatranscriptomics to elucidate (i) the pattern of casein hydrolysis by S. thermophilus 1275, (ii) the supply of peptides and glucose by S. thermophilus 1275 to Lb. brevis 145, (iii) the transportation of peptides in Lb. brevis and the degradation of peptides by this organism, which was reported to be nonproteolytic, and (iv) GABA production by Lb. brevis 145 under acidic conditions. Based on the widely reported contribution of lactic acid bacteria (LAB) and GABA to human health, the elucidation of interactions between the two groups of bacterial communities in the production of GABA-rich milk is important for promoting the development of functional dairy food and may provide new insight into the development of industrial GABA production., (Copyright © 2020 American Society for Microbiology.)

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must 'dock' to refill vacated release sites. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation, then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. After stimulation, the total number of docked vesicles across all synapses decreases by ~40%. Within 14 ms, new vesicles are recruited and fully replenish the docked pool, but this docking is transient and they either undock or fuse within 100 ms. These results demonstrate that the recruitment of synaptic vesicles to release sites is rapid and reversible.

Objective: To establish validity evidence for the Essentials in Minimally Invasive Gynecology laparoscopic and hysteroscopic simulation systems., Methods: A prospective cohort study was IRB approved and conducted at 15 sites in the United States and Canada. The four participant cohorts based on training status were: 1) novice (postgraduate year [PGY]-1) residents, 2) mid-level (PGY-3) residents, 3) proficient (American Board of Obstetrics and Gynecology [ABOG]-certified specialists without subspecialty training); and 4) expert (ABOG-certified obstetrician-gynecologists who had completed a 2-year fellowship in minimally invasive gynecologic surgery). Qualified participants were oriented to both systems, followed by testing with five laparoscopic exercises (L-1, sleeve-peg transfer; L-2, pattern cut; L-3, extracorporeal tie; L-4, intracorporeal tie; L-5, running suture) and two hysteroscopic exercises (H-1, targeting; H-2, polyp removal). Measured outcomes included accuracy and exercise times, including incompletion rates., Results: Of 227 participants, 77 were novice, 70 were mid-level, 33 were proficient, and 47 were experts. Exercise times, in seconds (±SD), for novice compared with mid-level participants for the seven exercises were as follows, and all were significant (P<.05): L-1, 256 (±59) vs 187 (±45); L-2, 274 (±38) vs 232 (±55); L-3, 344 (±101) vs 284 (±107); L-4, 481 (±126) vs 376 (±141); L-5, 494 (±106) vs 420 (±100); H-1, 176 (±56) vs 141 (±48); and H-2, 200 (±96) vs 150 (±37). Incompletion rates were highest in the novice cohort and lowest in the expert group. Exercise errors were significantly less and accuracy was greater in the expert group compared with all other groups., Conclusion: Validity evidence was established for the Essentials in Minimally Invasive Gynecology laparoscopic and hysteroscopic simulation systems by distinguishing PGY-1 from PGY-3 trainees and proficient from expert gynecologic surgeons.

Background: Adnexal surgery is believed to be more complex in patients with prior hysterectomy; however, there is little data regarding surgical outcomes. Understanding of individualized risks improves counseling, informed consent, and preoperative planning., Methods: We performed a retrospective cohort study with a control group; we evaluated 744 patients undergoing laparoscopic adnexal surgery at an academic tertiary care center from 2011 to 2015. Comparisons were made using Chi square, Fisher's exact, or Wilcoxon-rank sum tests. We used log-binomial regression to calculate risk ratio and 95% confidence interval., Results: Patients with prior hysterectomy were more likely to have intraoperative or postoperative complications at the time of laparoscopic adnexal surgery when compared to patients without prior hysterectomy [17.7% vs. 10.2%, p = 0.02, risk ratio (RR) 1.7, 95% confidence interval (CI) 1.1-2.7]. Patients with prior hysterectomy were four times more likely to have intraoperative complications (3.2% vs. 0.8%, p = 0.047, RR 4.0, 95% CI 1.1-14.7), and five times more likely to have conversion to laparotomy (5.6% vs. 1.1%, p = 0.004, RR 5.0, 95% CI 1.8-14.0). Patients with prior hysterectomy were more likely to need additional procedures, including lysis of adhesions (69.4% vs. 26.0%, p < 0.001), ureterolysis (15.3% vs. 4.8%, p < 0.001), and cystoscopy (28.2% vs. 8.1%, p < 0.001). They had longer operative time [101.5 min (IQR 59.5-135.0) vs. 78.0 min (IQR 53.0-109.0, p < 0.001)], and were less likely to have outpatient surgery (56.5% vs. 84.8%, p < 0.01). Postoperative complications were also more common (15.3% vs. 9.4%, p = 0.046)., Conclusions: Patients with prior hysterectomy were 70% more likely to have a complication at the time of laparoscopic adnexal surgery than patients without hysterectomy. Increased risk of complications in subsequent adnexal surgery may influence the informed consent process or decisions regarding ovarian conservation. Awareness of potential need for additional surgical procedures may guide availability of equipment, choice of operating site, or referral to an advanced pelvic surgeon.

