Pregnant women are an especially important population to monitor for environmental exposures given the vulnerability of the developing fetus. During pregnancy and lactation chemical body burdens may change due to the significant physiological changes that occur. Developmental exposures to some persistent organic pollutants (POPs) have been linked with adverse health outcomes.First trimester maternal and cord blood plasma concentrations of several POPs including polychlorinated biphenyls (PCBs), organochlorine pesticides (OCs), polybrominated diphenyl ethers (PBDE)s and perfluoroalkyl substances (PFASs) were measured in samples from 1983 pregnant women enrolled in the Maternal-Infant Research on Environmental Chemicals (MIREC) cohort. Predictors of exposure were also identified.In maternal plasma, there was >90 % detection for the perfluoroalkyl substances (PFASs) perfluorooctanoic acid (PFOA), perfluoroctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), and dichlorodiphenyldichloroethylene (DDE), oxychlordane and PCB 138 and 153. Cord blood plasma had much lower detection rates with low or very limited detection for most PCBs and PBDEs. The PFASs were the most frequently detected (23–64 %) chemical class in cord plasma. In a subset of 1st and 3rd trimester paired samples, PFAS concentrations were found to be strongly correlated and had ICCs ranging from 0.64 (PFOA) to 0.83 (PFHxS). The cord:maternal plasma concentration ratios ranged from 0.14 (PFOS) to 0.87 . Similar to other studies, we found parity, maternal age, income, education, smoking status, pre-pregnancy BMI and fish consumption to be significant predictors for most chemicals. Those participants who were foreign-born had significantly higher concentrations of organochlorinated pesticides and PCBs.In the MIREC study, multiple chemical contaminants were quantified in the plasma of pregnant women. In cord plasma PFOA had the highest detection rate. However, compared to other Canadian and international population studies, the MIREC participants had lower contaminant concentrations of these substances.The online version of this article (doi:10.1186/s12940-016-0143-y) contains supplementary material, which is available to authorized users. Persistent organic pollutants (POPs), including organochlorine pesticides (OCs), industrial chemicals and their by-products are chemicals that persist in the environment and tend to bioaccumulate, possess toxic properties, and resist degradation . SeveralPerfluoroalkyl substances (PFASs) are heat stable, non-flammable and able to repel water and oils. They are used in a wide variety of products including non-stick cookware, breathable all weather clothing, wiper blades, wire and cable insulation, fire retardant foams, lubricants, paper coatings, pharmaceuticals, and nail polish . They arPBDEs are a class of brominated flame retardants that are environmentally persistent, bioaccumulate and biomagnify in terrestrial food chains . StudiesPregnant women are an especially important population to include in biomonitoring and epidemiological studies given the susceptibility of the developing fetus , 23, 24 This article describes both maternal and cord plasma concentrations of several POPs including polychlorinated biphenyls (PCBs), OC pesticides, polybrominated diphenyl ethers (PBDE)s and perfluoroalkyl substances (PFASs) measured in the Canadian prospective cohort, Maternal-Infant Research on Environmental Chemicals (MIREC). We also explored maternal and infant characteristics to identify elevated exposure groups.The MIREC Study is a national-level pregnancy cohort of approximately 2000 women recruited from 10 cities across Canada and has been previously described . The MIRn = 48) PFASs were also measured in the 3rd trimester.In the MIREC study there were close to 300 chemical metabolites measured in maternal urine, blood, cord blood, meconium and breast milk which have been described elsewhere . These c13C12, PCB 153, -13C12, PCB 180-13C12, PCB 194-13C12, hexachlorobenzene-13C6, α-HCH-13C6, trans-nonachlor-13C10, oxychlordane-13C10, p,p’-DDE-13C12, Parlar 26-13C10, Parlar 50-13C10, PBDE-77-13C12) plus analogous internal standards  and proteins were denaturated with reagent grade alcohol. POP compounds were extracted with hexane from the aqueous matrix using a liquid-liquid extraction in the presence of a saturated ammonium sulfate solution for salting