Although GAD is intracellular, it is thought that autoimmunity to GAD65 may play a role in the development of SPS. To test this hypothesis, we immunized mice, that expressed enhanced green fluorescent protein (EGFP) under the GAD65 promoter, with either GAD65 (n = 13) or phosphate buffered saline (PBS) (n = 13). Immunization with GAD65 resulted in autoantibodies that immunoprecipitated GAD, bound to CNS tissue in a highly characteristic pattern, and surprisingly bound not only to GAD intracellularly but also to the surface of cerebellar neurons in culture. Moreover, immunization resulted in immunoglobulin diffusion into the brainstem, and a partial loss of GAD-EGFP expressing cells in the brainstem. Although immunization with GAD65 did not produce any behavioral abnormality in the mice, the induction of neuronal-surface antibodies and the trend towards loss of GABAergic neurons in the brainstem, supports a role for humoral autoimmunity in the pathogenesis of SPS and suggests that the mechanisms may involve spread to antigens expressed on the surface of these neurons.Stiff person syndrome (SPS) is a highly-disabling neurological disorder of the CNS characterized by progressive muscular rigidity and spasms. In approximately 60–80% of patients there are autoantibodies to glutamic acid decarboxylase (GAD), the enzyme that synthesizes  An autoimmune pathogenesis has been suggested in SPS because of its association with type-1 diabetes and GADAbs Stiff person syndrome (SPS), first described by Moersch and Woltman in 1956, is a highly disabling, progressive disorder of the central nervous system (CNS) characterized by muscle rigidity and spasms One of the criteria for establishing the autoimmune nature of a disease is the induction of a disease model by immunization of experimental animals with the purified antigen ad libitum in a non-specific pathogen free environment.Transgenic mice on a background of CBA/C57Bl6 that express enhanced green fluorescent protein (EGFP) under the GAD65 promoter were used to monitor the number of GABAergic neurons visualized by EGFP expression All procedures were carried out in accordance with the UK Home Office guidelines under a project license granted by the Home Office to AV . Compliance to rules and regulations and adherence to the 3Rs principles was monitored by Biomedical Services, University of Oxford and Home Office inspectors. Animals were sacrificed using an approved by the Home Office Schedule 1 procedure (exposure to carbon dioxide gas in a rising concentration).GAD-EGFP transgenic mice of 8 weeks of age were used. Because of limitations on the numbers, both females  and males  were immunized. The allocation to test or control was based on behavioral testing (see below). The mice were immunized subcutaneously with 20 µg of recombinant human GAD65  in PBS emulsified with complete Freund's adjuvant  or with PBS in CFA (see Supplementary figure 1 for details). Three boosts were given at 28-day intervals with the same amount of rhGAD65 in incomplete Freund's adjuvant (IFA). Lipopolysaccharide (LPS) (Sigma-Aldrich) 3 mg/kg was injected intraperitoneally 10 days after the second boost and 3 days after the third boost to permeabilize the blood brain barrier 125I-labelled GAD65 (RSR Ltd) was added and incubated overnight at 4°C. The antigen-antibody complex was precipitated over 2 hours at room temperature with anti-mouse IgG . The precipitate was centrifuged at 13000 rpm for 5 minutes, washed three times with PT× and radioactivity counted for 1 minute on a gamma counter . The GADAb titer in U/ml for the sample was read from the standard curve and multiplied by the appropriate serum dilution factor.Mouse sera were obtained at days 62, 97 and 155  and seriMice were allocated prior to immunization to balanced ‘Test’ or ‘Control’ groups by matching for their baseline burrowing performance, which is a species-typical behavior that is very sensitive to sickness and welfare problems. After immunization, they were observed for abnormalities in gait or other indication of stiffness or spasms. Behavioral tests included burrowing and the accelerating rotarod to evaluate general wellbeing and motor coordination; the light-dark box and the white open-field tests were used to evaluate the response to anxious and stressful environments Primary cultures of cerebellar neurons were prepared from transgenic GAD65-EGFP P2 pups, as previously described To confirm the reactivity of the sera with intact neurons, live unfixed wild-type cerebellar cultures from P6 rat pups Mice deeply anaesthetized by halothane inhalation were transcardially perfused with chilled PBS (25 ml/mouse) followed by a freshly made filtered solution of 4% PFA (Sigma-Aldrich) in PBS (150 ml/mouse). Brains and spinal cords were removed and post-fixed in the same fixative for 30 minutes. All tissues were cryoprotected overnight at 4°C in a solution of 30% sucrose in Tris-buffered saline . Brains and spinal cords were sectioned into regions and mounted in Tissue-Tek® mounting medium  within cryomoulds on dry ice and stored at −80°C. Coronal and sagittal cryostat sections (12 µm-thick) were cut on chrome gelatin-coated slides, dried overnight in a vacuumed chamber, wrapped in foil and stored at −20°C until used for antibody detection. 40 µm-thick sections were collected in PBS, and stored at 4°C until use.2O2 for 15 minutes. Following 3 rinses in TBS, the sections were incubated in 10% NGS (Vector Laboratories) in TBST (0.3% Triton X-100) for 2 hours to block non-specific binding. Sections were then incubated with biotinylated anti-mouse IgG  in TBST containing 10% NGS overnight at 4°C with constant gentle agitation on a platform shaker. The next day, sections were rinsed in TBS (3×5 min) and incubated for 2 hours in Vectastain ABC (avidin-biotin) solution (Vector Laboratories). Sections were further rinsed in TBS . Visualization was achieved by incubating sections in 0.05% DAB (Sigma-Aldrich) in TBS+6 µl H2O2 for 5–8 min protected from light. The reaction was terminated by transferring sections into cold TBS followed by further rinses in TBS (3×5 min). Sections were mounted on chrome gelatine coated slides and air-dried overnight at room temperature. Coverslips were applied over a xylene based mounting solution  and viewed under a light microscope (Nikon Eclipse E400). For identification of mouse GAD, a mouse monoclonal antibody that binds both human and mouse GAD65/67 was used .Endogenous peroxidases were inactivated by rinsing 40 µm-thick sections in TBS containing 1% HImmunostaining was carried out using the Shandon Sequenza slide racks with coverplate system . 12 µm-thick sections were washed with two runs of PBS. Non-specific binding was blocked with 10% NGS in 0.3% PBST at room temperature for 1 hour. Primary antibodies were applied for one hour in 10% NGS in 0.3% PBST. After 3 washes in PBS, the secondary antibodies were applied for 1 hour. Slides were washed with PBS (3×5 min) and coverslips applied on fluorescent mounting medium (Dako). Slides were allowed to dry at 4°C overnight before examination under confocal microscopy.GABAergic (GAD) neurons were identified by the green fluorescence emitted by the EGFP co-expression, and the neuron-specific mouse anti-NeuN antibody , or rabbit anti-calbindin antibody  for the cerebellum, were used to identify the total number of neurons. Five regions each in the cerebellum, hippocampus, brainstem, cervical, thoracic and lumbar cords were selected and labeled 1 to 5 and the tissue sections from each region photographed using the confocal microscope. Neurons were counted in the frame only if part or the entire nucleus was within the frame. To reproduce the results, further three mice were immunized with GAD65 or PBS and the analysis was repeated using 12 sections from each mouse brain starting from the midline and progressing laterally whilst collecting every third section and discarding two. In both experiments, the micrographs were analyzed using ImageJ cell counter software 1.36b . Neuronal counting was performed by two independent investigators, blinded to mice group allocation and the mean counts were used in the analysis.Immunostaining of brain sections was performed as described above for neuronal counting. Hamster anti-CD3  (T cells), rat anti-CD45  (human leucocyte antigen), rat anti-F4/80  (microglia), rabbit anti-GFAP  (astrocytes), and anti-mouse CD16/CD32 (FcγIII/II receptor)  were applied as primary antibodies to detect an inflammatory response in immunized mouse brain tissue. Rabbit anti-synaptophysin  were used to detect any loss of synapses whilst rabbit anti-AC3  and TUNEL test  were used to detect apoptosis. Secondary antibodies were specifically directed against the source of species of the primary antibodies and conjugated with fluorophores with excitation maxima compatible with the confocal microscope lasers. All secondary antibodies were from Alexa Fluor®568, Molecular Probes®, Life Technologies Ltd.Two-way ANOVAs were used for the behavioral tests to compare test and control results. For the neuronal counts, the test and control results in different brain and spinal cord regions were compared with unpaired t tests.The GAD65-immunized mice had high GADAbs titers by day 10 after the second boost , rangingOn immunohistochemistry, sera from GAD65-immunized mice demonstrated immunoreactivity on permeabilized rat brain sections that was not seen in the PBS-immunized mice , general well-being (burrowing) or anxiety (white open-field and light-dark box) tests were performed at baseline and four later stages during the immunization protocol on coded mice. When tested at day 65, one day after LPS injection, some aspects of exploratory activity were altered in both test and control mice, particularly in the white open field. This was not seen when exploratory activity was tested at day 92, 7 days after LPS. There were no differences between control and test groups at any time point  and EGFP-positive neurons were counted in regions of the hippocampus, cerebellum, brainstem and spinal cord . The EGFThe presence of GADAbs in patients with stiff person syndrome has been known for almost three decades but because GAD is an intracellular enzyme the pathogenic significance of these antibodies has been in doubt. Our data demonstrate two potentially important findings. Firstly, immunization against GAD, which induced very high GADAb levels similar to those found in patients with SPS, resulted in IgG reactivity not only against the intracellular compartment of the GAD-EGFP expressing neurons, where GAD65 is located, but also against the extracellular surface of unpermeabilized GAD-EGFP positive and negative cerebellar neurons in both mouse and rat cultures. Secondly, in two independent and coded experiments, there was a trend towards reduced numbers of GAD-expressing neurons in the brainstem of the immunized mice. The apparent loss of GAD-EGFP expressing neurons suggests that antibodies, either against GAD or against an extracellular epitope, or possibly GAD-specific T cells that would also have been induced by the immunizations, are involved in the pathogenesis of SPS.The antibody responses to immunization resulted in GADAb titers in mice that were comparable to or even higher than titers detected in SPS in humans. The high GADAb titers were sustained over a period of 5 months by boosts to allow a prolonged exposure of the mouse CNS to the immune response. The additional presence in the mouse sera of autoantibodies to surface epitopes of GABAergic neurons was a novel finding and interestingly similar reactivity has been observed in sera of SPS patients To try to maximize any potential effects, we used LPS to “permeabilize” the BBB. LPS has been recognized as a tool for this purpose for some time and was successful employed in a mouse model employing immunization against an epitope of the NMDA receptor hyrt mice with lower levels of GABAA receptors in the brainstem and spinal cord, exhibited stiff gait, hunched posture and stimulus-sensitive jerky movements, and on EMG demonstrated continuous motor unit activity (CMUA) at rest, reminiscent of SPS GABAergic neurons