Eisosomes are membrane furrows at the cell surface of yeast that have been shown to function in two seemingly distinct pathways, membrane stress response and regulation of nutrient transporters. We found that many stress conditions affect both of these pathways by changing plasma membrane tension and thus the morphology and composition of eisosomes. For example, alkaline stress causes swelling of the cell and an endocytic response, which together increase membrane tension, thereby flattening the eisosomes. The flattened eisosomes affect membrane stress pathways and release nutrient transporters, which aids in their down-regulation. In contrast, glucose starvation or hyperosmotic shock causes cell shrinking, which results in membrane slack and the deepening of eisosomes. Deepened eisosomes are able to trap nutrient transporters and protect them from rapid endocytosis. Therefore, eisosomes seem to coordinate the regulation of both membrane tension and nutrient transporter stability.

Objective: To characterize long-term national trends in surgical approach for hysterectomy after the U.S. Food and Drug Administration (FDA) warning against power morcellation for laparoscopic specimen removal., Methods: This was a descriptive study using data from the American College of Surgeons National Surgical Quality Improvement Program from 2012 to 2016. We identified hysterectomies using Current Procedural Terminology codes. We used an interrupted time-series analysis to evaluate abdominal and supracervical hysterectomy trends surrounding The Wall Street Journal article first reporting morcellation safety concerns and the FDA safety communication. We compared categorical and continuous variables using χ, t, and Wilcoxon rank sum tests., Results: We identified 179,950 hysterectomies; laparoscopy was the most common mode of hysterectomy in every quarter. Before The Wall Street Journal article, there was no significant change in proportion of abdominal hysterectomies (0.3% decrease/quarter, P=.14). After The Wall Street Journal article, use of abdominal hysterectomy increased 1.1% per quarter for two quarters through the FDA warning (P<.001), plateaued for three quarters until March 2015 (P=.65), then decreased by 0.8% per quarter through 2016 (P<.001). Supracervical hysterectomy volume continuously decreased after the FDA warning (1.0% decrease per quarter, P<.001) and after three quarters (0.7% decrease per quarter, P=.01), then plateaued from April 2015 through 2016 (0.05% decrease per quarter, P=.40). Mode of supracervical hysterectomy was unchanged from 2012 to 2013 (P=.43), followed by two quarters of significant increase in proportion of supracervical abdominal hysterectomies (11.7%/quarter, P<.001). This change in mode of supracervical hysterectomy then plateaued through 2016 (P=.06)., Conclusion: Despite early studies suggesting that minimally invasive hysterectomy decreased in response to safety concerns regarding power morcellation, we found that this effect reversed 1 year after the FDA safety communication. However, there was a sustained decline in supracervical hysterectomy, and the remaining supracervical hysterectomies were more likely to be performed using laparotomy.

