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.

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.

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.

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.

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.

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.

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%.

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.

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.

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.

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.

α5ß1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3.

Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5ß1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP-α5ß1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.

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.

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.

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.

Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results.

In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively <1% (1/147 studies) and 2% (3/147 studies). In case additional information (e.g. repeat assay, organ toxicity, metabolism, tissue exposure) would have been available for evaluation, a final conclusion could most probably have been drawn for most or all of these studies. All (46) negative in vivo comet assays submitted alongside with a negative in vivo micronucleus assay were accepted by the regulatory authorities to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. The survey results demonstrate the robustness of the comet assay and the regulatory acceptance of the current ICH S2 guidance.

The role of flotillins in regulating aß production, investigated using flotillin 1-/-, flotillin 2-/- double knockout mice.

Flotillin 1 and flotillin 2 associate in the plasma membrane to form microdomains that have roles in cell signaling, regulation of cell-cell contacts, membrane-cytoskeletal interactions, and endocytosis. They are thought to be involved in the trafficking and hence processing of the Amyloid Precursor Protein, APP. In this study we set out to obtain in vivo confirmation of a link between flotillins and cleavage of APP to release amyloidogenic Aß peptide, and to generate tools that would allow us to ask whether flotillins are functionally redundant. We used a mouse model for Aß-dependent cerebral amyloidosis, APPPS1 mice, combined with deletion of either flotillin 1 singly, or flotillin 1 and flotillin 2 together. There was a small but significant reduction in Aß levels, and the abundance of congo-red stained plaques, in brains of 12 week old mice lacking flotillin 1. A similar reduction in Aß levels was observed in the flotillin 1-/-, flotillin 2-/- double knockouts. We did not observe large effects on the clustering or endocytosis of APP in flotillin 1-/- mouse embryonic fibroblasts. We conclude that flotillins are likely to play some role in APP trafficking or processing, but the relevant cellular mechanisms require more investigation. The availability of flotillin 1-/-, flotillin 2-/- mice, which have no overt phenotypes, will facilitate research into flotillin function in vivo.

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.