arising from the lower medial brainstem (‘the inhibitory centre of Magoun’) and inhibitory interneurons within the medullary reticular formation have been shown to modulate axial muscle tone Our results do not exclude a role for GAD65-specific T cells which one would expect to have been induced initially and may have peaked earlier during the immunization, before we were able to investigate the brain pathology. One of our mice immunized against GAD65 demonstrated an inflammatory cell infiltrate that was conspicuously confined to subcortical and brainstem regions. It is likely that this mouse either had a more florid inflammatory response, or a slower clearance of the cellular infiltrate from the CNS, or both. Other evidence supports the contribution of T cells. Mice expressing a monoclonal GAD65-specific CD4+ T cell population developed a lethal encephalomyelitis-like disease in the absence of any other T cells or B cells, although they did not exhibit an SPS phenotype The clinical diversity of SPS includes variants such as stiff limb syndrome, progressive encephalomyelitis with rigidity and myoclonus, jerking SPS and paraneoplastic SPS. In addition, GAD antibodies at equally high titers have been found in patients with cerebellar We conclude that although immunization with GAD65 did not produce an overt clinical phenotype of SPS in mice and the loss of GABAergic neurons in the brainstem did not quite reach statistical significance, nevertheless these findings suggest an integral role for GAD65 immunity in the pathogenesis of SPS but a role which includes the development of novel cell-surface antibodies which might perhaps begin to provide a basis for the phenotypic diversity. Further studies are required to explore in more detail the roles of GAD-specific antibodies, the targets of the novel neuronal surface binding antibodies that we identified in this model, and in patients with GAD antibodies Figure S1Active immunization (A) Study profile and (B) time line for active immunization of GAD65-EGFP mice with rhGAD65. Note that 4 mice from the test group and 1 mouse from the control group had to be sacrificed because of ulcerating granulomas following immunization. tg = transgenic; BT = behavioral tests; LPS = lipopolysaccharide; SC = subcutaneous; CFA and IFA = complete and incomplete Freund's adjuvant; D = day.(TIF)Click here for additional data file.Figure S2GAD immunoreactivity in rat brain sections. (A) Whole rat brain saggital sections stained with healthy control and GAD monoclonal antibodies (×10). Note the contrast in immunoreactivity. (B) Examples of immunoreactivity in saggital sections through cortex, basal ganglia, hippocampus and cerebellum. First column represents normal healthy serum. Second and third columns represent GADmAb. Note (c) immunoreactive dots outlining pyramidal cells in the cortex (arrow heads); (e) intense immunoreactivity of lateral globus pallidus (LGP) relative to the striatum and thalamus (Th) in basal ganglia;  staining surrounding pyramidal cells in the hippocampal regions CA1-3; (l) the dense accumulation of immunoreactivity at the axon hillock  and puncta that outline the perikaryon and dendritic tree of Purkinje cells (PC), punctate staining in the molecular (ML) and granular layer, and (k) intense immunoreactivity in deep cerebellar nuclei (DCn). SO: stratum oriens; SR: stratum radiatum; DG: dentate gyrus.(TIF)Click here for additional data file.


The „one drug – one target – one disease“ paradigm in drug discovery has been reconsidered during the last decade. This paradigm change was mainly caused by high attrition rates in drug approvals due to toxicity and lack of efficacy. Computational techniques play an important role in prediction and recognition of novel targets for approved drugs. We will discuss two machine learning approaches – self organizing maps and inverse distance weighting – for polypharmacological profiling of bioactive compounds, exemplified by two prospective studies ,2.While the recognition of occasional polypharmacological behavior is an established task, the rational design of multitarget ligands remains challenging. Dual or multi-target ligands have several advantages compared with selective compounds, including improved efficacy and more simple pharmacokinetic and pharmacodynamic properties in comparison to the combination of several drugs. In this context we present two in silico approaches to design dual inhibitors of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). The first study contains the discovery of a benzimidazole-based dual 5-LO/sEH inhibitor by means of in silico screening . The str


The leatherback turtle (Dermochelys coriacea) in the Dominican Republic appeared to be isolated from Awala-Yalimapo, Cayenne, Trinidad and St. Croix but connected with other Caribbean populations. Two distinct nesting populations of hawksbill turtles (Eremochelys imbricata) were detected in the Dominican Republic and exhibited interesting patterns of connectivity with other nesting sites and juvenile and adult male foraging aggregations. The green sea turtle (Chelonia mydas) has almost been extirpated from the Dominican Republic and limited inference could be made from our samples. Finally, results were compared with Lagrangian drifting buoys and published Lagrangian virtual particles that travelled through the Dominican Republic and Caribbean waters. Conservation implications of sink-source effects or genetic isolation derived from these complex inter-connections are discussed for each species and population.Nesting by three species of marine turtles persists in the Dominican Republic, despite historic threats and long-term population decline. We conducted a genetic survey of marine turtles in the Dominican Republic in order to link them with other rookeries around the Caribbean. We sequenced a 740 Many marine vertebrates have complex life cycles requiring the use of different habitats that are often spread over wide spatio-temporal scales and usually result in a network of connections among different populations and between populations and distant feeding grounds Chelonia mydas), loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) turtles during the 1980s Eretmochelys imbricata) shell was exported to Japan between 1970 and 1986 The Dominican Republic has, for many years, presented a gap of knowledge in marine turtle biology and conservation. Although it has been suggested as an important nesting area for several marine turtle species The consequences of the accumulation of these threats to the Dominican Republic nesting populations were not properly addressed until a recent study mixed stock point of view nesting population point of view Genetic markers have been widely used to establish links between populations and feeding grounds and to infer relative exposure to threats Other approaches have involved the study of ocean currents, and have demonstrated that they may play a crucial role in defining hatchling and juvenile marine turtle dispersion Here we combine genetic data from the Dominican Republic turtle nesting population with published genetic data from other nesting populations and feeding grounds, as well as tracks of passive drifter buoys and satellite tracking of adult turtles to: 1) determine if Dominican Republic turtle nesting populations are isolated from others in the region; 2) determine if mtDNA sequences reflect population size reductions; and 3) determine which feeding grounds are used by Dominican Republic marine turtles.During the 2007 and 2008 nesting seasons (from March to October), samples from three nesting marine turtle species were collected within an extensive monitoring project to assess nesting marine turtle abundance and distribution in the Dominican Republic bp fragment of the mtDNA control region using the primers LCM15382 (5′-GCTTAACCCTAAAGCATTGG-3′) and H950 (5′GTCTCGGATTTAGGGGTTT-3′) bp (leatherback sea turtle), 491bp (green sea turtle) or 384bp/480bp (hawksbill sea turtle). Our 25 µL polymerase chain reaction (PCR) included the following: genomic DNA, 1× PCR Buffer, 2 mM MgCl2, 0.12 mM dNTP, 0.2, µm of each primer and 0.04 U/µL of Taq polymerase. After an initial 5 min denaturing step (94°C), our PCR protocol consisted of 35 cycles of the following temperature regime: 1 min at 94°C (denaturing), 1 min at 52°C  and 90 s at 72°C (extension). In addition, we included a final extension step of 10 min at 72°C. Following PCR, we removed single-stranded DNA by digesting 5 µL of PCR product with 2 µL of a combined Exonuclease I and Shrimp Alkaline Phosphatase solution (ExoSAP-IT®). The reaction mixture was incubated for 15 min at 37°C, followed by other 15 min incubation at 80°C to inactivate the two enzymes. We sequenced both forward and reverse strands using the BigDyeTM Primer Cycle Sequencing Kit (Applied Biosystems) run on an automated DNA sequencer (ABI PRISM 3100). For each sequencing reaction, we used 2 µL of our PCR product in a 10 µL reaction mix under the following conditions: 1 m denaturing step at 96a followed by 25 cycles consisting of an initial denaturing of 10 s at 96°C, 5 s at 50°C  and 4 m at 60°C (extension). Products were purified by ethanol precipitation before enter the sequencer.DNA was extracted using the QIAamp extraction kit (QIAGEN®) following the manufacturer’s instructions. We amplified a ∼740bp) haplotypes previously described for the leatherback turtle bp) haplotypes described in those manuscripts that used the same or similar primers in the leatherback turtle http://accstr.ufl.edu/) that includes all published haplotypes. Posterior statistical analyses were carried out with short, long sequences or both depending on the data found in the published literature.Sequences were aligned by eye using the program BioEdit 5.0.9 Differences in haplotype frequencies of samples from the same species at different locations within Dominican Republic were assessed using a Chi-square test. Values were compared to the distributions observed by randomizing individuals among populations using Monte-Carlo resampling h) and nucleotide diversity (π) st) using Arlequin 3.0 In order to assess the genetic diversity compared to the other Atlantic populations we calculated haplotype diversity  (bp) haplotype frequencies, as this analysis requires detailed information of haplotype frequencies of nesting populations and feeding areas and the dataset regarding putative feeding grounds available in the literature is most extensive for this fragment  . Additiofragment . Nestingfragment  or long fragment . As we cfragment  and S3.http://www.aoml.noaa.gov/). An initial query within the locations database was undertaken in order to select all locations plotted near (<50 km) either the Jaragua National Park (DRJ/DR) or Saona Island (DRS) nesting populations. Then, all buoys with at least one location obtained near one of these Dominican Republic nesting areas were selected as an evidence of passive arrival or departure from the area.In order to simulate hatchling turtle dispersal, data describing the tracks of satellite-tracked surface drifter buoys were obtained from the Global Drifter Program of the National Oceanic and Atmospheric Administration . These Lagrangian buoys are periodically released throughout the year at varying locations and are tracked by satellite , providing several positional fixes per day  and exhibited the haplotype CM-A5  which isbp fragment or the 711bp fragment (h (SD) = 0.176 (0.074), π (SD) = 0.0014 (0.0012)] with the exceptions of some populations outside the Caribbean like Awala-Yalimapo , Cayenne , French West Indies , Saint Croix  and Trinidad . No recent expansion was suggested for Dominican Republic leatherback population, independently of the length of the marker used . As previous studies with the 496bp fragment indicated bp fragment yielded similar results  and hence were treated as two independent units for all posterior analysis. Genetic variability of the two Dominican Republic populations was similar or higher than other nesting populations . No recent expansion was suggested for either populations irrespective of the length of the marker used . Analysis of genetic structuring of Dominican Republic nesting beaches in relation to other populations in the Caribbean showed deep levels of differentiation with the exceptions of the DRS population that showed no significant differences to the proximate nesting aggregation of Mona Island in Puerto Rico (PRV), the populations of Belize (BLZ), Barbados Windward (BWI) and U.S. Virgin Islands (USV)  . The simare test . The lacp 83.9%;  and the  79.61%;  fragment 79.61%; .rookery centred ‘many-to-many’ mixed stock analysis for Dominican Republic juveniles suggested that turtles originating from the two nesting areas are likely to have been distributed in foraging grounds across the Caribbean, although the 95% confidence intervals were very high   between Conservation planning for mobile species such as marine turtles, depends on robust spatio-temporal information about the RMU of interest ca. 260 green turtles nesting per year in the 1980s Green turtles in the Dominican Republic bp sequences did not change the number of haplotypes in the Dominican Republic in contrast to previous studies, where some 496bp haplotypes were subdivided into multiple 711bp haplotypes bp sequences to other nesting and feeding areas is needed in order to test if longer sequences improve resolution of population structuring as they have for the loggerhead turtle The leatherback turtle is thought to be the least philopatric of the marine turtles The connection between the Dominican Republic leatherback population with several larger populations within the Atlantic would favour putative recovery through immigration Previous studies in the Caribbean have shown that the hawksbill turtle exhibits a high degree of philopatry resulting in fine scale population structure bp)/EiA23 (720bp) haplotype, the second by the high frequency of the F (380bp)/EiA11 (720bp) haplotype and the last by the high frequency of the A (380bp)/EiA1 (720bp) haplotype . The nesting aggregation in Mona Island, Puerto Rico aplotype . Based oThe structuring of the Caribbean stocks has strong implications for the conservation of the hawksbill nesting populations of the Dominican Republic. The DRJ population is completely isolated, suggesting it is unlikely to receive females by immigration. The small population size Regardless of female philopatry, hawksbill turtles appear to be highly mobile during the juvenile developmental phase. Blumenthal et al. rookery centred mixed stock analysis, did not support this hypothesis, as it predicted an homogeneous distribution of juveniles originating in both Dominican Republic nesting populations. Such mixed stock analysis usually yields wide confidence intervals in marine turtles due to the existence of common haplotypes and results may be taken with caution. Furthermore, the population sizes of the two Dominican Republic nesting populations mixed stock centred analysis. For instance, the juvenile feeding ground located near DRJ present study), but it also receives turtles from Cuba. This means that any extreme mortality of this juvenile aggregation would have an impact on these populations.Considering all these results and the putative dispersal mechanisms of the species The contribution of both Dominican Republic nesting populations to the Puerto Rico male aggregation is very clear. Satellite tracks of adult males and females in Puerto Rico showed that adults move from Puerto Rico and arrive to the vicinity of DRJ and DRS and remained there, possibly to breed Historical measures of the genetic variability would be desirable in order to detect a recent loss of the genetic variability in the Dominican Republic nesting populations caused by the recent population decline One of the milestones in the conservation of endangered species is the detection and quantification of threats affecting declining populations. However, the contextualisation of these potential conservation sinks in a wider regional area is necessary when complex life histories and complex populations structures are present, such as in sea turtles Table S1Haplotype frequencies of Caribbean Hawksbill marine turtle nesting populations. Using the short (380 bp) fragment and including nesting population size (nests/year) used as a baseline for the Mixed Stock Analysis. Puerto Rico data from PRB (DOCX)Click here for additional data file.Table S2Haplotype frequencies of Caribbean Hawksbill marine turtle feeding grounds. Using the short (380 bp) fragment.(DOCX)Click here for additional data file.Table S3Haplotype frequencies of Caribbean Hawksbill marine turtle nesting populations. Using the long (720 bp) fragment and including nesting population size (nests/year) used as a baseline for the Mixed Stock Analysis. PS: Present Study.(DOCX)Click here for additional data file.


Hyperparathyroidism 2 (HRPT2) gene was found mutated in the majority of sporadic parathyroid carcinomas investigated, and studies regarding the protein product parafibromin proposed loss of nuclear parafibromin as a highly sensitive marker for the detection of parathyroid carcinoma. Recent studies have not fully reproduced these findings, as subsets of carcinomas display positive parafibromin immunoreactivity, and fractions of adenomas demonstrate absent expression. Overall, parafibromin is a marker of value to the endocrine pathologist, but it cannot be recommended as a sole indicator of parathyroid carcinoma. Additional markers such as protein gene product 9.5 (PGP9.5) and adenomatous polyposis coli (APC) could complement parafibromin when assessing malignant potential of parathyroid tumours.The diagnosis of parathyroid carcinoma requires an invasive growth pattern or metastases detected at histopathological examination; unfortunately, not all carcinomas exhibit visible malignant properties at the initial assessment. Therefore, immunohistochemical markers have been sought for the recognition of parathyroid malignancy. In 2003, the  Primary hyperparathyroidism (PHPT) originating from a parathyroid carcinoma is an infrequent finding in the clinical setting. Clinical manifestations indicative of parathyroid carcinoma include profound hypercalcemia with severe metabolic disease and a palpable mass in the anterior neck region . HistopaOptimally, the diagnosis of a malignant tumour is made prior to the onset of advanced malignant properties so that the tumour can be radically removed before an eventual metastatic spread. Extensive research has, therefore, been conducted to identify a potential molecular marker which could discriminate between malignant and benign parathyroid tumours prior to the onset of required histopathological requirements. Early studies focused on immunohistochemical analyses of well-established proteins controlling the cell cycle process as well as apoptosis, such as the retinoblastoma protein (pRb), p53, and cyclin D1 –6. AlthoHyperparathyroidism 2 gene (HRPT2), located at 1q25-q31 [HRPT2 mutations in the majority of malignant cases examined [HRPT2 mutations only have been detected in small subsets of cases [HRPT2 encodes a ~60 kDa protein termed parafibromin, a protein which bears sequence resemblance with the yeast protein Cdc73 [HRPT2 will augment cellular proliferation. Following the initial discovery of inactivating HRPT2 mutations in parathyroid carcinomas, studies were launched with the aim of characterizing parafibromin expression in parathyroid tumours. In the first study published by Tan et al. in 2004, the authors constructed a monoclonal parafibromin antibody (2H1) and investigated a large number of tumours for parafibromin expression using immunohistochemistry [HRPT2 mutations; 6 (67%) of these cases displayed total loss and 2 cases exhibited focal loss (22%) whereas one case was positive for parafibromin expression (11%). These findings suggest that the absence of parafibromin immunoreactivity might be indicative of HRPT2 mutations, which is further supported by the loss of parafibromin expression in the majority of carcinomas, a group known to frequently carry HRPT2 inactivating mutations.In 2002, Carpten et al. demonstrated that the hyperparathyroidism-jaw tumour (HPT-JT) syndrome is caused by a germline mutation in the tumour suppressor gene 1q25-q31 . Interesexamined , 13. Thiof cases . HRPT2 ein Cdc73 . Just liin Cdc73 , 15. In in Cdc73 –19  and HPT-JT-related tumours  as opposed to positive staining in benign tumours . The senHRPT2 mutations displayed positive parafibromin expression, suggesting that cases with HRPT2 aberrations, not as a rule, display alterations in parafibromin immunoreactivity [In a subsequent study, the authors assessed parafibromin immunoreactivity in a set of parathyroid tumours using monoclonal and polyclonal parafibromin antibodies targeting diverse epitopes scattered across the protein, including the previously used monoclonal parafibromin antibody 2H1 . Reducedactivity .HRPT2 gene mutations in which 21/22 sporadic adenomas were parafibromin positive whereas all 11 carcinomas were negative for parafibromin immunoreactivity using the 2H1 antibody [HRPT2 gene mutations. To counter these findings, another group has since detected positive parafibromin expression in the majority of parathyroid carcinomas stemming from secondary hyperparathyroidism [The discussion regarding the value of parafibromin as a discriminating marker took a new turn when a subsequent study showed excellent correlations between loss of parafibromin expression and antibody . The paryroidism , and an yroidism .HRPT2 gene aberrancies play an important role in the progression of parathyroid malignancy, and HRPT2 inactivating mutations have been established as a major event in sporadic parathyroid carcinomas [HRPT2 gene mutations and absent parafibromin expression using Western blot analysis as well as immunohistochemistry [HRPT2 gene alterations in these cases suggest that they have evolved sporadically. In addition, independent studies have also demonstrated reduced parafibromin immunoreactivity and/or HRPT2 mutations in a few cases of sporadic, parathyroid adenomas [HRPT2 mutations and downregulation of parafibromin are present in very small subgroups of parathyroid benign tumours.rcinomas , 13. Subrcinomas –23, 25. hemistry . Althougadenomas . These fHRPT2 gene mutation [HRPT2 gene [HRPT2 mutation analysis in the afflicted patient to exclude a possible hereditary background. This demonstrates one of the main benefits of utilizing parafibromin immunohistochemistry as a marker in the clinical context.Since parathyroid carcinomas are so uncommonly observed in the clinical settings as opposed to parathyroid adenomas, parafibromin immunohistochemistry will require almost near-perfect specificity to reduce the number of false positive cases. With the currently reported specificity, even a few parathyroid adenomas with negative parafibromin staining will outrank the “true” parafibromin negative cases in the infrequently encountered carcinoma group. As a consequence, loss of parafibromin immunoreactivity could either imply parathyroid adenoma or carcinoma, but may also indicate an underlying mutation . PositivPT2 gene , an obseHRPT2 germline and somatic mutations is of missense type rather than the more commonly observed nonsense or frameshift types [HRPT2 mutated cases if the antibody is not targeting the epitope of the corresponding missense alteration.Another issue regarding parafibromin immunohistochemistry stems from the different staining patterns detected when analyzing parathyroid malignant tumours. Variations in the number of tumour cells with parafibromin expression vary between studies, with authors reporting total absence as well as a mixed pattern of positive and negative nuclei in addition to 100% positive nuclei in different proportions. For example, Gill et al. classified parathyroid tumours with focal loss as positive and, therefore, only approved a total absence of parafibromin immunoreactivity as diagnostic of malignancy . Howeverft types . Missensubiquitin carboxyl-terminal esterase L1 (UCHL1) gene has been shown to be upregulated in the majority of parathyroid carcinomas based on gene expression profiling, and the results were verified by