out. Thereafter, the extracts were evaporated to dryness before they were redissolved in 0.5 mL of hexane. These extracts were further purified on activated florisil columns and which were eluted with a mixture of dichloromethane:hexane  prior to GC-MS analysis.Two mL of plasma was enriched with labeled internal standards  coupled to an Agilent 5973 Network or 5975C mass spectrometer (MS) . The GC was fitted with an Agilent 60 m DB-XLB column  for detection by MS and with an Agilent Ultra-1 50 m   column for the Electron Capture Detector (ECD). The carrier gas was helium and the injections were 3 μL in splitless mode. The mass spectrometer was operated in selected ion monitoring (SIM) mode, using negative chemical ionization (NCI) with methane (99.97 %) as the reagent gas. The ECD serves only to quantify PCB congeners 28 and 52 when the detection limits for these compounds were not achieved with the mass detector.Concentrations were reported in units of micrograms per liter (μg/L) and the limits of detection (LOD) were between 0.005 and 0.3 μg/L for POPs. LODs were determined by first estimating concentrations of analytes yielding a signal to noise ratio of 3. Plasma samples spiked with analytes in concentrations ranging from 4 to 10 times the estimated LODs were analyzed (10 replicates) and standard deviations were multiplied by three to obtain the LODs. The method recoveries for each analyte varied from 68 to 90 %. The intra-day precision (repeatability) and the inter-day precision (reproducibility) of the method were respectively between 1.2 to 8.3 % and 2.8 to 13 %. Total cholesterol (TC), free cholesterol (FC), triglycerides (TG) and phospholipids (PL) levels were also measured in these samples by enzymatic methods combined with colorimetry (in g/L) at the laboratory of Centre Hospitalier de l’Université Laval  and used to calculate the total lipid level as 1.677*(TC-FC) + FC + TG + PL .The PFAS analysis was performed as described in the Canadian Health Measures Survey (CHMS) Cycle 1 report . The LODThe internal reference materials used to control the quality of the analyses were the certified reference material SRM-1958 (POPs and PFASs) from the National Institute of Standards and Technology , the reference materials W-09-02 and W-10-06 (POPs) from the External Quality Assessment Scheme within the Arctic Monitoring and Assessment Program (AMAP) (Centre de toxicologie du Québec (CTQ), Institut National de Santé Publique du Québec (INSPQ), Québec, Canada) and some in-house reference materials for PFASs.The overall quality and accuracy for both analytical methods was monitored by participation in the interlaboratory program AMAP External Quality Assessment Scheme (Centre de toxicologie du Québec (CTQ), Institut National de Santé Publique du Québec (INSPQ), Québec, Canada) as well as the German External Quality Assessment Scheme .parametric methods, such as maximum likelihood (ML) estimation, or nonparametric methods, such as Kaplan-Meier (KM) and the generalized Wilcoxon test [As most contaminants had values below the limit of detection, censoring methods  were impxon test . Total lDescriptive statistics including sample size, detection limit, percentage of observations below the limit of detection (LOD), minimum, median, 95th percentile, maximum, geometric mean, geometric standard error and associated 95 % confidence interval were reported for maternal and cord plasma concentrations of POPs, both adjusted and unadjusted for total lipids, with the exception of PFASs which were not lipid-adjusted. Note that the highest detection limit is presented in tables when several detection limits were noted.For censoring methods, the geometric mean and 95 % confidence interval were calculated using the ML method and compared to the empirical median from the KM approach. The Greenwood estimate of variance was used for determination of Kaplan-Meier confidence intervals. Contaminants having the % < LOD of approximately 50 % were analyzed using the censored methods. Justification for including contaminants with up to 50 % censoring is provided in Helsel .Demographic variables which were considered as potential predictors of contaminant concentrations, including parity, maternal age group , smoking status , household income in Canadian dollars , pre-pregnancy body mass index (BMI) based on self-reported pre-pregnancy weight and measured height and categorized