Venous thromboembolism (VTE) is the leading cause of preventable healthcare-related death after surgery. Although there is a large body of research on VTE in the general population as well as risk-assessment tools, evidence specific to the current practices in gynecologic surgery is more sparse. This review article seeks to discuss current literature on VTE in gynecologic surgery, with a focus on minimally invasive surgery. Evidence on risk factors for VTE in gynecologic surgery is evaluated as well as current recommendations use of thromboprophylaxis for prevention of VTE. Despite data showing that minimally invasive gynecologic surgery independently decreases risk of VTE compared with laparotomy, current clinical risk assessment tools and guidelines do not incorporate mode of surgery into recommendations for perioperative VTE prevention., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the incidence of postoperative venous thromboembolism after gynecologic surgery by mode of incision., Methods: We conducted a retrospective cohort study of all patients who underwent gynecologic surgery from May 2006 to June 2015 at two tertiary care academic hospitals in Massachusetts. Billing and diagnosis codes were used to identify surgeries and cases of venous thromboembolism., Results: A total of 43,751 surgical encounters among 37,485 individual patients were noted during the study. The overall incidence of venous thromboembolism is 0.2% for all gynecologic surgeries, 0.7% for hysterectomy, and 0.2% for myomectomy. Compared with patients undergoing laparotomy, patients who underwent minimally invasive gynecologic surgery were less likely to develop venous thromboembolism (laparoscopy risk ratio 0.22, 95% CI 0.13-0.37; vaginal surgery risk ratio 0.07, 95% CI 0.04-0.12). This effect persisted when data were adjusted for other known venous thromboembolism risk factors such as age, race, cancer, medical comorbidities, use of pharmacologic thromboprophylaxis, admission status, and surgical time., Conclusion: Minimally invasive surgery is associated with a decreased risk of venous thromboembolism in patients undergoing gynecologic surgery, including hysterectomy and myomectomy. Although society guidelines and risk assessment tools do not currently account for mode of surgery when assessing venous thromboembolism risk and recommendations for prevention, there is a small but growing body of evidence in both general and gynecologic surgery literature that surgical approach affects a patient's risk of postoperative venous thromboembolism. Mode of surgery should be considered when assessing venous thromboembolism risk and planning venous thromboembolism prophylaxis for patients undergoing gynecologic surgery.

Sterilization is the most common form of contraception used worldwide and is highly effective in preventing unintended pregnancy. Each of the available sterilization methods has unique advantages and disadvantages that influence the choice of approach for each individual patient. Salpingectomy for sterilization has become more popular in recent years, with mounting evidence suggesting a protective effect against ovarian cancers originating in the fallopian tube. At the same time, Essure hysteroscopic sterilization has come under scrutiny because of increasing reports of possible adverse effects associated with its use. Here we review clinical updates in sterilization techniques, with a focus on salpingectomy and Essure hysteroscopic sterilization., (Copyright © 2017 American Association of Gynecologic Laparoscopists. All rights reserved.)

Synapse formation defines neuronal connectivity and is thus essential for neuronal circuit assembly. Trans-synaptic interactions of cell adhesion molecules are thought to induce synapse assembly. Here we demonstrate that a recently discovered and conserved short form of neurexin, γ-neurexin, which lacks canonical extracellular domains, is nonetheless sufficient to promote presynaptic assembly in the nematode C. elegans. γ- but not α-neurexin is required for assembling active zone components, recruiting synaptic vesicles, and clustering calcium channels at release sites to promote evoked synaptic transmission. Furthermore, we find that neurexin functions in parallel with the transmembrane receptor Frizzled, as the absence of both proteins leads to an enhanced phenotype-the loss of most synapses. Frizzled's pro-synaptogenic function is independent of its ligand, Wnt. Wnt binding instead eliminates synapses by inducing Frizzled's endocytosis and the downregulation of neurexin. These results reveal how pro- and anti-synaptogenic factors converge to precisely sculpt circuit formation in vivo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To estimate the prevalence of corpus uteri, cervix uteri, and ovarian malignancy in women undergoing hysterectomy or myomectomy for presumed benign indications., Methods: We conducted a secondary analysis of data from the 2014-2015 American College of Surgeons National Surgical Quality Improvement Program. Adult women undergoing hysterectomies and myomectomies without evidence for known or suspected cancer at the beginning of surgery were identified from the database. Our primary outcome measure was pathology-confirmed malignancy in the corpus uteri, cervix uteri, and ovary. We performed adjusted logistic regression analysis to examine the association of patient characteristics with the risk for malignancy., Results: Our sample included 24,076 women undergoing hysterectomy and 2,368 women undergoing myomectomy. Malignancy of the corpus uteri was found in 1.44% (95% CI 1.29-1.59%) of the women undergoing hysterectomy. The prevalence varied considerably across surgical routes with the rate being 0.23% (95% CI 0.06-0.58%) in laparoscopic supracervical hysterectomy and 1.89% (95% CI 1.65-2.14%) in total laparoscopic or laparoscopic-assisted vaginal hysterectomy. Older women were significantly more likely to have preoperatively undetected malignancy of the corpus uteri (adjusted odds ratio 6.46, 95% CI 4.96-8.41 for age 55 years or older vs age 40-54 years). Additionally, 0.60% (95% CI 0.50-0.70%) and 0.19% (95% CI 0.14-0.25%) of the women undergoing hysterectomy were found to have malignancy of the cervix uteri and the ovary, respectively. Among patients undergoing myomectomy, 0.21% (95% CI 0.03-0.40%) were found to have malignancy of the corpus uteri with no occult cervical or ovarian cancer identified., Conclusion: Prevalence of occult corpus uteri, cervical, and ovarian malignancy was 1.44%, 0.60%, and 0.19%, respectively, among women undergoing hysterectomy and it varied by patient age and surgical route.