immunohistochemistry [HRPT2 mutated parathyroid adenomas with loss of parafibromin expression have shown that APC is uniformly expressed in these tumours, thereby demonstrating a superior specificity which potentially could be of clinical use [As a result of the reduced sensitivity and specificity of parafibromin as well as the different staining patterns observed, additional molecular markers have been assessed to complement parafibromin in the screening process. For example, protein gene product 9.5 (PGP9.5), encoded by the hemistry . When anhemistry . Subsequical use . In addiical use , 33.HRPT2 gene aberrancies that could motivate germline HRPT2 testing to exclude possible familial disease. However, no clear consensus exists on how the different staining results should be interpreted  as well as how this interpretation should influence the overall treatment of the afflicted patient. Given the current literature, routine histopathology combined with immunohistochemical analysis of parafibromin cannot alone be recommended as a definite screening method for parathyroid malignancy, but should probably be regarded as an aiding tool for the pathologist when assessing parathyroid tumours which are not clearly benign. Additional discovered or yet undiscovered markers are likely to increase the sensitivity and specificity for the proper recognition of parathyroid carcinomas in the clinical setting.In present time, several pathology departments are known to habitually perform parafibromin immunohistochemistry for parathyroid tumours which are not clearly benign or that display atypical findings at the histopathological examination. Positive parafibromin expression clearly points towards benign disease, and reduced parafibromin immunoreactivity should probably indicate intensified followup, since this pattern is often encountered in parathyroid malignant disease. In addition, reduced parafibromin expression could indicate


Differences in the incidence and outcome of breast cancer among Hispanic women compared with white women are well documented and are likely explained by ethnic differences in genetic composition, lifestyle, or environmental exposures.A population-based study was conducted in Galicia, Spain. A total of 510 women diagnosed with operable invasive breast cancer between 1997 and 2010 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics were collected. The different breast cancer tumor subtypes were compared on their clinico-pathological characteristics and risk factor profiles, particularly reproductive variables and breastfeeding. Among the 501 breast cancer patients (with known ER and PR receptors), 85% were ER+/PR+ and 15% were ER-&PR-. Among the 405 breast cancer with known ER, PR and HER2 status, 71% were ER+/PR+/HER2- , 14% were ER+/PR+/HER2+ , 10% were ER−/PR−/HER2- , and 5% were ER−/PR−/HER2+ . A lifetime breastfeeding period equal to or longer than 7 months was less frequent in case patients with TNBC  compared to luminal A breast cancers. Both a low (2 or fewer pregnancies) and a high (3–4 pregnancies) number of pregnancies combined with a long breastfeeding period were associated with reduced odds of TNBC compared with luminal A breast cancer, although the association seemed to be slightly more pronounced among women with a low number of pregnancies .In case-case analyses with the luminal A cases as the reference group, we observed a lower proportion of TNBC among women who breastfed 7 or more months. The combination of longer breastfeeding duration and lower parity seemed to further reduce the odds of having a TNBC compared to a luminal A breast cancer. In the US, breast cancer impacts each racial group differently It has been reported that women diagnosed with estrogen receptor-positive (ER+)/progesterone receptor-positive (PR+) tumors are more responsive to hormonal treatment and have a better prognosis than those diagnosed with estrogen receptor-negative (ER-)/progesterone receptor negative (PR-) tumors, suggesting etiologic heterogeneity of hormone-receptor defined subtypes of breast cancer In this study, we describe the characteristics of breast cancer subtypes defined by ER, PR and HER2 receptor status and assessed the associations between reproductive factors and breastfeeding and tumor subtypes in a case series of female breast cancer patients from Galicia, a region located in the northwest part of Spain, whose history has been defined by mass emigration to Latin America To our knowledge, this is in one of the first studies to explore these relationships in this population.A population-based study, which is part of the Breast Oncology Galician Network (BREOGAN) Study, was conducted in the city of Vigo, Spain within a geographically defined health region that covers aproximately 437,000 inhabitants. The study involved 510 women with operable invasive breast cancer diagnosed and treated between 1997 and 2010 at the Clinical University Hospital of Vigo . Ethics approval for this study was obtained from the Galician Ethics and Research Committee  associated with the Complejo Hospitalario Universitario de Vigo from where all participants were recruited. Written informed consent was obtained for this study, which was conducted according to the Spanish law including adherence to the Helsinki Principles of 1975, as revised in 1983.Ella Binational Breast Cancer Study Risk factor information was collected through a risk factor questionnaire adapted from the Histopathological information was abstracted from computerized medical records by trained physicians. Immunohistochemistry (IHC) analyses on paraffin-embedded material have been previously performed following standard procedures in Galician hospitals to determine the status of ER and PR. In every tumor, 4-µm histological sections were cut and stained with hematoxylin and eosin for histopathological examination according to the criteria of the World Health Organization Immunohistochemistry (IHC) analyses were performed to determine HER2 status (Dako). No immunostaining (0) or weak membrane immunostaining (1+) was considered low HER2 expression (HER2-). Strong membrane immunostaining (3+) was considered HER2 overexpression (HER2+). Moderate membrane staining (2+) samples were further analyzed using fluorescence in situ hybridization techniques; they were considered to be HER2+ if the ratio of cerb-B2/centromere 17 copy number was >2.0.We classified breast tumors according to their expression of ER (n = 509), PR (n = 502) and joint status of both ER and PR expression (n = 501). For patients with data available on ER, PR, and HER2 (n = 405 case patients), we defined four tumor subtypes , shown in All statistical analyses were performed using the R statistical software (R_2.13.0). All reported test significance levels  were two-sided.A total of 501 breast cancer patients with known ER and PR and 405 with known ER, PR, and HER2 were identified. Among those with known ER and PR status, 85% were ER+/PR+ and 15% were ER-&PR- and among those with data for all three markers, 71% were ER+/PR+/HER2- , 14% were ER+/PR+/HER2+ , 10% were ER−/PR−/HER2- (TNBC), and 5% were ER−/PR−/HER2+ . The age of these patients ranged from 28 to 84 years, with a mean of 54.7±12.7 years. Detailed characteristics of the study population are presented in We further examined the joint effect of breastfeeding and parity and tumor subtype. No associations were shown between other breast cancer risk factors  and the different tumor subtypes .Regarding tumor characteristics, high grade  and medullar type breast cancer , were more frequent in case patients with triple-negative tumors compared to luminal A tumors .In a population-based study of breast cancer patients from Spain, we observed that the proportion of cases with TNBC versus luminal A breast cancer was lower in women who breastfed for 7 or more months than in those who did not breastfeed. Also, compared with luminal A breast cancers, TNBCs were more common in parous vs. non-parous women. Both a low (2 or fewer pregnancies) and a high (3–4 pregnancies) number of pregnancies combined with a long breastfeeding period were associated with reduced odds of TNBC compared with luminal A breast cancer; although the association seemed to be slightly more pronounced among women with a low number of pregnancies we lacked the statistical power to detect any difference. No other associations were detected between tumor subtypes and other reproductive/lifestyle breast cancer risk factors despite the accumulating evidence favoring distinct reproductive profiles among the differing tumor subtypes Several case-control and cohort studies have examined the association between parity and breastfeeding and the risk of TNBC. Although an inverse association of parity with risk of ER+ breast cancer has been found by many studies, studies of ER– breast cancer indicate a positive, risk-enhancing association with parity It is unclear why the inverse association between breastfeeding and TNBC tends to be more pronounced among Hispanics than NHWs GABRP, a type A receptor that is expressed in reproductive tissues), proliferate within the breast lobules and then are lost during breastfeeding. These investigators evaluated 203 newly diagnosed invasive breast cancers and showed that higher GABRP gene expression was more common in younger women with a limited history of lactation after pregnancy study GABRP gene expression was higher in Hispanic women compared with white controls. This investigation suggested that GABRP gene expression may be associated with high-grade breast cancer in Hispanic women; although this needs additional study.It is possible that genetic susceptibility to breast cancer may differ among Hispanics and other ethnicities. Major differences in gene expression between Hispanics and NHW have been detected BRCA1 screening study in familial breast cancer carried out in different centers in Spain, France and the United Kingdom, the missense mutation 330 A>G was independently identified in six families, all of them with Spanish/Galician ancestors BRCA1 330 A>G being a founder mutation of Spanish/Galician origin. Similarly, the BRCA1 185delAG mutation has been identified among several apparently non-Jewish families in Spain The present study was conducted in Galicia, a region located in the northwest part of Spain, whose history has been defined by mass emigration to Latin America , there could be the possibility of potential heterogeneity in reading stains and scoring of immunohistochemistry; however this limitation would be expected to bias the study results towards the null. Finally, the case-case study design has obvious limitations Although our results are mostly in agreement with those of other studies, limitations of our study should be discussed, notably its small sample size particularly in subset analyses stratified by hormonal receptor status ER, PR or HER2. Sample sizes for the less common subtypes were limited. In addition, the breastfeeding data were based on self reported information collected years later. In general, breastfeeding history has been shown to be accurate and reliable Our case-case design study can be particularly useful in assessing the relative correlation of established risk factors and the different tumor subtypes. We have included in our study detailed tumor marker information, such as ER, PR and Her-2 receptor status, which is needed to identify etiologic heterogeneity for established breast cancer risk factors and disease subtypes In conclusion, this analysis shows that associations between breast cancer and reproductive factors or breastfeeding vary by breast cancer tumor subtypes defined by ER, PR, and HER2 status, particularly luminal A and TNBC. These results are in concordance with emerging evidence that relationships for genetic susceptibility loci also vary by expression levels of markers in tumors Table S1Associations between other reproductive and lyfestyle breast cancer risk factors and different tumor subtypes.(DOC)Click here for additional data file.Table S2Associations between tumor characteristics and different tumor subtypes.(DOC)Click here for additional data file.


