N-Nitrosodimethylamine investigations in Muta™Mouse define point-of-departure values and demonstrate less-than-additive somatic mutant frequency accumulations.

The N-nitrosamine, N-nitrosodimethylamine (NDMA), is an environmental mutagen and rodent carcinogen. Small levels of NDMA have been identified as an impurity in some commonly used drugs, resulting in several product recalls. In this study, NDMA was evaluated in an OECD TG-488 compliant Muta™Mouse gene mutation assay (28-day oral dosing across seven daily doses of 0.02-4 mg/kg/day) using an integrated design that assessed mutation at the transgenic lacZ locus in various tissues and at the endogenous Pig-a gene-locus, along with micronucleus frequencies in peripheral blood. Liver pathology was determined together with NDMA exposure in blood and liver. The additivity of mutation induction was assessed by including two acute single-dose treatment groups (i.e. 5 and 10 mg/kg dose on Day 1), which represented the same total dose as two of the repeat dose treatment groups. NDMA did not induce statistically significant increases in mean lacZ mutant frequency (MF) in bone marrow, spleen, bladder, or stomach, nor in peripheral blood (Pig-a mutation or micronucleus induction) when tested up to 4 mg/kg/day. There were dose-dependent increases in mean lacZ MF in the liver, lung, and kidney following 28-day repeat dosing or in the liver and kidney after a single dose (10 mg/kg). No observed genotoxic effect levels (NOGEL) were determined for the positive repeat dose-response relationships. Mutagenicity did not exhibit simple additivity in the liver since there was a reduction in MF following NDMA repeat dosing compared with acute dosing for the same total dose. Benchmark dose modelling was used to estimate point of departure doses for NDMA mutagenicity in Muta™Mouse and rank order target organ tissue sensitivity (liver > kidney or lung). The BMD50 value for liver was 0.32 mg/kg/day following repeat dosing (confidence interval 0.21-0.46 mg/kg/day). In addition, liver toxicity was observed at doses of ≥ 1.1 mg/kg/day NDMA and correlated with systemic and target organ exposure. The integration of these results and their implications for risk assessment are discussed.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

A cross-industry survey on photosafety evaluation of pharmaceuticals after implementation of ICH S10.

A cross-industry survey was conducted by EFPIA/IQ DruSafe in 2018 to provide information on photosafety evaluation of pharmaceuticals after implementation of ICH S10. This survey focused on the strategy utilized for photosafety risk assessment, the design of nonclinical (in vitro and in vivo) and clinical evaluations, the use of exposure margins in risk assessment, and regulatory interactions. The survey results indicated that a staged approach for phototoxicity assessment has been widely accepted by regulatory authorities globally. The OECD-based 3T3 NRU Phototoxicity Test is the most frequently used in vitro approach. Modifications to this assay suggested by ICH S10 are commonly applied. For in-vitro-positives, substantial margins from in vitro IC50 values under irradiation to Cmax (clinical) have enabled further development without the need for additional photosafety data. In vivo phototoxicity studies typically involve dosing rodents and exposing skin and eyes to simulated sunlight, and subsequently evaluating at least the skin for erythema and edema. However, no formal guidelines exist and protocols are less standardized across companies. A margin-of-safety approach (based on Cmax at NOAEL) has been successfully applied to support clinical development. Experience with dedicated clinical phototoxicity studies was limited, perhaps due to effective de-risking approaches employed based on ICH S10.

Practical considerations of linear accelerator-based frameless extracranial radiosurgery for treatment of occipital neuralgia for nonsurgical candidates.

Occipital neuralgia generally responds to medical or invasive procedures. Repeated invasive procedures generate increasing complications and are often contraindicated. Stereotactic radiosurgery (SRS) has not been reported as a treatment option largely due to the extracranial nature of the target as opposed to the similar, more established trigeminal neuralgia. A dedicated phantom study was conducted to determine the optimum imaging studies, fusion matrices, and treatment planning parameters to target the C2 dorsal root ganglion which forms the occipital nerve. The conditions created from the phantom were applied to a patient with medically and surgically refractory occipital neuralgia. A dose of 80 Gy in one fraction was prescribed to the C2 occipital dorsal root ganglion. The phantom study resulted in a treatment achieved with an average translational magnitude of correction of 1.35 mm with an acceptable tolerance of 0.5 mm and an average rotational magnitude of correction of 0.4° with an acceptable tolerance of 1.0°. For the patient, the spinal cord was 12.0 mm at its closest distance to the isocenter and received a maximum dose of 3.36 Gy, a dose to 0.35 cc of 1.84 Gy, and a dose to 1.2 cc of 0.79 Gy. The brain maximum dose was 2.20 Gy. Treatment time was 59 min for 18, 323 MUs. Imaging was performed prior to each arc delivery resulting in 21 imaging sessions. The average deviation magnitude requiring a positional or rotational correction was 0.96 ± 0.25 mm, 0.8 ± 0.41°, whereas the average deviation magnitude deemed within tolerance was 0.41 ± 0.12 mm, 0.57 ± 0.28°. Dedicated quality assurance of the treatment planning and delivery is necessary for safe and accurate SRS to the cervical spine dorsal root ganglion. With additional prospective study, linear accelerator-based frameless radiosurgery can provide an accurate, noninvasive alternative for treating occipital neuralgia where an invasive procedure is contraindicated.

Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (ß-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Quantifying isocenter measurements to establish clinically meaningful thresholds.

A dataset range of isocenter congruency verification tests have been examined from a statistical perspective for the purpose of establishing tolerance levels that are meaningful, based on the fundamental limitation of linear accelerator isocentricity and the demands of a high-precision stereotactic radiosurgery program. Using a laser-defined isocenter, a total of 149 individual isocenter congruency tests were examined with recorded values for ideal spatial corrections to the isocenter test tool. These spatial corrections were determined from radiation exposures recorded on an electronic portal imaging device (EPID) at various gantry, collimator, and treatment couch combinations. The limitations of establishing an ideal isocenter were quantified from each variable which contributed to uncertainty in isocenter definition. Individual contributors to uncertainty, specifically, daily positioning setup errors, gantry sag, multileaf collimator (MLC) offset, and couch walkout, were isolated from isocenter congruency measurements to determine a clinically meaningful isocenter measurement. Variations in positioning of the test tool constituted, on average, 0.38 mm magnitude of correction. Gantry sag and MLC offset contributed 0.4 and 0.16 mm, respectively. Couch walkout had an average degrading effect to isocenter of 0.72 mm. Considering the magnitude of uncertainty contributed by each uncertainty variable and the nature of their combination, an appropriate schedule action and immediate action level were determined for use in analyzing daily isocenter congruency test results in a stereotactic radiosurgery (SRS) program. The recommendations of this study for this linear accelerator include a schedule action level of 1.25 mm and an immediate action level of 1.50mm, requiring prompt correction response from clinical medical physicists before SRS or stereotactic body radiosurgery (SBRT) is administered. These absolute values were derived from considering relative data from a specific linear accelerator and, therefore, represent a means by which a numerical quantity can be used as a test threshold with relative specificity to a particular linear accelerator.

Where will genetic toxicology testing be in 30 years' time? Summary report of the 25th Industrial Genotoxicity Group Meeting, Royal Society of Medicine, London, November 9, 2011.

A number of influences including legislation, industry and academia have encouraged advances in computational toxicology and high-throughput testing to probe more broadly putative toxicity pathways. The aim of the 25th United Kingdom Mutagen Society (UKEMS) Industrial Genotoxicity Group Annual Meeting 2011 was to explore current and upcoming research tools that may provide new cancer risk estimation approaches and discuss the genotoxicity testing paradigm of the future. The meeting considered whether computer modelling, molecular biology systems and/or adverse outcome pathway approaches can provide more accurate toxicity predictions and whether high-content study data, pluripotent stem cells or new scientific disciplines, such as epigenetics and adductomics, could be integrated into the risk assessment process. With close collaboration between industry, academia and regulators next generation predictive models and high-content tools have the potential to transform genetic toxicology testing in the 21st century.

Frameless image-guided linear accelerator (LINAC) stereotactic radiosurgery for medically refractory trigeminal neuralgia: Clinical outcomes in 116 patients.

