Purpose: A 2006 National Cancer Institute clinical announcement recommended the use of combined intravenous (IV) and intraperitoneal (IP) chemotherapy over IV chemotherapy alone for women with International Federation of Gynecology and Obstetrics (FIGO) stage 3 optimally debulked ovarian cancer due to significant survival benefit demonstrated in multiple randomized clinical trials. We examined uptake of IP chemotherapy in community practice before and after this recommendation.Methods: We identified 288 women with FIGO stage 2 or greater incident ovarian cancer diagnosed from 2003 to 2008 at three integrated delivery systems in the US. Administrative health plan data were used to determine patient characteristics and receipt of IV and IP chemotherapy within 12 months of diagnosis. We compared characteristics of women receiving IV chemotherapy alone vs. IP chemotherapy (with or without IV chemotherapy) and assessed temporal trends in IP chemotherapy use.Results: Overall 12.5% (n = 36) of women received IP chemotherapy during the study period. IP chemotherapy use was non-existent between 2003 and 2005. Use of IP chemotherapy occurred among 26.9% of women diagnosed in 2006 and plateaued at 20.4% of women diagnosed in 2008. IP recipients were younger  and more likely to have stage 3 ovarian cancer (77.8 vs. 50.4% p = 0.039) compared to their IV-only chemotherapy counterparts.Conclusion: Use of IP chemotherapy for newly diagnosed advanced stage ovarian cancer patients was uncommon in this community setting. Future research should identify potential patient, physician, and system barriers and facilitators to using IP chemotherapy in this setting. Approximately 22,280 new ovarian cancers were diagnosed in 2012, and an estimated 15,500 women died from the disease  released a clinical announcement that recommended combined IV and intraperitoneal (IP) chemotherapy for women with FIGO stage 3 optimally debulked ovarian cancer , 6. IP cThe purpose of our study was to examine patterns of IP and IV chemotherapy treatment in three sites of the Cancer Research Network (CRN), a consortium of research groups associated with integrated delivery systems in the United States with over 11 million enrollees . We examThe HMO CRN consists of the research programs, enrollee populations, and databases of 14 members of the HMO Research Network . An overN = 921). We included women with epithelial and other morphologies of ovarian cancer who had records of IV chemotherapy and/or IP chemotherapy following diagnosis (N = 381). We excluded morphologies for myomatous neoplasms (ICD-O-3 code 8890), malignant lymphoma (ICD-O-3 9590 and 9680), sex cord stromal , germ cell , and follicular and marginal lymphoma (ICD-O-3 9690) (N = 4). We further excluded women with FIGO stage I disease (N = 52) and women who started chemotherapy treatment more than 12 months after their cancer diagnosis (N = 37). Even though IP chemotherapy is only recommended for stage 3 ovarian cancers, we included stages 2 and 4 to evaluate whether treatment was administered outside of guidelines. Most women were treated by oncologists employed by their health plan . However, women may have received oncology care outside of the health plan (such as at a cancer center) based on their insurance coverage and preferences; treatment received outside of the health plans was collected for this analysis via claims. Our final sample size for analysis was 288 women.We identified all women with incident ovarian cancer diagnosed between January 1, 2003 and December 31, 2008 from each site’s local Surveillance Epidemiology and End Results (SEER) or tumor registry , which has been described in detail elsewhere . BrieflyWe collected data on chemotherapy administration (including claims) using validated VDW procedure codes –12. ChemWe compared the distribution of patient characteristics, tumor characteristics, and healthcare utilization between women who received any IP chemotherapy (with or without IV chemotherapy) and women who received IV chemotherapy alone. We used the two-sample Wilcoxon rank-sum test to evaluate differences in continuous variables  and Fisher’s exact test to evaluate differences in categorical variables . We calculated the cumulative count of first IP and IV procedures separately over time. If a woman had more than one IP or IV procedure, only the first one of each was counted. All analyses were descriptive and unadjusted. All statistical analyses were conducted in SAS software version 9.3 .p < 0.001). Women who received IP chemotherapy differed in disease stage compared with those who received IV chemotherapy alone (p = 0.039); a larger percentage of women receiving IP had stage 3 disease (77.8%) than women receiving IV chemotherapy alone (50.4%).A total of 288 women were identified with FIGO stage 2 or greater incident ovarian cancer between 2003 and 2008. Of these, 36 12.5%) women received IP chemotherapy with or without IV chemotherapy and 254 (87.5%) received IV chemotherapy only  or whether they had local access to properly trained surgeons with experience in catheter placement and care or IP chemotherapy administration. We did not collect information on treating physicians and have no way of identifying the small number of patients treated outside of their health plan through the treatment codes we used. The small number of IP users limits our ability to conduct sub- and multi-variable analyses. Between 20 and 40% of women presenting with advanced ovarian cancer will have suboptimal surgical debulking, which would make them ineligible for IP treatment . UnfortuDespite these limitations, our study has several strengths. To our knowledge, this was the first study to document the diffusion of IP chemotherapy in community-based clinical settings. We were able to conduct this analysis because of our data linkages between procedure data, tumor registry data, and other administrative health plan data using the CRN VDW, a validated source of information regarding community cancer patients. We also had complete and valid data on chemotherapy use from CPT and HCPCS codes, which have been validated in prior studies –12 and iIn conclusion, our study shows treatment with IP chemotherapy for women with advanced stage ovarian cancer is uncommon in our three community-based integrated delivery systems. This may be due to system, provider, and/or patient factors. However, the lack of uptake IP chemotherapy use is concerning as ovarian cancer patients may not be receiving therapy that would give them the best chance at survival. Additional research into the barriers and facilitators to IP chemotherapy treatment is needed.The authors contributed to this manuscript as follows: conception and design of the work ; acquisition, analysis, or interpretation of data for the work ; drafting the work or revising it critically for important intellectual content ; final approval of the version to be published .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite its wide use over six decades, warfarin therapy remains challenging due its narrow therapeutic index. The multitude of factors interacting with warfarin makes it difficult to maintain anticoagulation within the target International Normalized Ratio (INR) range  allow flexibility in selecting a starting dose of warfarin, suggesting 5–10 mg. Although the ACCP guidelines recommend lower (2.5–5 mg) doses recognizing the influence of age, comorbidities, nutritional status, and drug interactions, these recommendations do not tailor dosing to individual patients  and Vitamin K epoxide reductase complex1 . The influence of SNPs in CYP2C9 and VKORC1 on warfarin dose has been extensively assessed and reviewed  in two genes; Cytochrome P450 2C9 , VKORC1 (-1639/3673G>A), GGCX (rs11676382), and CYP4F2 (V433M) polymorphisms. The algorithm is freely available at www.warfarindosing.org and allows calculation of warfarin dose based on clinical and demographic factors alone (if genotype is not available). Incorporation of novel and potentially important genetic variants (such as CYP2C9*8) can further improve dosing prediction in African American patients , dosing based on clinical/demographic factors alone improves prediction of stable therapeutic dose of warfarin , specifically in patients that need =7 mg/day or =3 mg/day. Furthermore inclusion of Both pharmacogenetic algorithms .The Clinical Pharmacogenetics Implementation Consortium (CPIC) of the National Institutes of Health Pharmacogenomics Research Network has developed guidelines to assist clinicians in the interpretation and use of VKORC1, CYP2C9, and identified CYP4F2 as the main genes associated with dose  have been conducted. Among patients of European descent these studies have confirmed the influence of As these known variants in candidate genes account for a smaller percent of the variability in warfarin dose among Blacks, the IWPC is leading a GWA analysis in Blacks. Preliminary results of the ongoing GWAs meta-analysis were presented at the 2011 American Heart Association meeting  of the variability in warfarin dose remains unexplained. Perhaps emerging genotyping technologies such as exome sequencing or whole genome sequencing will reveal important clues that can explain the missing heritability. In addition to interrogating genetic variation ongoing and future studies must assess in detail environmental  and lifestyle  factors with similar rigor. It is very likely that this along with gene-environment interactions holds the key to explaining the majority of the variability in response.One group that remains unrepresented is the Hispanic/Spanish population. This is of great importance in the US as people of Hispanic descent accounted for more than half the growth in the population between 2000 and 2010 and account for 16.3% of the US population Can a genetic risk factor (genetic marker) associated with an adverse  outcome be a clinically useful predictor of that outcome? CYP2C9 and VKORC1 genotypes are clinically useful predictors of warfarin dose in clinical trials Can incorporation of the genetic factor predict risk of the outcome more accurately than existing clinical models? CYP2C9 and VKORC1 genotypes provided superior warfarin dose prediction compared with the clinical algorithm Will the outcome predicted for individuals be sufficiently different to warrant a change in treatment decisions? 1)CYP2C9 and VKORC1 genotypes improves prediction of warfarin doses as confirmed by randomized clinical trials proving excellent insight into the effectiveness of utilizing pharmacogenetics in a real-world setting improved anticoagulation control is being evaluated.Whether the benefits of pharmacogenetic guidance of warfarin dosing would translate into Anderson et al. showed that pharmacogenetic-dosing resulted in a higher percent time in target range  is greater than that achieved by specialty anticoagulation clinics . Importantly, these benefits in the pharmacogenetic cohort accrued in a setting where warfarin-treated patients were typically managed by standard protocol by an anticoagulation service/clinic.3)Observational cohort studies have demonstrated that possession of CYP2C9 variant allele increases the risk of hemorrhage  the test/intervention must demonstrate improvement in intermediate endpoints  and preferably hard-endpoints  in randomized clinical trials.When will this evidence be available? Are we there yet?A number of clinical trials studies have assessed whether dose prediction and anticoagulation control are superior in patients receiving pharmacogenetically guided dosing vs. standard dosing or on clinical factors excluding genotype. However trials to date have been limited by study design issues  and limited sample size. Nonetheless these studies have provided valuable information on effect sizes and genotype-specific expectations which was valuable in the design of large ongoing randomized trials.Clarification of Optimal Anticoagulation through Genetics  trial is a multicenter double-blind, randomized trial aims to determine whether the use of genetic and clinical information for selecting the dose of warfarin in 1022 patients during the initial dosing period will lead to improvement in stability of anticoagulation at 4 weeks relative to a strategy that incorporates only clinical information (without genetics).The EUropean Pharmacogenetics of AntiCoagulant Therapy  trials are single-blinded randomized controlled trials aiming to assess the safety and clinical utility of genotype-guided dosing of the three main coumarins used in Europe: acenocoumarol, phenprocoumon, and warfarin. The warfarin arm will recruit 900 (UK and Sweden) warfarin patients to determine if genotype-guided dosing vs. standard dosing improves PTTR at 3 months.The Genetic InFormatics Trial  of Prevent DVT is a randomized controlled trial aiming to determine the benefit of genotype-guided dosing vs. clinically guided dosing among 1600 participants (age 65 years or older) in reducing the composite endpoint: non-fatal VTE, non-fatal major hemorrhage, death, and an INR > 4.0 at 1 month.The Pharmacogenetic-dosing of warfarin: a controlled randomized trial is led by the Taiwan Warfarin Consortium which aims to determine whether genotype-guided dosing can improve safety (as measured by time to target INR and PTTR) of warfarin treatment in 600 participants compared to clinically guided dosing.Warfarin Adverse Events Reduction for Adults Receiving Genetic Testing at Therapy Initiation  is a randomized blinded interventional trial where 4300 patients (age > 65 years) are to be randomized to warfarin dosing based on the GenoSTAT test plus clinical factors, or clinical factors alone, using the warfarindosing.org website. The primary aim is to determine if genotype-guided therapy reduces the incidence of warfarin-related clinical events, including major hemorrhage and thromboembolic events at 30 days and in fewer hospitalizations and/or deaths compared to clinically guided therapy at 90 days compared to clinically guided therapy.www.clinicaltrials.gov. These trials will provide a robust data for efficacy/effectiveness and cost effectiveness analysis and will provide the foundation for policy development.Additional information on the trials below and others can be found at CYP2C9*2 polymorphism was published in 1994. With the discovery of VKORC1 in 2004 the field burgeoned with investigations in multiple populations across the world documenting the effect of CYP2C9 and VKORC1 on warfarin dose, anticoagulation control, and risk of hemorrhage. Twenty years from the report identifying the CYP2C9 *2 (10 years following the identification of VKORC1), the results of the first double-blinded randomized clinical trial  testing genotype-guided dosing intervention are expected to be available.Although detractors claim pharmacogenetics/genomics in general has not yielded information to justify the investment of effort and funds, progress in the genomics/genetics arena has been maintained a rapid pace compared to other fields in medicine. For warfarin, the first report identifying the Although the uptake of newly available oral anticoagulants is slow, a frequently raised question is “Will warfarin or warfarin pharmacogenetics matter in the coming years?”warfarin while just 12.6% were prescribed one of the two new oral anticoagulants , Rivaroxaban 2011), and Apixaban  is changing the landscape of anticoagulation therapy  to advance science and enable discoveries beyond what can be gaged solely by investments; past, current, and future.