ABLIM3, P = 1.3×10−10) for the sensitivity to cold pressor pain in males, but not in females. Significant associations were also observed with rs12548828, rs7826700 and rs1075791 on 8q22.2 within NCALD . Our results demonstrate the utility of a novel paradigm that integrates publicly available disease-specific gene expression data with clinical data curated from MEDLINE to facilitate the discovery of pain-relevant genes. This data-derived list of pain gene candidates enables additional focused and efficient biological studies validating additional candidates.Identifying human genes relevant for the processing of pain requires difficult-to-conduct and expensive large-scale clinical trials. Here, we examine a novel integrative paradigm for data-driven discovery of pain gene candidates, taking advantage of the vast amount of existing disease-related clinical literature and gene expression microarray data stored in large international repositories. First, thousands of diseases were ranked according to a disease-specific pain index (DSPI), derived from Medical Subject Heading (MESH) annotations in MEDLINE. Second, gene expression profiles of 121 of these human diseases were obtained from public sources. Third, genes with expression variation significantly correlated with DSPI across diseases were selected as candidate pain genes. Finally, selected candidate pain genes were genotyped in an independent human cohort and prospectively evaluated for significant association between variants and measures of pain sensitivity. The strongest signal was with rs4512126 (5q32,  The mechanisms underlying pain are incompletely understood, and are hard to study due to the subjective and complex nature of pain. From a genetics perspective, the discovery of genes relevant for the processing of pain in humans has been slow and genome-wide association studies have not been successful in yielding significantly associated variants. Targeted approaches examining specific candidate genes may be more promising. We present a novel integrative approach that combines publicly available molecular data and automatically extracted knowledge regarding pain contained in the literature to assist the discovery of novel pain genes. We prospectively validated this approach by demonstrating a significant association between several newly identified pain gene candidates and sensitivity to cold pressor pain. A significant number of diseases are associated with pain, thereby affecting the quality of life of many individuals. The Institute of Medicine's recent report titled, “Relieving Pain in America” presented pain as a public health challenge, and emphasized the need for an integrative approach to understand mechanisms underlying pain Studies in rodents and humans have established the importance of genetic factors in the processing of pain Pain is among the most difficult phenotypes to study due to its complex and subjective nature. The perception of pain is influenced by a multitude of variables including gender, age, mood, ethnicity and genetic factors Candidate gene studies have proven successful in the identification of pain genes. A particularly promising approach used gene expression microarray analysis to select candidate genes In this study, we describe an integrative approach exploiting publically available gene expression data for a large set of disease conditions to develop a disease-specific pain index (DSPI). This approach is based on the hypothesis that differences at the gene expression level correlating with pain indices would allow identifying novel pain gene candidates We built the disease-specific pain index (DSPI) using our literature-based approach, in which 2962 diseases were ranked according to their disease-pain ratio. As previously described, the raw microarray data for 311 diseases were extracted from public gene expression databases The sensitivity and accuracy of this strategy for capturing genes implicated in the processing of pain was first evaluated with the aid of the Pain Gene Database (PGD) DLG4 (PSD-95) and CHRNA4, were referenced in the PGD DLG4 and CHRNA4 were both found to have expression changes in 13 of 121 diseases that were positively correlated with pain indices  and retrieved gene significantly associated with this index. Using genes belonging to the Gene Ontology category “Inflammatory Response” (GO:0006954) as gold standard we computed the area under the ROC curve. ABLIM3 , PDE2A , CREB1 (cAMP responsive element binding protein 1), NAALAD2 , and NCALD   .ABLIM3 was selected as our top candidate as it showed the highest correlation with the DSPI. The cGMP-sensitive phosphodiesterase PDE2A localizes at the neuronal membrane in synapses and has been described as being regulated by TNFα, a known proinflammatory cytokine shown to sensitize primary nociceptors PDE2A provides a mechanism for nitric oxide-mediated cGMP synthesis to control intracellular concentrations of cAMP c-Fos, which are associated with nociceptive afferent activation NAALAD2 is highly similar in sequence to NAALAD1 and both hydrolyze N-acetyl-L-aspartate-L-glutamate (NAAG) to N-acetyl-aspartate and glutamate, a neuropeptide that activates and antagonizes neuronal N-methyl-D-aspartate (NMDA) receptors NAALAD1 was found to plays a role in maintaining mechanical allodynia after carrageenan injection NAALAD1 was also positively correlated with the DSPI but not to the same level of significance as NAALAD2. Finally, NCALD  is a calcium-binding protein abundantly and almost exclusively expressed in the central nervous system that has not previously been associated with pain The genotyping study was conducted in samples obtained from twins enrolled in an ongoing independent IRB-approved pharmacogenomic study testing subjects' sensitivity to experimental heat and cold pressor pain among other outcomes (see ABLIM3 (rs4512126) and NCALD  showed significant association with the cold pressor pain threshold after Bonferroni correction . The largest effect sizes (Cohen's d) were observed between T/T Males and A/A Males and Females . Effect sizes between T/T Males and the other groups were below the small effect size threshold (< = 0.2) with 0.16, 0.11 and 0.17 for A/T Males, A/T Females and TT Females respectively.Within the five selected genes, 251 tag SNPs were tested. Polymorphisms in rrection . Linkagerrection  and S3. rrection . Males wABLIM3 and NCALD with measures of pain sensitivity in an independent human cohort.The primary objective of this study was to demonstrate the utility and validity of a novel, data-driven approach for generating a list of pain gene candidates. Such a list could facilitate the discovery of pain genes. We first validated our approach by demonstrating a statistically significant sensitivity and specificity prioritization of known pain-related genes contained in the Pain Gene Database (PGD). In addition, further genotyping of a human cohort revealed a significant association between variants of the newly discovered pain gene candidates A major emphasis of this study was to document the utility of the principal approach and highlight its future potential. The ever growing amount of publically available molecular and clinical data should allow for expanding and refining this approach to generate more comprehensive and specific lists. For example, as more data becomes available, it may be possible to link gene expression of diseases to specific types of pain, such as neuropathic pain. Similarly, the outlined approach can be expanded to include proteomic data sets, which should provide additional insight into signaling pathways relevant to the processing of pain. Finally, the pain associated with a specific disease can be construed differently. For example, disease-specific pain ratings could be retrieved from databases of large health care organizations There are a few limitations in our approach and study. First, among the 47 candidate pain genes significantly correlating with the DSPI, only two are referenced in the PGD. While the PGD is a valuable resource of curated information and likely represents the best available reference, it is not yet a globally accepted master repository containing all pain genes, especially those resulting from human studies. The database is constrained by the fact that it only catalogs genes revealed by studies examining nociception in mechanistic – but not disease-related – models in knock-out mice. It should also be noted that gene expression data for diseases and matched controls were only available for 121 diseases. As a result only 130 of the 300 genes listed in the PGD could be explored in the current study.KCNS1, GCH1, COMT or OPRM1, each of which has been implicated in the processing of pain KCNS1, are thought to be important in specific types of pain like neuropathic pain, but might not participate genetically in determining pain of other etiologies represented in the 121 diseases. There is considerable potential for more refined approaches in the near future to resolve some of these limitations, as there are a constantly growing number of publicly available repositories containing molecular and phenotypic data sets.The presented paradigm did not capture genes such as ABLIM3 is a newly characterized protein-coding gene belonging to the actin binding LIM protein family, which is composed of 3 members (ABLIM1-3) and shows a high degree of conservation throughout evolution in vertebrates. ABLIM3 is expressed in various tissues, most prominently in muscle and neuronal tissue ABLIM protein family, conservation of key structural features suggests comparable biological function as linkers between the actin cytoskeleton, cell signaling pathways and transcription events ABLIM1 in C. elegans (UNC-115) has been implicated in axonal guidance during outgrowth through interaction with Receptor for Activated C Kinase (RACK-1) ABLIM3 in the perception or processing of pain is not apparent. ABLIM3 could potentially affect nociceptive signaling by regulating synaptic strength through actin rearrangement and modulation of synaptic