Background: Frameless image-guided radiosurgery (IGRS) is an effective and non-invasive method of treating patients who are unresponsive to medical management for trigeminal neuralgia (TN). This study evaluated the use of frameless IGRS to treat patients with medically refractory TN. Methods: We performed a retrospective review of records of 116 patients diagnosed with TN who underwent frameless IGRS using a linear accelerator (LINAC) over 10 years (March 2012-February 2023). All patients had failed medical management for TN. Facial pain was graded using the Barrow Neurological Institute (BNI) scoring system. Each patient received a BNI score before frameless IGRS and following treatment. Failure was defined as a BNI score IV-V at the last follow-up and/or undergoing a salvage procedure following IGRS. Results: All patients had a BNI score of either IV or V before the frameless IGRS. The mean follow-up duration for all 116 patients following IGRS was 44.1 months. Most patients (81 [69.8%]) had not undergone surgery (microvascular decompression [MVD] or rhizotomy) or stereotactic radiosurgery (SRS) for TN before frameless IGRS. A total of 41 (35.3%) patients underwent a salvage procedure (MVD, rhizotomy, or an additional IGRS) following frameless IGRS. The mean duration between the initial frameless IGRS and salvage procedure was 20.1 months. At the last follow-up, a total of 110 (94.8%) patients had a BNI score of I-III. No complications were reported after the frameless IGRS. The BNI score at the last follow-up was lower compared to the initial BNI for patients regardless of prior intervention (P < 0.001). Patients who failed IGRS had a higher BNI score at the last follow-up compared to those who did not fail IGRS (2.8 vs. 2.5, P = 0.05). Patients with pain relief had a shorter follow-up compared to those with pain refractory to SRS (38.0 vs. 55.1, P = 0.005). Conclusion: In this large cohort of patients with medically refractory TN, frameless IGRS resulted in durable pain control in the majority of patients without any toxicity.

Cryo-electron tomography of NLRP3-activated ASC complexes reveals organelle co-localization.

NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing.

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

Shulin packages axonemal outer dynein arms for ciliary targeting.

The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliate Tetrahymena to identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo-electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

Tensin 2 modulates cell contractility in 3D collagen gels through the RhoGAP DLC1.

Cytoskeletal proteins of the tensin family couple integrins to the actin cytoskeleton. They are found in both focal adhesions and the fibrillar adhesions formed between cells and the fibronectin matrix. There are four tensin genes which encode three large (approximately 200 kDa) tensin isoforms (tensin 1, 2, 3) and one short isoform (cten). However, the subcellular localization and function of the individual isoforms is poorly understood. Using human foreskin fibroblasts (HFFs), and imaging on both fixed and live cells, we show that GFP-tensin 2 is enriched in dynamic focal adhesions at the leading edge of the cell, whereas GFP-tensin 3 translocates rearward, and is enriched in fibrillar adhesions. To investigate the possible role of tensins in cell-matrix remodeling, we used siRNAs to knockdown each tensin isoform. We discovered that tensin 2 knockdown significantly reduced the ability of HFFs to contract 3D collagen gels, whilst no effect on fibronectin fibrillogenesis was observed. This inhibition of collagen gel contraction was associated with a substantial reduction in Rho activity, and it was reversed by depletion of DLC1, a RhoGAP that binds to tensin in focal adhesions. These findings suggest that focal adhesion-localized tensin 2 negatively regulates DLC1 to permit Rho-mediated actomyosin contraction and remodeling of collagen fibers.

Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies.

A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT).

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling.

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection and is sensed primarily by RIG-I-like receptors (RLRs). Oligomerization of RLRs following binding to cytosolic dsRNA activates and nucleates self-assembly of the mitochondrial antiviral-signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super-resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context, MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling, and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.

Are all bacterial strains required by OECD mutagenicity test guideline TG471 needed?

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to seek consensus on difficult or conflicting approaches to genotoxicity testing based upon experience, available data, and analysis techniques. At the 2017 IWGT meeting in Tokyo, one working group addressed the sensitivity and selectivity of the bacterial strains specified in the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471 to recommend possible modification of the test guideline. Three questions were posed: (1) Although TA100 is derived from TA1535, does TA1535 detect any mutagens that are not detected by TA100? (2) Among the options of Salmonella TA1537, TA97 or TA97a, are these strains truly equivalent? (3) Because there is a choice to use one of either E. coli WP2 uvrA, E. coli WP2 uvrA pKM101, or Salmonella TA102, are these strains truly equivalent? To answer these questions, we analyzed published bacterial mutation data in multiple strains from large (>10,000 compound) databases from Leadscope and Lhasa Limited and anonymized data for 53 compounds tested in TA1535 and TA100 provided by a pharmaceutical company. Our analysis involved (1) defining criteria for determining selective responses when using different strains; (2) identifying compounds producing selective responses based upon author calls; (3) confirming selective responses by visually examining dose-response data and considering experimental conditions; (4) using statistical methods to quantify the responses; (5) performing limited additional direct-comparison testing; and (6) determining the chemical classes producing selective responses. We found that few mutagens would fail to be detected if the test battery did not include Salmonella strains TA1535 (8/1167), TA1537 (2/247), TA102 (4/46), and E. coli WP2 uvrA (2/21). Of the mutagens detected by the full TG471 strain battery, 93% were detected using only strains TA98 and TA100; consideration of results from in vitro genotoxicity assays that detect clastogenicity increased this to 99%.