The molecular constituents of organic light emitting devices (OLEDs) are typically soluble in organic solvents. Consequently, OLEDs cannot be exposed to the key components of conventional photolithographic processes. The OLED industry relies instead on thermally evaporated thin organic films that are patterned by metal shadow masks. But shadow masks presently limit OLED displays to smaller substrate sizes than their liquid crystal display (LCD) counterparts.–3 SerialTraditional photolithographic techniques have been applied to organics by making use of their orthogonality with highly flourous materials.–12 They'2O resists to pattern features at the nanometer scale. Th Th17] Ther scale.–20 But wer scale., 22] In In2O resFigure2 mask lifting off is available in the Supporting Information.A typical process flow for phase-change lithography is shown in 2 as the phase-change resist. The phase diagram of CO2 is shown in Figure2 is reduced. For example, at our operating pressure of 10−6 Torr, the sublimation temperature of CO2 is roughly 90 K. Thus, for a stable lift-off mask, the substrate must be cooled to at least 85 K, which is still within the range of relatively inexpensive cooling with liquid nitrogen.To avoid degradation of our active materials, we employ inert CO2 resist is patterned by selective sublimation, it is important to control the partial pressure of CO2 in the chamber to prevent unwanted re-condensation of CO2 vapor on patterned regions of the substrate. It is also possible to freeze other impurity gases onto the substrate, notably H2O, whose phase diagram is shown in 2 films at 10−7 Torr, Gerakines et al. measured a water deposition rate of 2 nm h−1. Th ThJ, andld traps.In addition to addressing concerns of the organic layers' growth under cold conditions, the transistor backplane of active-matrix displays must also withstand the low temperatures of the process. To verify this, a small active-matrix OLED display was removed from a digital photo frame, pumped down to high vacuum and cooled with liquid nitrogen, and then replaced in its housing and connected to its driver. There was no noticeable difference in pixel brightness, uniformity or operation aside from the seal of the passivation glass coming loose—passivation being a manufacturing step strictly after full device fabrication.To conclude, the use of an in situ definable mask has been shown to be a viable alternative to patterning thin films of organic semiconductors and metals at the large scale. The dry resist material frozen directly to the surface of the substrate alleviates many of the issues of scaling up as fine metal masks have proven increasingly cumbersome with area. While pixel density as high as 325 pixels-per-inch has been demonstrated, there is no reason to believe this is a fundamental limit—especially knowing that nanometer-scale patterning has been demonstrated with an electron beam paired with frozen water resist.–20 Patte−7 to 10−6 Torr range for substrate temperatures between 20 K and 100 K. Cooling is achieved using either a liquid nitrogen reservoir or a repurposed cryogenic pump depending on the desired base temperature. With the substrate sufficiently cooled, CO2 gas  is introduced to the substrate via 1/4” copper tube attached to either a mass flow controller or variable leak valve depending on the desired flow rate; the flow rate also being a function of temperature and pressure; see Supporting Information.Patterning was successfully demonstrated within a vacuum in the 10−2 s−1 until the frozen CO2 formed the intended pattern observed from a camera mounted in situ.In the resistive heating experiments, 160 nm-thick ITO was patterned by traditional contact photolithography and etched with aqua regia. Copper foil tape was used to make contact from the ITO on the substrate to a ceramic power feedthrough. A sourcemeter (Keithley 2400) was used to drive 100 mA of current through 100 μm-wide lines resulting in heating at a rate of 225 mJ cm2 films is measured by double interferometry, as detailed in the Supporting Information., [, [2 filmormation.For experiments with temperatures less than the ∼80 K obtainable with a liquid nitrogen reservoir, a cryogenic pump was repurposed for use as a cooling source and all components are mounted onto it via an oxygen-free high-conductivity (OFHC) copper rod. All cold parts are machined out of OFHC copper and indium foil is sandwiched between all temperature-critical interfaces. A kapton encapsulated heater placed in between the substrate and substrate holder provides adequate local heating for encouraging lift-off without adding too much heat to the bulk thermal mass of the apparatus. A silicon thermal diode was attached to the copper substrate holder to approximately monitor the temperature of the sample and a cryogenic temperature controller (Lakeshore Cryotronics) is employed to manage operating temperature. Patterning is monitored using a digital microscope mounted on the stamp actuator. The repurposed cryogenic pump's compressor and cold head are briefly turned off during the actual stamping so that the vibrations do not interfere while the stamp and resist make contact.3) as the emitter were grown on ITO-coated glass substrates at similar temperatures as the stamping process, but active areas were defined using traditional shadow-masking techniques so as to evaluate the critical temperature-dependent parameter of using CO2 as a lift-off resist in producing organic optoelectronic devices. The substrates were detergent, solvent, and plasma cleaned prior to device fabrication. No CO2 was introduced in these experiments. N,N,N′,N′-tetrakis(4-methoxyphenyl)-benzidine (MeO-TPD), 4,4′,4″-tris(carbazol-9-yl)triphenylamine (TCTA), 2,2′,2″--tris(1-phenyl-1-H-benzimidazole) (TPBi), and lithium fluoride (LiF)/aluminum (Al) were used as the hole transport, host, electron transport and cathode layers respectively.We fabricated OLEDs using standard shadow-masking techniques to demonstrate compatibility with the low temperature substrates. Basic phosphorescent OLEDs employing fac tris(2-phenylpyridine) iridium (Ir(ppy)


Approximately 2.4 million people are living with HIV in India. This large disease burden, and potential for epidemic spread in some areas, demands a full understanding of transmission in that country. We wished to quantify the effects of key sexual risk factors for HIV infection for each gender and among high- and low-HIV risk populations in India.2 statistic. Publication bias was tested by Begg's test and funnel plots. Meta regression was used to assess effect modification by various study attributes.We conducted a systematic review of sexual risk factors for HIV infection from 35 published studies. Risk factors analyzed were: male circumcision/religion, Herpes Simplex Virus 2, syphilis, gonorrhoea, genital ulcer, multiple sexual partners and commercial sex. Studies were included if they met predetermined criteria. Data were extracted and checked by two researchers and random-effects meta analysis of effects was conducted. Heterogeneity in effect estimates was examined by Imen: 5.87; 95%CI: 2.46–14.03; ORwomen: 6.44; 95%CI: 3.22–12.86). The effect of multiple sexual partners was similar among men  and women  and when further stratified by HIV-risk group. The association between HSV-2 and HIV prevalence was consistently stronger than other STIs or self-reported genital ulcer. If the strong associations between HSV-2 and HIV were interpreted causally, these results implied that approximately half of the HIV infections observed in our study population were attributable to HSV-2 infection.All risk factors were significantly associated with HIV status. The factor most strongly associated with HIV for both sexes was HSV-2 infection (ORThe risk factors examined in our analysis should remain targets of HIV prevention programs. Our results confirm that sexual risk factors for HIV infection continue to be an important part of Indian HIV epidemic 26 years after it began. Approximately 2.4 million people live with HIV/AIDS in India today Previous studies on HIV in India have identified heterosexual sex  as the primary driver of HIV incidence in the population The Indian HIV epidemic exhibits a large amount of geographic heterogeneity in terms of HIV risk N/Ath 2011. We searched (and exploded) the terms: “HIV”, “delta retrovirus”, “Disease Transmission” , “Risk Factors”, “sexually transmitted infection” or disease, “herpes” or HSV and “India”. We combined these terms by merging results for HIV/delta retrovirus, any combination of the subsequent search terms, save India, and the last term. Results were limited to English language studies (due to logistic constraints) and those published from 1986 onwards (the year that HIV was first identified in India).Published studies were identified by searching electronic databases. The protocol for this review is unregistered. We searched three databases: Web of Science, EMBASE and Medline on June 9et. al. a priori, were assessed using meta-regression. Where possible stratification of study results by these factors was done . Seven specific characteristics were used to determine sufficient quality for inclusion. These factors included: biological confirmation of STIs, biological confirmation of HIV status following WHO guidelines, clear description of HIV-risk population, description of study population selection methods, provision of raw numbers of subjects exposed and unexposed by HIV status, separate reporting of unadjusted and adjusted estimates of effect and reporting a measure of variance around the effect estimate .Quality review was done using an approach recommended by the MOOSE group guidelines for meta-analysis of observational studies Data was abstracted and entered twice (two people) into a standard Excel template and cross-checked by each data extractor. Where required data were likely available but not presented in a published included study, the study authors were contacted for the relevant data.Case (HIV-positive) status was determined by referring to WHO guidelines for screening in developing countries (two reactive enzyme-linked immunosorbent assays) (24). We included both prevalent and incident HIV cases. In the final complement of 35 studies, only four reported on incident HIV cases.versus 0–1 partners). Where lifetime sexual partnerships were not available, current/recent number of sexual partners was used instead. In India, male circumcision is almost exclusively practiced