spine density ABLIM3 with pain is a novel finding that is based on a data-driven approach but is not anchored in our current understanding of pain biology. While this approach may offer the advantage of making unexpected and important discoveries, it requires establishing the biological relevance of such discoveries in subsequent experimental steps. The SNP rs4512126 (5q32) is located in the second and largest intron of ABLIM3. This variant was found in weak linkage disequilibrium (>0.6) with five other SNPs and in perfect linkage disequilibrium with rs4546368 located in the same intronic region of ABLIM3. All were non-coding SNPs. Similar to ABLIM3, NCALD has never been reported to be associated with pain. However, several polymorphisms in the 3′ UTR have been associated with mRNA instability and diabetic nephropathy NCALD and pain cold was modest, demonstrated in only a single cohort, and barely above the Bonferroni corrected threshold. The parallel approach using quantile normalized phenotypical pain measures did not sustain the association for NCALD , Disease and Syndrome (T047) and Neoplastic Process (T191) MeSH is a comprehensive vocabulary thesaurus organized in a hierarchical structure allowing the indexing of publications with various levels of specificity. MeSH terms are used by trained human curators to annotate publications referenced in MEDLINE. We first built a thesaurus of 3743 disease-related MeSH terms using the Unified Medical Language System (UMLS); restricting ourselves to terms belonging to the following semantic type: Aches, Burning pain, and others. MEDLINE searches were automated using the EUtils programming tools available from the NCBI (http://eutils.ncbi.nlm.nih.gov). The ratio of these two counts formed the disease-pain ratio, as shown in equation (1).Searching MEDLINE for each of these MeSH terms gave us the number of publications published on each disease. For each disease returning a result, we conducted a second search in MEDLINE to count the number of papers published that were also annotated with the MeSH heading term “pain”[mh]. Searching MEDLINE for “pain”[mh] includes publications annotated with any terms hierarchically below “pain” in MeSH, such as The comprehensive disease-specific pain index (DSPI) was established by ranking all 2962 diseases by their respective disease-pain ratio . ImpliciA similar approach was followed to establish the Disease-Specific Inflammation Index (DSII) by searching for publication annotated with the MeSH term “Inflammation”[mh].We annotated all datasets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and the European Bioinformatics Institute (EBI) ArrayExpress (AE) public databases with UMLS identifiers for diseases as previously published One-hundred twenty-one of the 311 diseases retrieved from GEO and AE were also present in the DSPI list, and thus could be associated with disease-specific pain indices see . BecauseV over the total number of hypothesis rejected R (2) The Spearman rank correlation was then computed for each gene using the fold-change values against the DSPI ranking. We computed the positive false discovery rate (pFDR) values for each gene by permuting the diseases rank and re-computing the Spearman correlation for each gene. The operation was repeated 1000 times to obtain a null distribution of the correlation coefficients. The pFDR values were calculated as the ratio of the expected proportion of false positive www.jbldesign.com/jmogil/; accessed October 2010) www.ncbi.nlm.nih.gov/homologene). All 300 mouse genes were translated to unique human genes except 8 genes lacking homologs. These eight genes were not further considered. We established the confidence intervals for the ROC curve using a leave-one-out resampling method by repeatedly recalculating the pain-gene rankings with nine-tenths of the 121 diseases. These alternative pain-gene lists were then used to compute the confidence interval of the standard error of our pain-gene ranking. We used the ROCR package from R to compute the ROC curves We evaluated the sensitivity and accuracy of our method in prioritizing pain genes using Receiver Operating Characteristic (ROC) curves. We compared the DSPI-based pain-gene list against a list of known pain genes from the PGD (Gene extracted using the DSII were compared to a gold standard made from the genes belonging to the “Inflammatory response” Gene Ontology category (GO:0006954). The gene list was retrieved from the Molecular Signature Database Our study used samples and data from a pre-existing large pharmacogenomic study in twins examining the heritability of various opioid effects In total, 228 healthy pain-free twins (114 twin pairs) of diverse ethnicities were genotyped and phenotyped. We considered only individuals with self-declared European ancestry, which represented 179 individuals with an age range of 18–68 years. Zygosity status of identical (MZ) and fraternal (DZ) twins was assessed by genotyping concordance using a panel of 47 SNPs Experimental pain measurements were performed in the Human Pain Laboratory of the Department of Anesthesia at Stanford University School of Medicine. Heat pain was induced with a thermal sensory analyzer . A 3×3 cm thermode was placed in contact with skin at the volar forearm. Starting at 35°C, the thermode temperature was increased at a rate of 1°C/s. Study participants pushed a button of a hand-held device at the onset of pain. This procedure was repeated 4 times with an inter-stimulus interval of 30 seconds. The average temperature (C°) eliciting pain was recorded as the pain threshold.Cold pressor pain is thought to mimic important qualities of clinical pain, since verbal descriptors for both types of pain are strikingly similar 2 = 1) based on the HapMap CEU population using the Tagger software from the Broad Institute (http://www.broadinstitute.org/mpg/tagger/) The twin cohort was genotyped for 251 SNPs across 5 genes selected from the list of candidate pain genes . We seleOut of 251 SNPs assayed only 216 yielded successful results following Illumina quality controls. Some successfully genotyped loci had missing genotypes for a small number of twins and were imputed using the homozygous wildtype allele from the population. We filtered out all SNPs with a minor allele frequency <5% or those whose genotype frequency departed from Hardy-Weinberg equilibrium at p<0.01. In summary, 207 SNPs were tested against heat pain threshold, cold pain threshold and cold pain tolerance.While twin individuals were not required to test association of our candidate genes with pain, twins allowed us to control for environmental variability in pain measurements. Rather than utilize methods to correct for the relatedness of observations coming from the two members of the same twin pair, we used a model in which each pair was treated as a single observation by using within-pair genotype and phenotype averages. Genotypes were transformed to numbers according to the allele frequency, 0, 1, 2 for homozygous wildtype, heterozygous and homozygous rare, respectively. Then, the genotypes of twins were averaged within each pair. The genotype of single twin was not altered. Categorical covariate data such as sexes were discretized to −1 and 1 for male and female respectively and then averaged. DZ twin pairs of different sex were coded 0.y, against the genotype for the SNP considered while controlling for depression of the individual (Beck Depression Index) and sex (3).To test association of measures of pain sensitivity with each SNP, we used a generalized least square (GLS) regression model ABLIM3) polymorphism remained significant for cold pain threshold (ABLIM3) showed a significant association with cold pain tolerance (ABLIM3) showed a significant association with heat pain threshold  and the log of both the cold pressor pain threshold and cold pressor pain tolerance. P-values were corrected for multiple hypotheses testing using the Bonferroni correction. One twin with the T/T allele for rs4512126 reached the maximum allowed time in the cold threshold test (3 min). This result was considered an outlier and was removed from the analysis. In parallel, we also evaluated the effect of the pain phenotype normally distributed through quantile normalization. This analysis revealed similar results. The rs4512126  t-tests with unequal sample size and unequal variances (Welch two sample t-test).We computed the Cohen's Figure S1Receiver Operating Characteristic (ROC) curve. The ROC curve depicts the performance of our algorithm to identify known inflammatory genes belonging to the Gene Ontology “Inflammatory response” category. The area under the ROC curve is 73.2%, which is significantly different from random chance.(TIF)Click here for additional data file.Figure S2Linkage Disequilibrium comparative representation of the ABLIM3 SNPs. (A) LD structure (r2) of genotyped SNPs within our twin cohort. (B) LD structure of the same SNPs using HapMap II+III population with European ancestry. Blue star indicates rs4512126 SNP location.(TIF)Click here for additional data file.Figure S3Linkage Disequilibrium comparative representation of the NCALD SNPs. (A) LD structure (r2) of genotyped SNPs within our twin cohort. (B) LD structure of the same SNPs using HapMap II+III population with European ancestry. Blue star indicates rs12548828, rs7826700 and rs1075791 SNPs location.(TIF)Click here for additional data file.Figure S4Manhattan plot for ABLIM3 polymorphisms when pain phenotype are quantile normalized. (A) Represent association p-values for SNPs in ABLIM3 with cold pain pressor threshold. (B) Association of ABLIM3 polymorphisms with cold pain tolerance. (C) Association analysis of ABLIM3 polymorphisms with heat pain threshold.(TIF)Click here for additional data file.Table S1Disease-specific pain index (DSPI) ranking all 2962 diseases by their respective disease-pain ratio.(XLS)Click here for additional data file.Table S2List of 121 diseases retrieved from GEO and AE that were also present in the DSPI.(XLS)Click here for additional data file.