Co-storage and release of insulin-like peptide-5, glucagon-like peptide-1 and peptideYY from murine and human colonic enteroendocrine cells.

OBJECTIVE: Insulin-like peptide-5 (INSL5) is an orexigenic gut hormone found in a subset of colonic and rectal enteroendocrine L-cells together with the anorexigenic hormones glucagon-like peptide-1 (GLP-1) and peptideYY (PYY). Unlike GLP-1 and PYY, INSL5 levels are elevated by calorie restriction, raising questions about how these hormones respond to different stimuli when they arise from the same cell type. The aim of the current study was to identify whether and how INSL5, GLP-1 and PYY are co-secreted or differentially secreted from colonic L-cells. METHODS: An inducible reporter mouse (Insl5-rtTA) was created to enable selective characterisation of Insl5-expressing cells. Expression profiling and Ca2+-dynamics were assessed using TET-reporter mice. Secretion of INSL5, PYY, and GLP-1 from murine and human colonic crypt cultures was quantified by tandem mass spectrometry. Vesicular co-localisation of the three hormones was analysed in 3D-SIM images of immunofluorescently-labelled murine colonic primary cultures and tissue sections. RESULTS: INSL5-producing cells expressed a range of G-protein coupled receptors previously identified in GLP-1 expressing L-cells, including Ffar1, Gpbar1, and Agtr1a. Pharmacological or physiological agonists for these receptors triggered Ca2+ transients in INSL5-producing cells and stimulated INSL5 secretion. INSL5 secretory responses strongly correlated with those of PYY and GLP-1 across a range of stimuli. The majority (>80%) of secretory vesicles co-labelled for INSL5, PYY and GLP-1. CONCLUSIONS: INSL5 is largely co-stored with PYY and GLP-1 and all three hormones are co-secreted when INSL5-positive cells are stimulated. Opposing hormonal profiles observed in vivo likely reflect differential stimulation of L-cells in the proximal and distal gut.

Frameless image-guided radiosurgery for trigeminal neuralgia.

BACKGROUND: Frameless image-guided radiosurgery (IGRS) is a safe and effective noninvasive treatment for trigeminal neuralgia (TN). This study evaluates the use of frameless IGRS to treat patients with refractory TN. METHODS: We reviewed the records of 20 patients diagnosed with TN who underwent frameless IGRS treatments between March 2012 and December 2013. Facial pain was graded using the Barrow Neurological Institute (BNI) scoring system. The initial setup uncertainty from simulation to treatment and the patient intrafraction uncertainty were measured. The median follow-up was 32 months. RESULTS: All patients' pain was BNI Grade IV or V before the frameless IGRS treatment. The mean intrafraction shift was 0.43 mm (0.28-0.76 mm), and the maximum intrafraction shift was 0.95 mm (0.53-1.99 mm). At last follow-up, 8 (40%) patients no longer required medications (BNI 1 or 2), 11 (55%) patients were pain free but required medication (BNI 3), and 1 (5%) patient had no pain relief (BNI 5). Patients who did not have prior surgery had a higher odds ratio for pain relief compared to patients who had prior surgery (14.9, P = 0.0408). CONCLUSIONS: Frameless IGRS provides comparable dosimetric and clinical outcomes to frame-based SRS in a noninvasive fashion for patients with medically refractory TN.

Ligand-Occupied Integrin Internalization Links Nutrient Signaling to Invasive Migration.

Integrin trafficking is key to cell migration, but little is known about the spatiotemporal organization of integrin endocytosis. Here, we show that α5ß1 integrin undergoes tensin-dependent centripetal movement from the cell periphery to populate adhesions located under the nucleus. From here, ligand-engaged α5ß1 integrins are internalized under control of the Arf subfamily GTPase, Arf4, and are trafficked to nearby late endosomes/lysosomes. Suppression of centripetal movement or Arf4-dependent endocytosis disrupts flow of ligand-bound integrins to late endosomes/lysosomes and their degradation within this compartment. Arf4-dependent integrin internalization is required for proper lysosome positioning and for recruitment and activation of mTOR at this cellular subcompartment. Furthermore, nutrient depletion promotes subnuclear accumulation and endocytosis of ligand-engaged α5ß1 integrins via inhibition of mTORC1. This two-way regulatory interaction between mTORC1 and integrin trafficking in combination with data describing a role for tensin in invasive cell migration indicate interesting links between nutrient signaling and metastasis.