by MuslimsTreponema pallidum) and gonorrhoea (Neiserria gonorrhoea infection). Information on stage of syphilis infection was not available for most studies. Genital ulcer was recorded as self-reported history of genital ulcer or history (ever) of diagnosis.We were interested in four broad sexual risk factors for HIV. These were STIs, male circumcision, genital ulcers and sexual behaviour. Sexual behaviour was sub-categorized into: paid sex  and lifetime numbers of sex partners  were calculated for each risk factor and meta-analysis was conducted. To account for heterogeneity between study effect estimates, summary ORs were calculated using the random effects methods of DerSimonian and Laird. This method was chosen because we wished to make inferences about the effect of exposures beyond the population of studies observed in our analysis and account for between study variations in effect estimates Our search strategy initially yielded 1195 results. The final dataset contained 35 studies. The final number of studies was arrived at as follows  significantly reduced the probability of HIV infection  pooled across both genders and risk populations . Among mHaving two or more lifetime sexual partners was significantly associated with HIV positivity  pooled across both genders and risk populations . Among m. This effect was slightly different for high-risk and general population men . Only two study estimates were available to estimate association between being paid for sex and HIV status among women, both suggesting a very high probability of infection  (data not shown).Among men, paying for sex was significantly associated with HIV positivity  pooled across both risk populations Among 11 studies, HSV-2 infection increased the probability of HIV infection  pooled across both genders and risk populations . Among m. These findings were true for both genders and risk populations. The association between syphilis and HIV was much stronger among general-population groups and this pattern was seen for both genders.Syphilis infection was associated with a quadrupling of likelihood of HIV infection  pooled across both genders and risk populations . Gonorrhoea was significantly associated with increased probability of HIV infection  and this effect size was similar for both men and women.Only three studies were included that reported on the association between biologically confirmed history of gonorrhoea infection and HIV and these were all in high-risk populations . This effect magnitude was also observed for each sex. History of genital ulcer was more strongly associated with HIV among high-risk groups however this difference was not significant. Among women, all included studies (n = 6) were in high-risk populations except for one.Self-reported history of genital ulcer was significantly associated with increased probability of HIV infection  pooled across both genders and risk populations Egger's test = 0.04) . Heterogeneity in effect estimates (I2) are reported in .Evidence of significant publication bias (protective effect) was only observed for male circumcision status/female Muslim religion  were not associated with gender, study year, study design or state. The associations between HSV-2, syphilis and multiple partnerships with HIV status were stronger among low-HIV risk groups and HIV test method (western blot versus other) was associated with larger effect size for multiple sex partners.The effects of study characteristics as analysed by random-effects meta-regression are presented in We have previously estimated that HIV transmission probability during partnership between discordant couples in India is low (30% to 46%) Male circumcision has been shown in randomized controlled trials in the African continent to significantly reduce risk of HIV infection in men by approximately half and indirectly, through their own risk reduction, in their partners and wives Circumcision was expected to be protective against HIV infection among men and not surprisingly among women as well. Muslim religious status among Indian women had a larger (though not significantly so) protective effect against HIV than in men and was the largest summary effect size among women in our study. This may be due to a combination of reduced exposure to HIV through their Muslim male partners and to behavioural reductions in risk. Among high risk women, which included FSWs, this protective effect could represent sorting of clients based on religion or participation in sex work based on location (Muslim FSWs being more likely to work in Muslim neighbourhoods and have Muslim clients). Among women in our study population, the single largest contributor to HIV risk was non-Muslim religion. This supports the prevailing theory that male sexual activity outside of regular partnerships is a key driver of the HIV epidemic in India.Sexually transmitted infections are a risk factor for HIV acquisition but can also increase onward HIV transmission and are therefore hypothesized to play an important role in HIV transmission dynamics in India HIV infection among men was most strongly associated with HSV-2 infection. Among men in our study population, HSV-2 had the largest summary association measure with HIV infection and was estimated to be causally associated with almost half of their HIV infections. In women HSV-2 positivity was the strongest risk factor examined for HIV infection for which there were a sizeable number of study estimates. There is a high degree of variation in HSV-2 prevalence estimates in India, particularly in high risk groups. HSV-2 prevalence has been reported between 1.0% and 18.9% from general population-based surveys,versus bacterial STIs play a more significant role and the difficulty in teasing apart independent effects The prevalence of syphilis in India is unknown but estimates from antenatal clinic attendees suggest a prevalence around 1.5%Meta-regression results suggested that the association of syphilis with HIV was stronger among low-HIV-risk population (p<0.001). This was mostly due to syphilis being substantially more strongly associated with HIV status among low-risk women  than high-HIV-risk women. This likely represents the effect of markedly different background prevalence of exposure. Among high-HIV-risk groups, syphilis was more prevalent generally in both cases and controls whereas in low-HIV-risk groups syphilis was generally rare (as was HIV infection) but still associated with HIV leading to a stronger association.We expected genital ulcer to be more strongly and consistently associated with male risk of HIV. This would have been congruent with other epidemiologic findings It is suggested that the Indian HIV epidemic is driven by heterosexual sex and particularly by male use of commercial sex work Compared to paying for sex, all STI exposures and reporting multiple sexual partners were more strongly associated with HIV infection and this was true even when restricted to men from high-risk populations. We have previously estimated that a sizable minority (10 to 20%) of HIV infections in married couples in India are introduced by the female partner Our study had several limitations. Firstly, only 35 studies were included in the final analysis of seven risk factors with two stratification levels. This limited our statistical power to find weaker associations and the extent to which we could examine other factors thought to influence the relationship between our seven exposures of interest and HIV status through stratification or meta-regression. The range of study years for each risk factor was limited prohibiting robust analysis of trends in effect sizes over time. Several studies did not provide sex-specific effect estimates and this limited our ability to explore effects between genders. While we received sex-stratified tables from several authors, we were unable to get these data for all studies.The HIV epidemic in India is geographically heterogeneous with 75% of reported HIV cases in four large southern states that house 30% of the country's population 2 = 0.0–98.2% – among summary effects for each gender) of heterogeneity in effect of exposures across studies. However the I2 measure does not work well for cohorts and in prevalence studies generally yields higher values. Some of this heterogeneity may be due to smaller studies which were included or studies in which few exposed cases or controls were observed. Random effects methods were used to help account for this higher level of heterogeneity.There was a wide range  in populations that were categorized as being from the general population (and assumed to be at low-risk for HIV infection). This suggests that some individuals in the general population samples were not at low risk of HIV and perhaps were more accurately categorized as high-risk. Under-reporting by women would have biased the summary effect estimate of multiple partnerships towards the null assuming that women with and without HIV were equally likely to under-report non-regular partnerships. In general, men have been shown to over report numbers of sex partners in sexual behaviour surveys . a) Male circumcision or Muslim religion b) Multiple sexual partners c) Paid for sex (men) d) Genital ulcer e) Syphilis f) HSV-2 (TIFF)Click here for additional data file.Figure S2Footnote: Each estimate represents the estimated summary odds ratio after the removal of the given study estInfluence plots for summary odds ratio by risk factor. a) Male circumcision/female religion status b) History of paying for sex (men) c) Multiple sex partners d) HSV-2 e) Syphilis f) Genital ulcer (PPT)Click here for additional data file.Table S1Footnote: Circ./Muslim = male circumcision or Muslim religion; HSV-2 = Herpes Simplex Virus 2; State: KN = Karnataka, MH = Maharashtra, AP = Andhra Pradesh, S.India =  South India; Population type: STI = Sexually transmitted infection clinic attendees; General = general population survey, FSW = female sex worker, MSM = Men who have sex with men, ANC = antenatal clinic attendees, IDU = injecting drug users; Design: CS = cross-sectional, CHRT = cohort, CCTRL = Case-control; HIV risk group: H = high, L = low (see methods).Summary of study population characteristics. (PDF)Click here for additional data file.Table S2Footnotes: 1. State =  Four large south Indian states versus all others. 2. F-test for categorical variablesResults of random effects meta-regression for six study characteristics. (DOC)Click here for additional data file.