The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS. The anti-phospholipid syndrome (APS) is a systemic autoimmune disease characterized by an adaptive immune response against self membrane anionic phospholipids or associated plasma proteins resulting in the generation of anti-phospholipid specific antibodies (aPL) 3Cys the activation threshold was markedly lowered indicating a synergistic activation pathway of aPL and microbial products also in PMN. These results suggest that PMN as important innate immune effector cells are directly activated by aPL under inflammatory conditions and therefore may be an important contributor to the pathophysiology of APS.To address this question, we analyzed the impact of a purified human monoclonal aPL Citrated blood of healthy volunteer donors was purified with Polymorphprep  using an established protocol Salmonella typhimurium  was utilized in a final concentration of 100 ng/ml or titrated  as indicated. Titrated concentrations  of Palmitoyl-3-Cys-Ser-(Lys)4  were employed as TLR2 ligand. Recombinant human lipopolysaccharide binding protein  was utilized in a final concentration of 10 ng/ml. Human monoclonal aPL (clones HL5B and HL7G) were utilized in a final concentration of 10 µg/ml and have been described previously Lipopolysaccharide (LPS) from PMN were incubated with L-Selectin (CD62L)-APC or CD11b-PE in PBS with 1% BSA and 0.05% sodium azide  on ice. To remove unbound mAbs, cells were washed twice with FACS buffer. All FACS-analyses were performed with a FACSCanto or a LSRII Flow Cytometer and FACSDiva software .Hydrogen peroxide activity was detected using oxidation of dichlorofluorescein diacetate  to DCF shown in green fluorescence. Kinetics were measured with a fluorescence reader  as described previously For detection of IL-8 release supernatants of PMN culture were harvested after 2 or 6 hours of stimulation as indicated and analyzed by enzyme-linked immunosorbent assay  according to the manufacturer’s instruction.5 per well) were stimulated as indicated in the presence of 5×106 microbeads for 90 min at 37°, then kept on ice, washed twice in FACS buffer, and fixed in 1% paraformaldehyde in PBS. Analysis was performed using FACS. The amount of phagocytosis was quantified as percentage of PE-positive events in the PMN region as defined by light scattergram.The phagocytic activity was evaluated by ingestion of PE-labelled polystyrene microspheres  as described previously et al. 5 per well) were resuspended in staining buffer , incubated for 2 hr at 4°C and analysed by FACS.Apoptotic cells were quantified by detection of DNA fragmentation, evaluating hypodiploid nuclei according to a modified protocol described by Nicoletti P values less than 0.05 were considered statistically significant.Statistical analyses of the data were performed Tukey’s test. PMN are the primary innate immune effector cells combating pathogenic microorganisms, such as bacteria and fungi. Inflammatory signals, i. e. IL-1 or TNF-α, or the recognition of microbial patterns prime the activation of the NADPH oxidase and induction of oxidative burst. This leads to the formation of toxic reactive oxygen species (ROS) in vitro and monitored ROS formation over time. To assess an optimal experimental setup, we first titrated aPL and chose 10 µg/ml as the final concentration utilized in all experiments  expression level is important and activation induced shedding is widely used as a marker for PMN activation This once again suggests a synergistic mode of activation for the shedding of L-Selectin.in vitro cultures of purified PMN and incubated them either with aPL, LPS or the combination and as a control with human IgG, as described before. After 2 or 6 hours of culture, supernatants were collected and tested in an IL-8 specific ELISA . Thus an activated phenotype of PMN is characterized by the increased production and release of IL-8. Therefore we asked in a next step whether IL-8 is induced upon contact of PMN with aPL or the combination of LPS and aPL. To this end, we performed ic ELISA . As expeic ELISA  as well ic ELISA . The conThese data clearly demonstrate that the inflammatory cytokine IL-8 is produced and released in the presence of aPL and LPS.+ cells 100- to 1000-fold in vitro PMN stimulation we incubated the purified cells with LPS in the presence or absence of LBP and additionally compared LBP+LPS with aPL+LPS stimulation. As depicted in Cells of the innate immune system make use of receptors recognizing common patterns of invading microorganisms. LPS as well as lipoproteins and others need to be bound and transported. Therefore the lipopolysaccharide-binding protein (LBP) and additional transport proteins participate as a first defence of the host immune response These results indicate that aPL may functionally play a similar role as LBP.3Cys led to enhanced burst activity in the case of low amounts of TLR2 ligand (100 ng/ml and 1 µg/ml).The activation pathway via TLR4 illustrates the initiation of an immune response for invading Gram-negative microorganisms. Components of Gram-positiv bacteria are mostly recognized via TLR2 3Cys concentrations we were unable to detect an additional or synergistic activity for this particular effector function.In the phagocytosis assay  aPL alon3Cys and aPL in a concentration dependent manner, depicted in 3Cys and aPL even at an early time point (2 hours) after stimulation  were incubated with LPS (100 ng/ml) together with medium or titrated hIgG or aPL . Detection of specific fluorescence index (SFI) over time was calculated by subtraction of the background fluorescence of labelled cells incubated in medium.(TIFF)Click here for additional data file.Figure S2Stimulation of PMN with aPL has no impact on induction of apoptosis. Human PMN (2×105 cells per well) were incubated with LPS (100 ng/ml) with or without the addition of aPL or hIgG (10 µg/ml) Apoptotic cells were quantified by detection of DNA fragmentation via FACS. Data shown are from one representative experiment out of 3 independent with 2 replicates per group.(TIFF)Click here for additional data file.


The spindle assembly checkpoint is crucial for the maintenance of a stable chromosome number. Defects in the checkpoint lead to Chromosomal INstability (CIN), which is linked to the progression of tumors with poor clinical outcomes such as drug resistance and metastasis. As CIN is not found in normal cells, it offers a cancer-specific target for therapy, which may be particularly valuable because CIN is common in advanced tumours that are resistant to conventional therapy.Drosophila and then screened the set of kinase and phosphatase genes by RNAi knockdown to identify those that induce apoptosis only in the CIN cells. Genes identified include those involved in JNK signaling pathways and mitotic cytoskeletal regulation.Here we identify genes that are required for the viability of cells with a CIN phenotype. We have used RNAi knockdown of the spindle assembly checkpoint to induce CIN in The screen demonstrates that it is feasible to selectively kill cells with CIN induced by spindle checkpoint defects. It has identified candidates that are currently being pursued as cancer therapy targets (e.g. Nek2: NIMA related kinase 2), confirming that the screen is able to identify promising drug targets of clinical significance. In addition, several other candidates were identified that have no previous connection with mitosis or apoptosis. Further screening and detailed characterization of the candidates could potentially lead to the therapies that specifically target advanced cancers that exhibit CIN. Chromosomal INstability (CIN) is a common feature of nearly all solid tumors The most common errors seen in CIN cancer cells are lagging chromosomes and chromosomal bridges. The mechanisms proposed to be responsible for these errors include defects in: sister chromatid cohesion The SAC is the only mechanism by which cells can detect aberrant kinetochore attachments in metaphase and delay the entry to anaphase until the problem is resolved, or otherwise induce apoptosis Drosophila melanogaster in which we induced tissue-specific chromosomal instability in a wild type organism. We generated CIN by knocking down the SAC protein Mad2, which shortens metaphase, giving cells less time to correctly orient their chromosomes before the onset of anaphase Here we have used depletion of the SAC to induce CIN. We have then carried out a systematic genome-wide screen for kinase and phosphatase genes that, when depleted, can trigger apoptosis only in these genetically unstable cells, but not in normal cells. Our rationale is that for a therapy to be effective, cell death should be restricted to the tumour, i.e. the cells that exhibit CIN. We set up an assay system using Kinases and phosphatases are key regulatory enzymes controlling vital processes such as cell growth, differentiation, and survival Drosophila. Ubiquitous expression of mad2 dsRNA in the whole organism gives ∼85% reduction of Mad2 expression as compared to control lacZ dsRNA  and 63% (±3%) respectively of the wild type RNA levels when measured by qPCR. This partial knockdown was expected for essential genes like bsk and asp, because our original screen selected for candidate RNAi lines that were not lethal in normal cells. CIN cells must be highly sensitive to dosage variations in these candidates to give such strong phenotypes following modest candidate depletion, emphasizing the significant role these candidates have in cellular responses to CIN.Acridine Orange staining of larval wing discs showed significantly elevated levels of cell death when the candidates were knocked down in CIN cells e.g. , consistmad2 and a selected candidate  we observed apoptosis in the affected area  depleted CIN cells was significantly higher than aPKC and Nek2  and significantly decreased the level of apoptosis in wing discs  wing discs, showing that, in this case, the apoptosis induced by asp knockdown in CIN cells is largely P53 independent (bsk (Jun kinase) gave a modest reduction in the level of cell death when P53 was depleted (data not shown), suggesting the involvement of both p53 dependent and independent mechanisms in inducing cell death in this case.Because P53 is commonly lost in tumours and has been implicated in their CIN tolerance ng discs , suggestependent . Other cDrosophila as a model, we were able to induce CIN in a genetically stable background by depleting ∼85% of Mad2, which weakens SAC function and shortens metaphase To identify targets that could be depleted to induce apoptosis in cells with chromosomal instability, we carried out RNAi screening of kinases and phosphatases in a CIN model system. We targeted CIN because it is common in cancers and CIN makes these cancers resistant to current therapies The screen identified a significant group of candidates, including Nek2, JNK and Asp, that are directly or indirectly linked to the centrosome . The cenMembers of the JNK pathway, which is known to promote apoptosis, DNA damage response, proliferation, migration and differentiation asp mad2 double depletions. This suggests that the apoptosis we observed was not simply a result of losing a negative regulator of p53. Further work will be required to determine what triggers cell death in this case.Abnormal spindle (Asp) binds at the minus end of microtubules, and is required for centrosome attachment Taken together, these results suggest that CIN cells are highly sensitive to centrosome disruption, responding by apoptosis to treatments that have no effect on normal dividing cells. One plausible hypothesis to explain this sensitivity