Background: Langerhans cell histiocytosis (LCH) is a rare localized or systemic disease characterized by proliferation of myeloid-derived dendritic cells. Vulvar lesions might be the herald symptom of LCH and might mimic other cutaneous lesions. Prognosis varies widely on the basis of the extent and spread of disease., Case: An 8-year-old girl with a 4-month history of vulvar lesions resistant to topical steroids was referred by her pediatrician. Vulvar biopsy was diagnostic for LCH. Imaging studies revealed a left hip lesion consistent with LCH. The patient was subsequently treated for multisystem LCH with vinblastine and prednisone., Summary and Conclusion: Although rare, LCH might be diagnosed by gynecologic providers and should be included in the differential diagnosis of genital lesions. We recommend having a low threshold for performing biopsy of vulvar lesions., (Copyright © 2017 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.)

Pituitary tumors are frequently associated with mutations in the AIP gene and are sometimes associated with hypersecretion of growth hormone. It is unclear whether other factors besides an enlarged pituitary contribute to the hypersecretion. In a genetic screen for suppressors of reduced neurotransmitter release, we identified a mutation in Caenorhabditis elegans AIPR-1 (AIP-related-1), which causes profound increases in evoked and spontaneous neurotransmitter release, a high frequency of spontaneous calcium transients in motor neurons and an enlarged readily releasable pool of vesicles. Calcium bursts and hypersecretion are reversed by mutations in the ryanodine receptor but not in the voltage-gated calcium channel, indicating that these phenotypes are caused by a leaky ryanodine receptor. AIPR-1 is physically associated with the ryanodine receptor at synapses. Finally, the phenotypes in aipr-1 mutants can be rescued by presynaptic expression of mouse AIP, demonstrating that a conserved function of AIP proteins is to inhibit calcium release from ryanodine receptors.

Unc13 proteins are required for vesicle docking and priming during exocytosis. In this issue of Neuron, Lai et al. (2017) demonstrate that Unc13 ensures that the SNAREs assemble into functional subcomplexes. In a second manuscript, Michelassi et al. (2017) identify a previously unknown autoinhibited state for Unc13 mediated by the tandem C1 and C2 domains., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The heterotrimeric G protein G q positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of G q that acts in parallel to the canonical G q effector phospholipase C. Here, we examine how Trio and Rho act to stimulate neuronal activity downstream of G q in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the G q -Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of G q in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal G q -Rho signaling in C. elegans ., (Copyright © 2017 by the Genetics Society of America.)

Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless-steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single-molecule localizations of synaptic vesicle and active zone proteins in three dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging.

The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing., (Copyright © 2016 Edelman et al.)