A ground-glass appearance of the cornea is noted immediately after cataract surgery (figure) and there is a +3 anterior chamber reaction. What condition do you suspect?What is the most likely diagnosis?□ Endophthalmiitis□ Mechanical trauma to the cornea□ Intraoperative introduction of a toxic substance into the eye□ Fuchs' corneal dystrophyAnswer: Introduction of a toxic substance into the eye. Mechanical trauma to the endothelium during surgery is considered to be the most significant factor influencing postoperative corneal oedema; however, toxic substances may inadvertently be introduced into the eye, causing immediate corneal oedema. This may come from intraocular irrigation solutions or topical and intracameral anaesthesia. Intraocular medications that have resulted in corneal toxicity include epinephrine (now available preservative free), various preparations of lidocaine, benzalkonium chloride-preserved viscoelastic, vancomycin at doses greater than 1 mg/mL, and inadvertent exposure of the endothelium to 5% povidone-iodine.Bacterial contamination of the cleaning bath detergent for surgical instruments may also cause acute corneal oedema following cataract surgery. Oedema in these cases can be abrupt, resulting in immediate corneal swelling.Toxic anterior segment syndrome (TASS) is an acute, sterile anterior segment inflammation following generally uneventful cataract and anterior segment surgery. Rapid onset of corneal oedema and absence of a hypopyon are the distinguishing factors in differentiating toxic corneal oedema from an infectious endophthalmitis. Most patients with TASS will develop symptoms and signs within 12 to 24 hours of the operation.Reproduced by kind permission of the Ophthalmic News and Education (ONE®) Network of the American Academy of Ophthalmology. Visit www.aao.org/one


ATP1A2 gene are implicated in familial hemiplegic migraine type II and increase the severity of ischemic brain injury in animal models. To further explore these observations, we assessed the association between ATP1A2 polymorphisms, migraine, and the risk of ischemic stroke in participants of the Genetics of Early-Onset Stroke Study, a population-based case–control study of ischemic stroke among men and women aged 15–49. Using responses to a headache symptoms questionnaire, subjects were classified as having no migraine, or migraine with or without visual aura. Evaluating a total of 134 ATP1A2 polymorphisms genotyped using a combination of Illumina platforms (Cardiovascular Gene-centric 50 K SNP Array and HumanOmni1-Quad_v1-0_B Bead Chip), only one polymorphism (rs2070704) demonstrated a nominally significant association with stroke in an age-, gender-, ethnicity-adjusted model  and in a vascular risk factor model adjusting for age, gender, ethnicity, hypertension, diabetes, smoking, and myocardial infarction . Ethnicity-stratified analyses demonstrated a significant association for rs2070704 among African-Americans  but not Caucasians . These associations were unchanged when migraine subtypes were included as co-variates. We did not observe an association between ATP1A2 polymorphisms and migraine. While our results do not demonstrate a strong relationship between ATP1A2 polymorphisms and migraine associated stroke risk, the results are hypothesis generating and indicate that an association between ATP1A2 polymorphisms and stroke risk may exist. Additional studies are required.In a recent meta-analysis migraine was associated with a two-fold increase in stroke risk. While the mechanism driving this association is unknown, one intriguing hypothesis is that migraineurs are genetically predisposed to developing ischemic stroke. Mutations in the The online version of this article (doi:10.1186/2193-1801-2-46) contains supplementary material, which is available to authorized users. Although the evidence supporting an etiologic relationship between migraine and ischemic stroke risk is substantial (Cole & Kittner EDN1) were associated with increased risk of stroke risk as well as migraine , migraine, and the risk of ischemic stroke in a previously collected biracial case–control sample of young-onset ischemic stroke.Familial hemiplegic migraine (FHM) is a rare inherited form of migraine that presents with asymmetric neurological deficits  are summarized in Table ATP1A2 SNPs  but not among European-Americans . Correcting for multiple comparisons including all 134 SNPs evaluated, and assuming a p-value = 0.0004 (0.05/134) as significant, none of our results remain significant.Implementing the same regression models we then evaluated the rs2070704 by stroke subtype in the combined population only demonstrated an association among strokes of undetermined etiology . Ethnicity stratified analyses demonstrated no subtype specific associations among Caucasians. Among African-Americans the protective effect of rs2070704 was greatest in the large-artery atherosclerotic subtype  as compared to cardio-embolic , small vessel  and strokes of undetermined etiology .Analyses of rs2070704 SNP and stroke, adding migraine as a co-variate should eliminate this association. However, when migraine was included in the model, the association between rs2070704 and stroke  remained essentially unchanged. We then performed a restricted analysis adding either migraine without aura or migraine with aura as a co-variate. Again, there was no change in the association; migraine without aura  and migraine with aura , indicating that migraine status does not mediate the relationship between this SNP and stroke risk.Utilizing the fully adjusted model we added migraine status as a co-variate. The prediction for this analysis was that if migraine mediates the association between the ATP1A2 SNPs and migraine status was tested using a fully adjusted logistic regression model accounting for the same risk factors. We did not observe an association between any ATP1A2 polymorphisms and migraine as defined by this study.Lastly, the association between ATP1A2 gene demonstrated one polymorphism that was nominally associated with ischemic stroke; however this association did not persist with correction for multiple testing, nor was this association mediated by migraine status. Our study was motivated by the fact that prior studies have shown that the ATP1A2 gene is implicated in rare forms of migraine  which has features typical of migraine with aura in addition to features consistent with transient brain ischemia  which provided insufficient power to detect causal variants with small effect sizes (e.g. less than 80% power to detect genetic risk effect less than 1.23). Alternatively, one can argue that the early-onset form of ischemic stroke may be more likely to be caused by rare variants with larger effects (and higher penetrance). Such rare variants are generally not well covered on currently available genotyping platforms and, thus, would be difficult to detect using the common variants as provided by GWAS data. Additionally, in our study population, a greater percentage of subjects had migraine with aura compared to migraine without aura which differs from prior epidemiological studies  Study was used to determine if SNPs in the ATP1A2 gene confer susceptibility to stroke and if this susceptibility is mediated by migraine. Subjects included 830 cases of first ever ischemic stroke and 907 age, ethnicity and gender-matched controls that were free of ischemic stroke. Study recruitment and data collection occurred in three waves with the initial recruitment for females occurring between 1992 and 1996, and again between 2001 and 2003. Male subjects were recruited between 2003 and 2007. In all three waves, cases were hospitalized with a first cerebral infarction identified by discharge surveillance from one of 59 hospitals in the greater Baltimore-Washington area and direct referral from regional neurologists. The methods for discharge surveillance, chart abstraction, case adjudication, and assignment of probable and possible underlying causes have been previously described elsewhere  reported ever seeing spots, lines, or flashing lights around the time of their probable migraine; or (2) if they reported ever experiencing loss of vision and also reported a frequency of probable migraine with visual aura of at least twice per year. Subjects were identified as having probable migraine without visual aura if they reported no history of visual aura and reported nausea, vomiting, or sensitivity to light during a probable migraine and probable migraine frequency of at least 5 times per year. Traditional stroke risk factors and other study variables, including age, ethnicity, history of hypertension, diabetes, myocardial infarction (MI), and current smoking status (defined as use within one month prior to event for cases and a comparable reference time for controls), were also collected during a standardized interview. The abstracted hospital records of cases were reviewed and adjudicated for ischemic stroke subtype by a pair of vascular neurologists as previously described : 1:160,085,519 - 160,113,380). Our initial analyses included the 13 ATP1A2 SNPs distributed throughout the gene .Shortly after completing our analyses, additional t-test or Χ2 test using Sigma Plot v11.0 software. For primary analyses, the entire population and then an ethnicity-stratified, minimally adjusted additive logistic regression model was used taking into account age and gender to test the association between each SNP and stroke (CC = SNP + age + gender where CC is case/control status). Each SNP was then also evaluated utilizing a fully adjusted model that included vascular risk factors; hypertension, prior myocardial infarction, diabetes, oral contraceptive use, and smoking. Analyses stratified by stroke subtype  were performed including age, ethnicity, and gender as covariates. SNPs demonstrating an association with stroke were then further analyzed incorporating a variable for the presence or absence of migraine, as well as the presence or absence of migraine with aura. A fully adjusted model was also used to test the association between ATP1A2 polymorphisms and migraine irrespective of the presence or absence of stroke. All logistic regression analyses were performed using SAS v9.2 software. Odds Ratios (OR) are listed for minor alleles and a p < 0.05 was considered nominally significant. Multiple comparisons were assessed using a Bonferroni correction among the initial 13 SNPs and then among the larger panel of 134 SNPs. Power calculations  indicated that our sample size of 830 cases and 907 controls provided 80% power to detect ORs ranging 1.23–1.49 for allele frequencies ranging 5–50% at an α = 0.05.Demographic and risk factor data between cases and controls were compared with either a


Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents.Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both  The molecular chaperone heat shock protein 90 (HSP90) is of particular interest as a therapeutic target in cancer owing to its role in maintaining the correct conformation and stability of a number of key oncogenic client proteins such as receptor and non-receptor tyrosine kinases  and serine/threonine kinases , including especially those with oncogenic abnormalities and drug-resistant alleles , 2.The natural product HSP90 inhibitors radicicol, geldanamycin and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) exert their effects by specifically interacting with the N-terminal ATP binding domain and inhibiting the intrinsic ATPase activity of HSP90 which is critical for its chaperone function -5. SubseWe and others have previously demonstrated that 17-AAG induces depletion of key regulators of signal transduction in many human tumor models, including colon and breast cancer -10. The BAX+/− cells are heterozygous for the BAX gene and express the BAX protein. In contrast, HCT116 BAX−/− cells have had the remaining BAX allele knocked out by homologous recombination resulting in complete loss of BAX protein expression, as confirmed here in Figure Previous reports have suggested that the loss of BAX is sufficient to confer resistance to apoptosis including that induced by several pharmacological agents . To deteBAX−/− cells have been shown previously to be resistant to apoptosis induced by the non-steroidal anti-inflammatory drug sulindac sulfide, as determined by fluorescence microscopy of DAPI-stained cells and analysis of caspase-9 activation [BAX+/− 93.2μM ± 4.9 SEM, HCT116 BAX−/− 109.2μM ± 8.5 SEM P > 0.05).HCT116 50 sulindac sulfide (as determined by 96 hours SRB assay) and the cleavage status of poly-ADP ribose polymerase (PARP) was analyzed by immunoblotting. PARP is a highly expressed nuclear protein that is a substrate for the apoptotic protease caspase-3 and PARP cleavage is a marker of apoptosis. Intact PARP was present in vehicle-treated control HCT116 BAX+/− cells whereas only the 89kDa caspase-3 cleaved form was detected in response to sulindac sulfide, consistent with apoptosis  present after 96-hour drug exposure, and hence does not distinguish between viable and nonviable cells or between cytostasis and cell death. HCT116 cells which become detached following 17-AAG treatment have previously been demonstrated to be apoptotic using morphology, sub-G1 distribution on flow cytometry and PARP cleavage analysis . Therefos Figure . In conts Figure .−/−BAX  cells a decrease in apoptotic response may not translate into increased sensitivity overall when measured by conventional cell proliferation assay [Our results are consistent with published data demonstrating that BAX expression is required for the induction of apoptosis in response to sulindac sulfide but also indicate that in HCT116 50 values for both members of the HCT116 isogenic cancer cell line pair . Because of the possible discrepancy between measuring inhibition of cell proliferation by SRB and cell death, as seen above for sulindac sulfide, an MTT assay was also used. The MTT assay is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase [50 values determined for the isogenic pair using the SRB assay, no significant difference in the overall sensitivity to 17-AAG was observed by MTT assay between the two cell types . We also determined the sensitivity of the isogenic HCT116 cancer cell pair to the HSP90 inhibitors radicicol and CCT18159 [As seen with sulindac sulfide, 96 hour SRB cell proliferation assays with 17-AAG gave significantly similar GIrogenase ; therefoCCT18159 , which aIt was important to establish that there was comparable inhibition of HSP90 in both members of the isogenic HCT116 cancer cell pair. Numerous studies have shown that inhibition of HSP90 activity by 17-AAG causes depletion of HSP90 client proteins CRAF and CDK4 , 20. ThiBAX+/− and BAX−/− HCT116 cells CRAF was depleted and HSP72 induced in a concentration-dependent manner, the extent of which was generally similar in both cell types  and 7.6 (± 1.6 SEM) fold greater total cell death compared to HCT116 BAX knockout cells when treated with 5x and 10x GI50 17-AAG respectively . A very similar level of inhibition  was also demonstrated by the measurement of final tumor weights at the end of the experiment  and caspase-3 dependent cleaved PARP. Neither of these effects were detected in HCT116 BAX−/− cells following treatment with 17-AAG.Our analysis of the cell death response to 17-AAG revealed that HCT116 ll death . This apBAX+/− and BAX−/− cells. Therefore, it is unlikely that HSP72 was involved in the differential apoptotic response to 17-AAG observed between these two cell types.As observed consistently in the literature , 12, 20,BAX+/− and BAX−/− cells in response to 17-AAG, indicating that the difference in BAX-mediated apoptosis is not due to differential CRAF degradation. Moreover, the similar depletion of CRAF and induction of HSP72 following 17-AAG treatment provide evidence of comparable HSP90 target engagement in the isogenic HCT116 cell line pair.The HSP90 client CRAF may also exert an anti-apoptotic role by its ability to modulate the activity of pro-apoptotic BAD . PhosphoBAX knockout cells and the overall level of cell death was considerably reduced, we report here, to our knowledge for the first time, that the lower level of cell death that is observed in the BAX−/− cells occurs via a necrotic mechanism. This interesting switch in cell death mechanism was demonstrated by the presence of cellular swelling, extensive cytoplasmic vacuolization and detection of the necrotic signature of PARP cleavage in response to 17-AAG treatment. The necrotic signature includes a 62kDa fragment which has been shown to be a consequence of PARP cleavage by lysosomal proteases such as cathepsins, especially B and G [Interestingly, although we showed that apoptotic cell death in response to 17-AAG was blocked in the isogenic  B and G . These e B and G . Cleavag B and G .in vitro sensitivity determined by SRB or MTT assays was no different between BAX+/− and BAX−/− HCT116 human colon cancer cells. The same lack of differential overall effect was seen with the chemically distinct HSP90 inhibitors radicicol and CCT18159 [Despite the block in apoptosis and greatly reduced total cell death in the absence of BAX, we observed that the overall CCT18159 .BAX+/− and BAX−/− HCT116 cells when grown in vivo as solid tumor xenografts in immune-compromised mice, as measured by tumor volume or final tumor weight. This was despite a 4-fold induction of cleaved caspase-3 positive cells in BAX+/− HCT116 tumors, comparable to the increase seen in vitro, and a lack of apoptosis in BAX−/− HCT116 tumors, as also seen in cell culture. This suggests that the in vivo response of the bulk tumor is likely dictated by the drug-induced decrease in Ki67-positive proliferative cells which was similar in BAX +/− and BAX −/− HCT116 tumors. As seen before with the parental HCT116 cells [BAX+/− and BAX−/− HCT116 cells. This suggests that the antiproliferative effect is mediated by a general slowing of progression through the cell cycle in HCT116 cells.Similar to the above results measuring overall cell proliferation by SRB or MTT assays in vitro, we saw no difference in responsiveness to 17-AAG between 16 cells  17-AAG dBAX+/− and isogenic BAX−/− cells, the proportion of apoptotic cells in HCT116 BAX+/− cells after 17-AAG treatment remained relatively small, accounting for only 3.6% of the total population in vitro and 2.9% in vivo. We conclude that the modest induction of apoptotic cell death observed in this study and elsewhere [Despite the clear differences in apoptosis induced by 17-AAG between HCT116 lsewhere  in respoBAX−/− tumor xenografts. However, there is increasing evidence suggesting that acute necrosis and the subsequent antitumor immune response generated could have clinical benefit in certain cancers [It was not possible to determine if necrotic cell death was contributing to the overall response of HCT116  cancers , 38. Unl cancers , 38. The cancers . An intrXL, MCL1, BCL-W) to inhibit their interaction with BAX and BAK [During the finalization of the present paper, He et al published complementary data to ours. They also demonstrated that BAX is necessary for the apoptotic response to 17-AAG in vitro but did not characterize the effects of BAX knockout on the activation of alternative cell death mechanisms or the effects of BAX in vivo. The activation of BAX in response to 17-AAG was shown to be via p53-dependant induction of PUMA, which is upstream of BAX . Knockou and BAK . Our ownBAX gene as a result of failure of DNA mis-match repair systems and microsatellite instability [Our findings have significant clinical relevance, especially because certain cancers, including colon and gastric, are predisposed to loss of BAX expression owing to frameshift mutations in both alleles of the tability . This geBAX+/− HCT116 colon tumor xenografts by co-administration of TRAIL [Efforts to increase the anticancer effectiveness and selective tumor cell killing by HSP90 inhibitors include the use of drug combinations . We receof TRAIL .In conclusion, we demonstrate that BAX is a critical requirement for the induction of apoptosis in a human colon carcinoma model in response to 17-AAG. In the absence of BAX the overall level of cell death is reduced. Moreover, the lower level of cell death proceeds via a necrotic rather than apoptotic mechanism. Interestingly, overall bulk tumor response to 17-AAG is independent of BAX status, both in vitro and in vivo, which is likely due to the predominance of a cytostatic antiproliferative response combined with the induction of an alternative cell death mechanism when apoptosis is blocked. Our results suggest that BAX status may not alter clinical response to HSP90 inhibitors and indicate that additional drugs may be required in combination to increase tumor-selective cell killing by these clinically promising drugs, such as combination with TRAIL  or HSP70BAX+/− and HCT116 BAX−/− were a generous gift from Professor Bert Volgelstein  and have been described elsewhere [BAX+/− cells are heterozygous for the BAX gene, but express BAX protein from the remaining intact allele. HCT116 BAX−/− cells have had the remaining intact BAX allele removed by homologous recombination, and hence do not express any BAX protein. Both cell types were cultured in DMEM (Sigma) supplemented with 10% FBS, 1x nonessential amino acids and 5mM L-glutamine (Invitrogen) in a humidified atmosphere of 5% CO2, 95% air at 37°C. Cells in mid-log phase of growth were exposed to 17-AAG (Axxora) for 72 hours or the cyclooxygenase inhibitor sulindac sulphide  for 48 hours. Viability of cells was assessed by trypan blue staining and counting on a hemocytometer. BAX status was confirmed by immunoblottingThe isogenic human colon adenocarcinoma cell lines HCT116 60Co source with a source-to-flask distance of 40cm3 and a dose rate of 1.5Gy min−1Cells were exposed to radiation using a 3 cells/well) and left to attach for 36 hours. A range of concentrations of sulindac sulphide, 17-AAG, CCT18159 [Cells were seeded into 96-well microtiter plates , fixed and embedded [Detached cells were harvested 72 hours after treatment with 5×GIembedded . For ligCells were collected and lysed in cell lysis buffer as previously reported . Sample BAX+/− and HCT116 BAX−/− human colon adenocarcinoma cell lines were established bilaterally in the flanks of NCr athymic mice. When tumors were of 5 to 6mm in mean diameter, mice were treated with either vehicle  or 80 mg/kg once daily 17-AAG on days 0-4, 7-11 and 14. Tumor growth was monitored by caliper measurement. For biomarker analysis, tumor samples were taken from a subset of mice on day 5. Protein lysates were prepared as previously described [Procedures involving animals were approved by The Institute of Cancer Research's ethics committee and were consistent with published guidelines . Subcutaescribed .Formalin-fixed paraffin-embedded (FFPE) xenograft tumors were immunostained with Ki67 (MIB-1) monoclonal mouse antibody (Dako) at a 1:2000 dilution and cleaved caspase-3 (Asp175) rabbit polyclonal antibody  at a 1:200 dilution. Standard polymer and avidin–biotin–peroxidase complex techniques were used respectively for immunohistochemistry, which was carried out on 4-μm sections of FFPE tissue. Heat antigen retrieval in citrate buffer at pH 6.0 was applied. Negative controls were included in every run. Slides were counterstained with Harris hematoxylin. Slides stained for Ki67 and cleaved caspase-3 were scanned with an Aperio ScanScope XT (Aperio) using a 20x objective. Whole slide images were analyzed with the Aperio nuclear IHC algorithm (Aperio).Statistical significance was calculated using Student's two-tailed t-test.