was that the centrosome disruption by itself caused a certain rate of CIN, which when added to the CIN from Mad2 depletion, took the cells over a threshold of instability beyond which they were unviable tefu, lok, bsk, and pp1a), suggesting a role for the DNA damage response pathway in responding to CIN. This is not surprising given the recent work showing that anaphase errors result in DNA damage Several interesting candidates identified in our screen, however, are not localized on centrosomes (e.g. Pask and aPKC). Furthermore, some candidates have no reported connection of any kind with mitosis, indicating that we may have detected novel pathways that sense segregation defects and provide stability to cancer cells against CIN. For example, Pask is a serine/threonine kinase involved in sensing and regulating cellular energy homeostasis aPKC knockdown in CIN cells. Furthermore our highest levels of DNA damage were seen in Pask mad2 double depletions, which gave no more apoptosis than aPKC. It is striking that none of our candidates alone, nor mad2 alone, gives significant levels of DNA damage. Our interpretation of this is that the cellular DNA damage response can keep the level of damage below our detection sensitivity in any of the single depletions, and it is only when multiple checkpoint and repair mechanisms are depleted that we see unrepaired damage and widespread apoptosis.It seems clear that the DNA damage we observed in our candidates in the presence of CIN is not simply part of the apoptotic program: we see strong apoptosis and little DNA damage in hemipterous/JNKK, Slpr: slipper/JNKKK, Pak3/Pak2 and Pvr: PDGF/VEGF receptor), may have potential as good CIN-specific targets.Depletion of Asp or Bsk gives some P53 independent apoptosis in CIN cells, which could make them desirable therapeutic candidates in a clinical setting, where P53 is often absent Drosophila to demonstrate the principle that it is possible to selectively kill CIN cells. Our RNAi knockdown identified candidates not previously known to have mitotic roles but whose depletion has the potential to kill proliferating CIN cells. Further characterization of screened candidates and their pathways may help to identify mechanisms by which cancer cells can tolerate the adverse effects of CIN and aneuploidy which in turn may lead to the identification of novel targets that can specifically kill advanced, drug resistant, CIN tumor cells without harming normal cells.In summary, we have used a new model for CIN in Candidate RNAi (kinase and phosphatase) lines, were obtained from the Vienna Drosophila RNAi Centre. The mad2-RNAi line used was v47918. Ubiquitous expression was driven using daughterless (da)-Gal4 and posterior wing disc expression was driven using engrailed (en)- or hedgehog (hh)-Gal4 (Bloomington stock center).All UAS-RNAi lines used, including 485 UAS-  knockdown of candidates : UAS>mad2-RNAi/CyO; da>Gal4/TM6 tub>Gal80ts × UAS> candidate-RNAi. The temperature of crosses was adjusted to give the best numbers of progeny while still getting effective knockdown (mostly 23°C). The ratio of the average viable fly count of double knockdown (mad2 and candidate) over single knockdown (candidate only) progeny gave the level of viability of that candidate in CIN flies. Depletion of candidates that gave >75% lethality when mad2 was co-depleted, compared to the candidate knockdown alone, were retested and, if reproducible, considered for further assays. This was measured by counting the number of surviving Cy versus non-Cy progeny from each cross, selecting those with a ratio of at least 4∶1.In viability screening we have tested ubiquitous For each genotype, thirty brains or sixty imaginal wing discs from third instar larvae were dissected in 1X PBS and quickly transferred and homogenized into pre-cooled Trizol reagent (Invitrogen). This was done in three biological replicates. Samples were then frozen in liquid nitrogen and kept in −80 C until RNA extraction. Total RNA was extracted with chloroform and precipitated with ethanol. RNA was further purified using the RNeasy Mini Kit (Qiagen).Drosophila.The qPCR assays were carried out using protocols described in mad2 F/R: GGCGACCAAAAACTGCATCA/GGTAAATTCCGCGTTGGAAGA, bsk F/R: GAATAGTATGCGCCGCTTACGA/ATTCCCTATATGCTCGCTTGGC, asp F/R: AGGCAAAGGCGGTAAACTCTGT/ACTCCGAACACCACATGAGCAG and (House-keeping gene) rp49 F/R: ATCGATATGCTAAGCTGTCGCAC/TGTCGATACCCTTGGGCTTG Loss of tissue in the posterior compartment of adult wings was scored by measuring the distance from where the fifth vein met the margin, to the fourth vein. Wings of adult females were dissected in ethanol and mounted in Aqua Poly/Mount . Levels of CIN were tested by counting defective anaphases in fixed, Hoechst 33342 stained brain squashes as described en>CD8GFP), using ImageJ software. Before normalization, background noise was subtracted from all the images by setting the rolling ball radius at 10 pixels.Cell death in 3rd instar larval imaginal wing discs was measured using a vital stain (Acridine Orange). Third instar larvae were selected and dissected, and imaginal discs were collected carefully in PBS. Imaginal wing discs were then incubated for 2 min in a 1 µM Acridine Orange solution and briefly rinsed in PBS before immediately mounting and imaging. The discs were transferred to a slide having double sided sticky tape on either side of the sample. This was done to prevent the squashing of discs when we placed a cover slip on top. Acridine staining was normalized by subtracting the mean intensity value of the wild type anterior compartment from the mean intensity value of the mutant posterior compartment . Quantitation of DNA damage staining (anti-P-H2AvD) was also done on normalized mean intensity value. Normalization was done by subtracting the mean intensity value of the wild type anterior compartment from the mean intensity value of the mutant posterior compartment (marked with en>CD8GFP), using ImageJ software. Before normalization, background noise was subtracted from all the images by setting the rolling ball radius at 5 pixels. All microscopy was done on a Zeiss Axioplan2 with Semrock Brightline filters and measurements and quantitation were done using Axiovision software (Carl Zeiss). Images were compiled using Axiovision (Carl Zeiss), Photoshop and Illustrator (Adobe) software.Cleaved caspase 3 immunostaining was performed on dissected wing imaginal discs. Single and double knockdown third instar larvae were selected and dissected in PBS and fixed in 4% formaldehyde for 20 minutes. Fixed discs were extensively washed with PBST (1xPBS+0.2% Tween) and then blocked by PBST containing 5% fetal calf serum (PBSTF) for 30 minutes. Discs were then incubated in primary antibody solution  for 2.5 hrs at room temp or left overnight at 4°C followed by 2–3 quick washes with PBSTF and then 30 minutes in PBSTF. For secondary antibody staining, discs were incubated for 2 hrs in the dark at room temperature with 1∶75 Anti rabbit CY-3 Antibody from Abacus/Jackson in PBST followed by 2–3 quick washes with PBST and then 30 minutes incubation in PBST. Mounting was done with 80% glycerol-PBS. DNA damage staining was done using the same method with rabbit anti-H2AvD P-Ser137  which is the Figure S1a. Loss of tissue in adult wings.Engrailed-driven single (candidate-RNAi only) and double (candidate and mad2-RNAi) knockdown in adult Drosophila wings. This driver depleted genes only in the posterior compartment of the wing, the lower half of each wing in this figure. We measured the loss of tissue by measuring the shortest distance from where the fifth longitudinal vein met the margin, to the fourth longitudinal vein (arrows). Depletion of Pask shows posterior wing margin notching (arrowheads) along with shorter inter-vein distance . Negative control (1118W) showed no significant tissue loss with (black bar) or without CIN. The Y-axis starts from 250 µm, to improve resolution. dWNK was an outlier not included in this graph, showing an inter-vein distance without and with mad2 RNAi of 245 µm and 60 µm respectively. Error bars represent 95%CIs, n≥8 in all cases.ance see , when co(PDF)Click here for additional data file.Figure S2a:Quantitation of Acridine Orange staining on larval wing discs. Graph shows quantitation of Acridine Orange staining of control and candidate-RNAi imaginal wing discs. Quantification shows arbitrary grey value units normalized by subtracting the mean grey value of the wild type (anterior) region from the mean grey value of the affected (posterior) region for each disc. Negative control (LacZ RNAi) and candidate RNAi alone are represented in light grey bars and the double knockdowns of candidates with mad2 are represented by dark grey bars, while double knockdown of mad2 with the LacZ negative control is shown in black. Error bars indicate 95%CIs, n≥8 in all cases. P-values are calculated by two-tailed t-tests with Welch’s correction: p<0.001 = ★★★, p 0.001−0.01 = ★★, p 0.01−0.05 = ★. All t-tests compare candidate-RNAi mad2-RNAi with lacZ-RNAi mad2-RNAi.  All wing discs are stained with Acridine Orange and the dotted line shows the en>CD8GFP marked posterior compartment or test region in which the genes were depleted. The other half of each disc expressed no transgenes. Single knockdowns of each candidate are arranged on the right and the double knockdowns with mad2 are on the left side. Representative discs for each genotype are shown; the level of variation for each genotype can be seen in (PDF)Click here for additional data file.Figure S3DNA damage staining quantitation. Graph shows a quantitative analysis of DNA damage (anti-P-H2AvD staining). The Y-axis represents the level of P-H2AvD staining in the affected half normalized by subtracting the level in the control half for each disc. Light grey bars represent the candidate knockdown in wild type background and dark grey bars represent the double (candidate and mad2) knockdown. Error bars indicate 95%CIs, n≥8 in all cases. P-values are calculated by two-tailed t-tests with Welch’s correction: p<0.001 = ★★★, p 0.01−0.05 = ★ and p>0.05 = ns (not significant).(TIF)Click here for additional data file.Figure S4Cell death assay for the validation of Mad2 results with BubR1. Dotted line shows the en>CD8GFP region or tester region and the other half expresses no transgenes (a-f) Images of wing discs stained with Acridine Orange. (a) mad2 RNAi (d) bubR1 RNAi. (b & e) Pask RNAi. Double knockdowns are (c): Pask RNAi and mad2 RNAi and (f): Pask RNAi and bubR1 RNAi.(TIF)Click here for additional data file.Figure S5CIN levels. Graph represents the frequency of defective anaphases in knockdowns of LacZ, Asp, Bsk, aPKC, Pask and Mad2 in brain cells. LacZ was used as a negative RNAi control and Mad2 is used as positive control to compare the level of CIN. None of the candidates show significantly elevated levels of CIN above the LacZ control. Error bars show 95%CIs, n>40 in all cases. P-values are calculated by two-tailed Fisher’s exact test: p<0.001 = ★★★ (extremely significant) and p>0.05 = ns (not significant).(TIF)Click here for additional data file.Table S1List of kinases and phosphatases tested in the viability screening of our induced-CIN model. Columns show: the gene identifiers; number of replicate crosses carried out; total numbers of CyO (kinase-RNAi only) and non-CyO  progeny; Ratio of the average number of Cy/non-Cy progeny per cross used to rank the table; and probability of finding a cross this diverged or more diverged from a 50/50 ratio (the null hypothesis), out of this number of crosses (936), purely by chance.(XLSX)Click here for additional data file.