The discovery of novel drugs against animal parasites is in high demand due to drug-resistance problems encountered around the world. Herein, the synthesis and characterization of 27 organic and organometallic derivatives of the recently launched nematocidal drug monepantel (Zolvix ® ) are described. The compounds were isolated as racemates and were characterized by 1 H, 13 C, and 19 F NMR spectroscopy, mass spectrometry, and IR spectroscopy, and their purity was verified by microanalysis. The molecular structures of nine compounds were confirmed by X-ray crystallography. The anthelmintic activity of the newly designed analogues was evaluated in vitro against the economically important parasites Haemonchus contortus and Trichostrongylus colubriformis. Moderate nematocidal activity was observed for nine of the 27 compounds. Three compounds were confirmed as potentiators of a known monepantel target, the ACR-23 ion channel. Production of reactive oxygen species may confer secondary activity to the organometallic analogues. Two compounds, namely, an organic precursor (3 a) and a cymantrene analogue (9 a), showed activities against microfilariae of Dirofilaria immitis in the low microgram per milliliter range., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To perform genotype-phenotype analysis in an infant with congenital arthrogryposis due to a de novo missense mutation in the NALCN ion channel and explore the mechanism of pathogenicity using a Caenorhabditis elegans model., Methods: We performed whole-exome sequencing in a preterm neonate with congenital arthrogryposis and a severe life-threatening clinical course. We examined the mechanism of pathogenicity of the associated NALCN mutation by engineering the orthologous mutation into the nematode C elegans using CRISPR-Cas9., Results: We identified a de novo missense mutation in NALCN, c.1768C>T, in an infant with a severe neonatal lethal form of the recently characterized CLIFAHDD syndrome (congenital contractures of the limbs and face with hypotonia and developmental delay). We report novel phenotypic features including prolonged episodes of stimulus-sensitive sustained muscular contraction associated with life-threatening episodes of desaturation and autonomic instability, extending the severity of previously described phenotypes associated with mutations in NALCN. When engineered into the C elegans ortholog, this mutation results in a severe gain-of-function phenotype, with hypercontraction and uncoordinated movement. We engineered 6 additional CLIFAHDD syndrome mutations into C elegans and the mechanism of action could be divided into 2 categories: half phenocopied gain-of-function mutants and half phenocopied loss-of-function mutants., Conclusions: The clinical phenotype of our patient and electrophysiologic studies show sustained muscular contraction in response to transient sensory stimuli. In C elegans, this mutation causes neuronal hyperactivity via a gain-of-function NALCN ion channel. Testing human variants of NALCN in C elegans demonstrates that CLIFAHDD can be caused by dominant loss- or gain-of-function mutations in ion channel function., (© 2016 American Academy of Neurology.)

Bisphenol-A (BPA) is an environmentally ubiquitous estrogen-like endocrine-disrupting compound. Exposure to BPA in utero has been linked to female reproductive disorders, including endometrial hyperplasia and breast cancer. Estrogens are an etiological factor in many of these conditions. We sought to determine whether in utero exposure to BPA altered the global CpG methylation pattern of the uterine genome, subsequent gene expression, and estrogen response. Pregnant mice were exposed to an environmentally relevant dose of BPA or DMSO control. Uterine DNA and RNA were examined by using methylated DNA immunoprecipitation methylation microarray, expression microarray, and quantitative PCR. In utero BPA exposure altered the global CpG methylation profile of the uterine genome and subsequent gene expression. The effect on gene expression was not apparent until sexual maturation, which suggested that estrogen response was the primary alteration. Indeed, prenatal BPA exposure preferentially altered adult estrogen-responsive gene expression. Changes in estrogen response were accompanied by altered methylation that preferentially affected estrogen receptor-α (ERα)-binding genes. The majority of genes that demonstrated both altered expression and ERα binding had decreased methylation. BPA selectively altered the normal developmental programming of estrogen-responsive genes via modification of the genes that bind ERα. Gene-environment interactions driven by early life xenoestrogen exposure likely contributes to increased risk of estrogen-related disease in adults.-Jorgensen, E. M., Alderman, M. H., III, Taylor, H. S. Preferential epigenetic programming of estrogen response after in utero xenoestrogen (bisphenol-A) exposure., (© FASEB.)

Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a "glycolytic metabolon," that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior. VIDEO ABSTRACT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cell-specific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3- to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications., (Copyright © 2016 by the Genetics Society of America.)

The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

Under most circumstances, GABA activates chloride-selective channels and thereby inhibits neuronal activity. Here, we identify a GABA receptor in the nematode Caenorhabditis elegans that conducts cations and is therefore excitatory. Expression in Xenopus oocytes demonstrates that LGC-35 is a homopentameric cation-selective receptor of the cys-loop family exclusively activated by GABA. Phylogenetic analysis suggests that LGC-35 evolved from GABA-A receptors, but the pore-forming domain contains novel molecular determinants that confer cation selectivity. LGC-35 is expressed in muscles and directly mediates sphincter muscle contraction in the defecation cycle in hermaphrodites, and spicule eversion during mating in the male. In the locomotory circuit, GABA release directly activates chloride channels on the muscle to cause muscle relaxation. However, GABA spillover at these synapses activates LGC-35 on acetylcholine motor neurons, which in turn cause muscles to contract, presumably to drive wave propagation along the body. These studies demonstrate that both direct and indirect excitatory GABA signaling plays important roles in regulating neuronal circuit function and behavior in C. elegans., (Copyright © 2015 the authors 0270-6474/15/352803-14$15.00/0.)