Hyperplastic polyps (HP) and sessile serrated adenomas (SSA) share morphological similarities. In this immunohistochemical study we chose a panel of potential relevant and promising biomarkers including α-methylacyl-coenzyme A racemase , which is involved in the degradation of branched chained fatty acids derivates, and analysed a cohort of HPs and SSAs in order to identify different immunophenotypes in relation to lesion localisation.Ink4a, Ki67, α-methylacyl-coenzyme A racemase , BRAF, CK 20, MLH1 and β-catenin was performed and and immunoexpression was compared among proximal and distal HPs as well as SSAs.154 specimen were carefully selected and a micro tissue array (TMA) was constructed. Immunohistochemistry of p16Ink4a (p = 0.028) in distal HPs compared to proximal HPs. In addition AMACR overexpression was associated with increased p16Ink4a immunoexpression (p < 0.001).None of the markers revealed a differential expression among HPs and SSAs. However, the study demonstrates a significant overexpression of AMACR (p = 0.004) and p16Ink4a in HPs in relation to their localisation. Distal HPs were characterized by AMACR and p16Ink4a overexpression in contrast to proximal HPs, although morphological identically. Thus AMACR overexpression points towards a pathobiological relevance of the protein in distal HPs. In context of recently published data this suggest distal HPs as potential precursor lesions of certain adenoma subtypes. However, at this point of time this finding remains speculative and needs to be confirmed by further studies.In this study we describe differential AMACR and p16http://www.diagnosticpathology.diagnomx.eu/vs/1836116001066768The virtual slide(s) for this article can be found here:  Historically hyperplastic polyps have been considered benign and harmless lesions for several decades . MeanwhiMolecular key features of the classical adenoma-adenocarcinoma include KRAS and p53, whereas in the serrated pathway BRAF mutations appear to be an early event ,7-9. In The aim of this study was to analyse several immunohistochemical markers in order to 1) identify additional markers, that may allow a more clearly separation of HPs and SSAs and 2) to compare proximal and distal HPs and to work out potential different immunophenotypes of proximal versus distal HPs.Ink4a, which is an important factor in carcinogenesis and was shown to be linked to oncogene-induced senescence in the serrated route to colon cancer [The antibodies used in this study were the following: 1) p16n cancer ; 2) Ki67n cancer  and has n cancer ; 4) BRAFInitially 891 biopsy specimen from 2009 to 2012 representing HPs and SSAs were retrieved from the files of the Institute of Pathology, Recklinghausen. The slides of these specimen were re-evaluated by the authors KJS and ND. All biopsies, that did not allow optimal evaluation of the complete lamina propria (including the base of the crypts) were excluded from further investigation (n = 737). In the remaining 154 specimen, lesion localisation was defined as proximal, if retrieved from coecum or ascending colon. Distal localization included specimen derived from descending colon, sigmoid or rectum. Table SSA and HP areas were carefully selected and marked on the respective H&E (haematoxylin and eosin-stained) slides. TMA construction was performed as previously described . The TMAThe primary antibodies used as well as the technical details are summarized in Table AMACR: AMACR expression was scored according to the intensity of solely basal crypt cytoplasmic staining pattern. AMACR negative (0): no basal staining; AMACR positive (1+): cytoplasmic immunostaining of basal located crypt epithelial cells, independent of the intensity.P16: p16 expression was scored according to the intensity of solely basal crypt nuclear and cytoplasmic staining patterns. p16 negative: missing basal immunostaining; p16 positive: basal positive nuclear and cytoplasmic staining.Ki67: Ki67 expression was scored according to the positivity of epithelial cells in the upper third of the crypts. Positive cells were counted.Cytokeratin 20: all cases showed an upper crypt staining and scattered basal stained cells. Cases were scored according the Cytokeratin 20 staining pattern: usual pattern (staining confined to upper crypt zone) and unusual pattern .BRAF, MLH1 and β-catenin: MLH1 immunostaining revealed no morphological differences among different groups (HP versus SSA) or different localizations . A weak MLH1 staining was noticed in all specimen. BRAF immunostaining was weak to negative in all cases . β-catenin immunostaining resulted in strong cytoplasmic and nuclear staining of the complete basal crypt zone of all specimen. BRAF, MLH1 and β-catenin were excluded from further analysis.2 analysis. The relationship between categorical data (e.g. SSA versus HP) and numeric data (number of Ki67 positive cells) was determined using the Kruskal Wallis test.All immunostainings were assessed by KJS and ND in a blind trial fashion. In case of disagreement, slides were re-evaluated by both investigators until agreement was reached. All data were converted to a PC and statistically analysed using SPSS version 20 for Macintosh . Relationships between ordinal parameters were investigated using the two-tailed χTable 2 analysis showed, that distal HPs exhibited statistically significantly positive AMACR expression . In addition a comparison of all SSAs versus all HPs revealed no differences in AMACR expression (p = 0.448).P16 immunostaining was located at the basal crypt zone in the cytoplasm and nucleus of crypt cells Figure . If all Further we analysed the association of AMACR and p16 expression within the group of HPs. There was a highly significant association of AMACR overexpression with p16 positivity in HPs . The Immunohistochemical analysis included the following markers: AMACR, p16e cancer . Moreovee cancer .Likewise SSAs, HPs have been shown to harbour specific genetic alterations such as somatic BRAF mutations and CPG island methylator phenotype (CIMP) ,13. ThisFurthermore this study describes a significant overexpression of AMACR in left-sided distal HPs when compared to right-sided proximal HPs. Thus although morphological identically these lesions exhibit a varying AMACR immunophenotype. It is unlikely, that these distal HPs are at higher risk to progress to SSAs, since SSAs are typically located proximal in the right colon. Given the known function of AMACR regarding fat metabolism, it may be possible, that in the distal parts of the colon the mucosa is exposed to an increased amount of branched-chain fatty acids and fatty acid derivates.Another possible explanation would be a specific supportive role of AMACR in the development of precursor lesions in the colorectal cancer carcinogenesis. This hypothesis is supported by recent data demonstrating increased AMACR expression in high grade dysplasia compared to low grade dysplasia in conventional adenomas .Until now only few studies focussed on AMACR expression in gastrointestinal tumours. In a recent study on 1315 colorectal cancers AMACR overexpression was found to be associated with left-side tumour localisation in colorectal cancer . In detaInk4 in proximal and distal HPs. Similar to AMACR, p16Ink4 was overexpressed in distal HPs and significantly associated with AMACR overexpression. p16Ink4 is a well characterized protein with an important role in oncogene-induced cell aging [Ink4a was recently shown to function as an senescence barrier in the serrated route to colon cancer [In the present study in addition to differential expression of AMACR in HPs, we were able to detect significant differences in the expression of p16ll aging ,25. Upren cancer .Ink in distal HPs points toward a pathophysiological relevance of both these proteins. It is now necessary to identify in which processes AMACR and p16Ink in distal HPs are involved and if their dysregulation is implicated in the development of certain subgroups of adenomas.Taken together the overexpression of AMACR and p16Highly interesting in this context is a very recent study from Zhang et al. that analyses AMACR expression in normal mucosa, adenoma and colorectal carcinoma both on immunohistochemical and genetic level .Zhang et al. reported AMACR negativity in normal colonic mucosa and tubular adenoma with low grade and intermediate dysplasia as well as in poorly differentiated carcinoma. In contrast AMACR overexpression was found in villous adenoma and high and moderately differentiated colon cancer. These results are in concordance with those of Marx et al. Using Laser-capture-microdissection Zhang et al. discovered different deletions in the AMACR promotor CpG Island, depending upon the underlying tissue: In normal colonic glands and tubular adenomas with low AMACR expression they detected a somatic double-deletion at CG3 and CG10, that was absent in villous adenomas and all colon cancers with variable AMACR levels. In contrast they identified a high prevalence (89%) of deletion of CG12-16 in moderately differentiated colon cancers with strong AMACR overexpression, whereas these deletions existed in only 14% of poorly differentiated colon cancer. This deletion of CG12-16 was shown to be a constitutional allele with a frequency of 43% in the general population.Taken together, AMACR protein expression may be regulated by somatic or constitutional genetic alterations in AMACR Promoter CPG islands. Keeping the above mentioned findings in mind, it is now tempting to speculate, that distal HPs with increased AMACR expression might constitute precursor lesions of a pathway leading to the development of villous adenomas and/or subtypes of low grade colorectal cancers.This study demonstrates differential expression of AMACR and p16 in morphologically similar looking HPs in relation to their localisation. Distal located HPs exhibit AMACR and p16 overexpression compared to proximal HPs. The reasons and pathobiologically significance of this varying immunophenotype needs be elucidated in further studies.The authors declare that they have no competing interests.ND and KJS drafted the manuscript, ND analysed Immunohistochemical data and performed TMA construction and data collection. HAB and KWS participated in the design of the study, have made substantial contributions to conception and design as was data analysis and have given final approval of the version to be published. KJS has performed statistical analysis. All authors read and approved the final manuscript.


CEP290  gene in Leber congenital amaurosis (LCA) cause early onset visual loss but retained cone photoreceptors in the fovea, which is the potential therapeutic target. A cone-only mouse model carrying a Cep290 gene mutation, −/−rd16;Nrl, was engineered to mimic the human disease. In the current study, we determined the natural history of retinal structure and function in this murine model to permit design of pre-clinical proof-of-concept studies and allow progress to be made toward human therapy. Analyses of retinal structure and visual function in CEP290-LCA patients were also performed for comparison with the results in the model.Mutations in the −/−Rd16;Nrl mice were studied in the first 90 days of life with optical coherence tomography (OCT), electroretinography (ERG), retinal histopathology and immunocytochemistry. Structure and function data from a cohort of patients with CEP290-LCA  were compared with those of the model.CEP290-LCA patients retain a central island of photoreceptors with normal thickness at the fovea ; the extent of this island declined slowly with age. The −/−rd16;Nrl model also showed a relatively slow photoreceptor layer decline in thickness with ∼80% remaining at 3 months. The number of pseudorosettes also became reduced. By comparison to single mutant −/−Nrl mice, UV- and M-cone ERGs of −/−rd16;Nrl were at least 1 log unit reduced at 1 month of age and declined further over the 3 months of monitoring. Expression of GNAT2 and S-opsin also decreased with age.CEP290-LCA patients and the −/−rd16;Nrl murine model. Pre-clinical proof-of-concept studies for uniocular therapies would seem most appropriate to begin with intervention at P35–40 and re-study after one month by assaying interocular difference in the UV-cone ERG.The natural history of early loss of photoreceptor function with retained cone cell nuclei is common to both  RPE65  gene mutations can have retained photoreceptors RPE65-LCA was paved by critical proof-of-concept research with large and small animal models of the human disease Leber congenital amaurosis (LCA) is a molecularly heterogeneous group of severe early-onset human diseases CEP290 -LCA, patients over a wide age range were shown to retain a central island of dysfunctional cone photoreceptors CEP290-LCA has been documented CEP290-LCA a worthy target for a macular gene augmentation therapy. Murine models of the disease recapitulate the rapid rod cell loss of the human disease Cep290 mutation, the double mutant −/−rd16;Nrl, showed dissociation of cone structure and function (as in the human disease); this determination was made in 3–4-months-old mice only In another molecular form of LCA with severe visual consequences, CEP290-LCA? What is the natural history of the cone disease in this murine model? What would be the optimal timing of therapeutic intervention in the model? What are the expectations from such treatment and how long should we wait for a postulated effect? The present work studies the natural history of cone structure and function in the −/−rd16;Nrl mouse, confronts the complexity of the photoreceptor dysmorphogenesis well known to −/−Nrl mice; determines the window of timing for testing therapies; and establishes outcomes for determining efficacy. The results should expedite the pre-clinical steps toward eventual translation to clinical trials.Many questions still need to be answered to enable proof-of concept studies for therapy to be performed in this model of human CEP290 mutations (some phenotypic features of 14 of the patients have been published) The human studies were approved by the University of Pennsylvania Institutional Review Board . For adult subjects, written informed consent was obtained. For all children, written parental permission was obtained. Written assent was obtained from children ages 12 to 17; oral assent was obtained from children ages 7 to 11; children under the age of 7 years were enrolled with written parental permission. These consent/assent procedures were approved by the Institutional review board and the procedures adhered to the tenets of the Declaration of Helsinki. The study included 15 patients with LCA due to CEP290-LCA OCT data that were mostly (14 of 15) collected during previous studies Retinal cross-sectional imaging with 9-mm (30-degree) line scans along the horizontal meridian crossing the fovea was mainly performed with a spectral-domain OCT unit ; images from a subset of patients were acquired using a time-domain OCT unit . Two novel parameters of foveal structure were derived from −/−rd16;Nrl double mutant mice and C57BL/6 control mice  were maintained in the University of Florida Health Science Center's animal care facilities in controlled ambient illumination on a 12-hour light/12-hour dark cycle . Access to food and water was ad libitum.All experiments were performed in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and the USDA’s Animal Welfare Act and Animal Welfare Regulations, and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida (IACUC Protocol # 201304739) and the University of Pennsylvania (IACUC Protocol #804387). Data will be made freely available upon request. The −2 white) ERGs in response to a Xenon UV flash  and to a green flash produced with LEDs . The energy of the UV flashes was adjusted to evoke responses matched in waveform to those elicited by the green stimulus in WT mice. The stimuli were presented in a ganzfeld lined with aluminum foil ERGs were performed with published methods −/−rd16;Nrl and control mice were acquired with a spectral domain (SD) OCT system . Animals were anesthetized and pupils were dilated as for ERG recordings. Corneas were lubricated during the imaging session . The optic nerve head (ONH) was centered within a 1.5 mm diameter field of view under fast fundus mode, using a raster consisting of 200 parallel b-scans of 200 longitudinal reflectivity profiles (LRPs) per scan line Retinal OCT imaging was performed as previously described Post-acquisition processing of OCT data was performed with custom programs  and commercial software . Four repetitions of the high-resolution scans were averaged using the manufacturer’s software. Vertical scans with ONH at the center, and those superior and inferior to the center of the ONH were montaged by custom programs. LRPs of the merged OCT images were aligned by manually straightening the retinal pigment epithelium (RPE) reflection en face reflectance images to enable counts of pseudorosettes were generated using 350 by 350 horizontal raster scans centered within the 1.5 mm diameter field of view. Spatial distribution of pseudorosettes around the ONH was revealed by integrating the OCT backscatter intensity between two boundaries that envelope all rosettes in the OCT raster images. The light-scattering pseudorosettes appear as white spots in the en face image. Total numbers of pseudorosettes in the sampled area were counted manually using ImageJ (http://rsb.info.nih.gov/ij/). Comparisons between groups were performed using unpaired, two-tailed t-tests. Significance is defined as a p value <0.05. Density of pseudorosettes in different sectors within the central retinal region was also quantified (n = 8 eyes for two age groups).Retinal −/−rd16;Nrl mice were enucleated at postnatal day 21 (P21), P40, P60 and P80 and immediately placed in 4% paraformaldehyde for 24 hours. P60 WT mice were used as controls. Following incubation in 30% sucrose/1X PBS (2 hrs), tissue was prepared for cryoprotection and sectioning as previously described 2O. Harris’s hematoxylin solution  was applied for 6 minutes followed by a 1 minute wash in running tap water. 0.2% ammonia “bluing” solution  was applied for 45 seconds, followed by a wash in running tap water. This was followed with ten successive immersions in 95% EtOH and a counterstain in eosin Y solution  for 6 minutes. Sections were then dehydrated with 95% EtOH and 2 immersions in absolute EtOH for 5 minutes each. Following immersion in deionized H2O, AQUA-MOUNT mounting media was applied and slides were coverslipped. Sections designed for immunohistochemistry were processed as follows. After rinsing with 1X PBS, sections were incubated with 0.5% Triton X−100 for 1 hour followed by a 30 min incubation with a blocking solution of 1% bovine serum albumin (BSA). Retinal sections were then incubated overnight at 4°C with rabbit polyclonal antibodies raised against cone transducin alpha (GNAT2)  or S cone opsin  and lectin PNA conjugated to Alexa Fluor 594  diluted in 0.3% Triton X−100/1% BSA. The following day, sections were rinsed with 1X PBS and incubated for one hour at room temperature in anti-rabbit IgG secondary antibody Alexa-fluor 488  diluted in 1X PBS at 1∶500. After a wash, sections were cover slipped with SlowFade Gold antifade reagent with DAPI . Retinal sections were imaged at 5X using a fluorescent Axiophot microscope . 20X images were captured with spinning disk confocal microscopy (Nikon Eclipse TE2000 microscope equipped with Perkin Elmer Ultraview Modular Laser System and Hamamatsu O-RCA-R2 camera). Exposure settings were consistent across images at each magnification.Eyes of −/−rd16;Nrl mice each from the different age groups; comparison with any regional differences from OCT was not possible because attention to exact dorsal-ventral orientation of the tissue did not occur. ONL thickness was measured using Volocity software (Perkin Elmer) in two peripheral retinal cuts from three −/−rd16;Nrl animals. Counts were made in four equally-spaced quadrants in each far peripheral section. Peripheral ONL thickness measurements and pseudorosette counts were averaged and compared across ages using unpaired, two-tailed t-tests. Significance is defined as a p value <0.05.Pseudorosettes were counted in sections from cuts through the globe representing the far peripheral retina in three CEP290 mutations can show a wide spectrum of visual acuity results but, in general, most of the measurable values are severely reduced CEP290 alleles retains a central island of ONL in the cone-rich fovea that is within normal limits for thickness, although the inner and outer segment laminar architecture is abnormal  and this was not significantly different from 0 (p = 0.49). In other words, there was no decrease in foveal ONL thickness associated with lower acuities. This is in contrast to the data from a group of patients with other retinal degenerative diseases, collectively termed RP , suggesting that a slow degenerative process is likely ongoing in these patients, as it is in all retinal degenerations.Does the island of cone photoreceptor nuclei remain stable with age in patients . The wid−/−rd16;Nrl mice. Representative vertical OCT scans across the ONH are shown from a wild-type (WT) mouse at age 90 days (P90), and −/−rd16;Nrl mice at P31 and P83  are readily discernible in WT cross-sectional images, as is the RPE hyperreflection −/−rd16;Nrl mice present a different pattern of reflectivities with an indistinct OLM and thus no clear delineation of the laminar architecture of the IS and OS of these cone cells  Optical imaging provided the opportunity to study the retinal morphological changes at different ages in the  and P83 . The scaPhotoreceptor layer thickness across the ONH along the vertical meridian  is quantified for two age groups of mutant mice, ages 31–41 and 83–89 days . The pro−/−Rd16;Nrl show pseudorosettes, limited IS/OS material and apparent retinal thinning over this time period  for the two age groups of mutant mice, expressed as a fraction of the earlier timepoint . Histoloe period . Two tim regions .−/−rd16;Nrl mice of different ages indicate the presence of pseudorosettes at both earlier and later timepoints  and S-cone opsin decrease over time in the −/−rd16;Nrl mice. Loss of expression was evident in both central and peripheral retina , remained intact at all ages studied  at 31 days to 22 μV  by age 83 days. The natural history of function loss is consistent with an underlying exponential decay which shows linear reduction of amplitudes on log-linear plots . Four weeks thereafter, at P65, there would be functional assessment with bilateral ERGs. At this age, responses in the untreated eye would be expected to be lower but clearly distinguishable from noise levels: mean 25 μV (1.4±0.4 log μV). Interocular asymmetries (±0.3 log) −/−Nrl mice or −/−rd16;Nrl double mutants. −/−Nrl mice have been used to engineer cone-only mouse mutants with other genetic abnormalities −/−Nrl mice with subretinal AAV −/−;Cnga3−/−Nrl double mutant What are other uncertainties in this theoretical experiment? Subretinal injections and resulting retinal detachments are well-known to have negative effects on photoreceptor outer segments as a result of trauma −/−miceNrlRPE65-LCA trials −/−rd16;Nrl mouse mutant, it would seem worthy to attempt, if surgically possible.Another issue to be addressed is the dysplastic phenotype, i.e. the presence of pseudorosettes, that we quantified in this study. From the present results, the superior retina showed less numbers of pseudorosettes at the early and later ages sampled. The finding of less degeneration in the superior retina would be consistent with results of previous studies suggesting a relationship of the dysplastic phenotype and eventual degeneration in mouse mutants including −/−rd16;Nrl mice? Choice must be dictated at least by the ability of vector to transduce the target cell (photoreceptors) and mediate persistent transgene expression. If delivery of full length Cep290 is necessary (cDNA = ∼7.4 kb), vector must also accommodate a relatively large genetic payload. Lentivirus can accommodate full length Cep290 cDNA. Equine infections anemia virus (EIAV)-based lentivirus, used in concert with various photoreceptor-specific promoters, showed modest expression of reporter gene in photoreceptors of mice subretinally injected at postnatal day 5 (P5), a timepoint prior to photoreceptor differentiation ABCA4 (another relatively large cDNA) restored the disease phenotype of −/−Abca4 mice following subretinal injection at P4–P5 Which vector platform will be useful for proof-of-concept gene replacement studies in Adeno-associated virus (AAV) has proven useful for driving persistent therapeutic transgene expression in postmitotic photoreceptors CEP290-LCA was created using morpholinos to generate an altered cep290 splice product that creates a premature stop codon at amino acid 988, modeling the most common CEP290-LCA mutation rd16 mouse carries a mutation close to the C’ terminus of Cep290 (in frame deletion of amino acids 1599–1897) rd16 deletion mutation alters Cep290 folding, preventing the N’ terminus from performing an essential function.What if the entire CEP290 protein (2479 amino acids) was not required for treatment? Recently, a zebrafish model of −/−rd16;Nrl mouse.It is known that the C’ and N’ termini of CEP290 can interact with themselves and one another


HOM) leads to decreased cholinergic tone, autonomic imbalance and a phenotype resembling cardiac dysfunction. In order to further understand the molecular changes resulting from chronic long-term decrease in parasympathetic tone, we undertook a transcriptome-based, microarray-driven approach to analyze gene expression changes in ventricular tissue from VAChT KDHOM mice. We demonstrate that a decrease in cholinergic tone is associated with alterations in gene expression in mutant hearts, which might contribute to increased ROS levels observed in these cardiomyocytes. In contrast, in another model of cardiac remodeling and autonomic imbalance, induced through chronic isoproterenol treatment to increase sympathetic drive, these genes did not appear to be altered in a pattern similar to that observed in VAChT KDHOM hearts. These data suggest the importance of maintaining a fine balance between the two branches of the autonomic nervous system and the significance of absolute levels of cholinergic tone in proper cardiac function.Autonomic dysfunction is observed in many cardiovascular diseases and contributes to cardiac remodeling and heart disease. We previously reported that a decrease in the expression levels of the vesicular acetylcholine transporter (VAChT) in genetically-modified homozygous mice (VAChT KD In many cardiovascular diseases an over-activation of sympathetic tone coupled with a decrease in parasympathetic tone is observed These results are in agreement with a number of recent publications which indicate that increased levels of ACh can be protective and increase survival in experimental models of heart failure. For example, vagal stimulation improves outcome in experimental models of heart failure in rats HOM and wild-type (WT) mice. Our goal was to determine whether long-term decrease in ACh levels leads to alterations in gene expression profiles that could contribute to heart dysfunction.In order to gain insight into potential molecular changes that may occur in the heart under conditions of chronically decreased cholinergic neurotransmission, we undertook a microarray-driven, transcriptome-based analysis of hearts from VAChT KDWe demonstrate here that chronic reduction in cholinergic neurotransmission in VAChT mutant mice is associated with altered gene expression in the heart. Of the changes observed, increased levels of the enzyme purine nucleoside phosphorylase could contribute to increased reactive oxygen species (ROS) levels. We found indeed that cardiomyocytes of mutant mice present an increase in ROS. Interestingly, we demonstrate that the alterations in gene expression observed in ventricles from VAChT mutant mice are distinct from those observed in a model of cardiac dysfunction with increased sympathetic drive, indicating the importance of absolute levels of cholinergic tone in regulating cardiac function.HOM mice were generated as previously described ad libitum.VAChT KDFor the isoproterenol infusion experiments, 3-month-old male wild-type mice from the colony were treated with isoproterenol  at a dose of 60 mg/kg/day or saline for two weeks using the Alzet Osmotic Pumps  and maintained in the Animal Care Facility for an additional two weeks. All animals used in these studies were maintained at the University of Western Ontario  and experiments were performed following the guidelines and protocols approved by the University Council on Animal Care (UCAC) for animal research.http://www.lrgc.ca). RNA quality was assessed using the Agilent 2100 Bioanalyzer  and the RNA 6000 Nano kit . Single stranded complimentary DNA (cDNA) was prepared from 2.0 µg (ventricle extracts) of total RNA as per the Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual . 5.5 µg of single stranded DNA was synthesized, end labeled and hybridized, for 16 hours at 45°C, to Mouse Gene 1.0 ST arrays to probe a set of 33,000 genes. All liquid handling steps were performed by a GeneChip Fluidics Station 450 and GeneChips were scanned with the GeneChip Scanner 3000 7G  using Command Console v1.1. Probes were summarized to gene level data in Partek Genomics Suite v6.5  using the RMA algorithm http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37458).All sample labelling and GeneChip processing was performed at the London Regional Genomics Centre  according to the manufacturer’s protocol. Total RNA from whole hearts was eluted in 80 µl of Elution Solution. Quantification and quality analysis of RNA in the extracted samples was done by microfluidic analysis using the Agilent 2100 Bioanalyzer . All RNA samples used for the reverse transcription reaction had an RNA integrity number of ≥8.0. 20 µl of cDNA was synthesized from 500 ng of total RNA using the High Capacity cDNA Reverse Transcription Kit  following the manufacturer’s instructions. cDNA was subsequently subjected to qPCR on a CFX-96 Real Time System (Biorad) using the iQ SYBR GREEN SUPERMIX (Biorad). The PCRs were cycled 40 times after initial denaturation  with the following parameters: 95°C for 10 s, annealing and extension at 60°C for 30 s. For each experiment, a non-template reaction was used as a negative control. Relative quantification of gene expression was done with the DDWhole hearts were placed in modified ice-cold RIPA buffer and homogenized using a motorized Dounce homogenizer. 40–80 µg of protein lysates were separated using SDS-PAGE and transferred onto PVDF membranes. Antibodies, dilutions and their sources are as follows: anti-acetyl-CoA carboxyase , anti-ATP citrate lyase , anti-fatty acid synthase , anti-purine nucleoside phosphorylase , anti-α-tubulin , and anti-α-actinin . α-tubulin or α-actinin was used as a loading control for all samples and the protein was quantified using densitometry analysis.2+-free modified Tyrode solution until the blood was cleared. Hearts were then perfused with Tyrode solution containing 100 µM CaCl2 along with 1.4 mg/ml collagenase  and 0.04 mg/ml protease  until they were soft (∼10 min). The hearts were removed from the perfusion apparatus, minced into ∼1- mm chunks, and stirred for 4 min in Tyrode solution containing 0.1 mM CaCl2, 0.7 mg/ml collagenase, and 0.02 mg/ml protease. Cells were filtered through a 200- µm mesh to remove tissue chunks, and extracellular Ca2+ concentration was raised to 0.5 mM over 10 min through three centrifuge cycles.Adult ventricular myocytes were isolated as previously described MitoSOX Red (Invitrogen) was used to measure mitochondrial reactive oxygen species (ROS) production. Isolated cardiomyocytes were loaded with MitoSOX Red (3 µmol/l) in DMEM for 20 min at room temperature, followed by washout. Confocal images were obtained by excitation at 514 nm and measuring the emitted light at 585 nm in cardiomyocytes bathed in normal Tyrode’s solution. The confocal imaging was performed with a Zeiss LSM 510META confocal microscope .Results of qPCR, immunoblotting and MitoSOX experiments are provided as mean ± SEM. The student’s t-test was used to assess statistical differences between two experimental groups using SigmaStat software. P<0.05 was considered statistically significant.HOM mice with reduced cholinergic neurotransmission develop cardiac dysfunction HOM and age-matched WT mice. Ventricular tissue was used for the microarray analysis due to the fact that the previous experiments with these mice revealed major alterations related to cardiac dysfunction in isolated, ventricular cardiomyocytes . The gene ontology (GO) analysis did not reveal robust alterations in any specific pathway in the mutant ventricles. However, a total of 71 genes showed differential expression between VAChT KDHOM and WT mice, of which 52 genes were significantly up-regulated and 19 down-regulated.We have previously demonstrated that VAChT KDHOM mice , a result which confirms our microarray data. Further analysis of the ventricular microarray suggested a significant upregulation in the expression of two purine nucleoside phosphorylases, Pnp and Pnp2. Both Pnp and Pnp2 are involved in generating hypoxanthine, a metabolite of adenosine HOM mice, the increased expression of these enzymes was further confirmed via qPCR . Importantly, this transcriptional upregulation in Pnp/Pnp2 led to a significant increase in the protein content of Pnp/Pnp2 in ventricular tissues obtained from VAChT mutant mice .To examine the robustness of our microarray results, we performed quantitative real-time PCR (qPCR) on four randomly chosen genes which demonstrated a significant increase  and two genes which demonstrated a significant decrease  in the microarray analysis. The qPCR results confirmed that the expression of Tsen15, Gas5, Kpna2 and Socs4 were significantly increased and that of Rnase4 and Ogdhl were significantly decreased in the ventricles obtained from VAChT KDHOM mice , a resulHOM mice , a resulHOM hearts showed a significant increase in superoxide levels as compared to wild-type control cells , suggesting greater levels of ROS production in VAChT KD cardiomyocytes as compared to WT mice.The increased expression of Pnp/Pnp2 in the hearts of mutant mice might predict a greater production of ROS (as determined via superoxide formation) in these mice. Interestingly, cardiomyocytes from VAChT KD. However, assessment of protein expression levels using immunoblotting revealed no significant differences in the protein levels of these enzymes between WT and VAChT mutant mice .In addition to changes in ROS, which may contribute to phenotype in these mice, previous work has suggested a decrease in fatty acid oxidation during heart failure can also contribute to further decline in cardiac function . HoweverHOM mice were observed in a different model of cardiac dysfunction, we chronically treated wild-type mice with isoproterenol to induce cardiac remodeling. Isoproterenol-treated mice demonstrated a significant increase in heart weight to tibia length ratio as compared to saline-treated mice . Furthermore, isoproterenol-treated mice demonstrated a significant increase in the expression of two markers of cardiac stress; namely, β-myosin heavy chain  were significantly increased in the VAChT mutant mice. Both Pnp and Pnp2 are important enzymes responsible for the conversion of inosine to hypoxanthine and have also been shown to metabolize adenosine into adenine, especially under conditions of cardiac stress The present study examined whether the decrease in parasympathetic tone in VAChT KDHOM mice may lead to the increased production of hypoxanthine in mutant hearts HOM mice. Oxygen free radicals contribute to declining cardiac function during heart failure via many different mechanisms and result in damage to the myocardium The increased levels of Pnp and Pnp2 in VAChT KDHOM mice show increased levels of Pnp which has previously been shown to metabolize adenosine into adenine It is important to note that ADP levels are upregulated in heart failure suggesting that failing cardiac tissue utilizes greater amounts of ATP Significant alterations in substrate metabolism have been observed during the progression of heart failure and it is suggested that these changes contribute to cardiac remodeling and dysfunction observed during disease progression To further examine transcriptional alterations in VAChT mutant hearts that may not have been identified in the microarray, we chose genes related to the lipid biosynthetic pathway . These pathways have been previously found to be altered in heart failure HOM animals exhibit a global decrease in VAChT levels and, therefore, decreased cholinergic tone. This is significant because it has recently been proposed that cardiomyocytes possess the machinery  for de novo production of ACh It is important to note that VAChT KDTable S1HOM mutant mice.Genes which show transcriptional alterations in ventricles from VAChT KD(PDF)Click here for additional data file.