as Underweight to Normal (BMI < 25); Overweight (25 < = BMI < 30); and Obese (BMI > =30), place of birth , fasting status , maternal education , sample collection year (2008–2011), consumption of: any kind of fish , bacon , hamburger , pork , poultry  and steak , use of non-stick cooking vessels (only for PFASs) and infant gender for cord plasma. Hypothesis tests were performed for contaminants with at least 50 % of observations above LOD  using thIn addition to performing the analysis described above, we also considered evaluation of infant cord plasma based on three demographic variables: infant gender, maternal smoking status and season of cord plasma collection. The maximum likelihood estimation (MLE) method was employed to account for left–censored repeated measures (twins and triplets), using nonlinear mixed models, analogous to Jin et. al.  and ThieFurthermore, prediction intervals were computed for geometric mean contaminant concentrations by the three demographic variables of interest, using Empirical Bayes estimates of the random effects . InterprLinear associations among all unadjusted chemicals with >70 % detection were examined using the correlation coefficient. Spearman’s rho (ρ) was calculated since the contaminant concentrations were not normally distributed. Observations below the LOD were imputed by LOD/2. A heat map was plotted to represent Spearman correlation coefficient matrix.For the 48 participants who had PFAS measurements in both the 1st and 3rd trimester we calculated intra-class correlation coefficients (ICCs). ICCs were calculated using a one-way random effects model (Proc Mixed) to estimate the between- and within-subject variability across both time points. The ICC measures the ratio of between-subject variance to total variance. It ranges from 0 to 1, with 0 meaning no within person reproducibility and 1 meaning perfect reproducibility.cm = Cuc / Cm where Cuc is the umbilical cord concentration and Cm is the maternal concentration. Ratio calculations are only presented for those chemicals that had ≥10 pairs above the LOD [Placental transfer was described by the calculation of the concentration ratios between paired cord and maternal samples for each compound on unadjusted and lipid adjusted concentrations (excluding the PFASs) using the formula R the LOD .Statistical analysis was performed using software packages SAS  Enterprise Guide 4.2 and R (R Core Development Team). For the censoring methods, functions from the R packages NADA and SURVIVAL were used for analysis. Unless otherwise indicated, a 5 % significance level (α = 0.05) was implemented throughout.We found high detection rates in maternal blood for a number of chemicals but limited detection in cord blood. The PFASs were found to be highly reproducible in a small subset of samples that had PFASs measured in the 1st and 3rd trimester. Associations were found between and number of covariates, including parity, maternal age, income, and fish consumption in line with other studies. Foreign born participants appear to have higher levels of some chemicals. The maternal:cord plasma ratios are described and all fall below 1.There were a total of 1983 pregnant women in the MIREC study . The meaTables Table In Table n = 865) while the highest median ratios (lipid adjusted) were for oxychlordane and PBDE47.Note that these ratios are only presented for pairs of maternal and cord plasma samples that are both above the LOD. The highest number of pairs was for PFOA  with a maximum of 33. In cord plasma, very few chemicals were detected with a mean and median of 2 chemicals per cord plasma sample and a maximum of 19 chemicals. Figure Demographic variables were explored for contaminants having at least 50 % of the observations above the LOD and included: β-HCH, DDE, oxychlordane, PBDE153, PBDE47, PCB118, PCB138, PCB153, PCB180, trans-nonachlor, PFHxS, PFOA, and PFOS. Sufficient detection permitted consideration of PCB 170 levels by parity and place of birth only  in foreign-born mothers compared to Canadian-born mothers for all contaminants except PBDE 47 and the PFASs (PFHxS and PFOS), where higher concentrations were seen in Canadian-born mothers.Fasting status demonstrated significant differences for DDE, oxychlordane, PCB 138 and trans-nonachlor with and without lipid adjustment. Contaminant residue levels were higher among those mothers who did not fast. For the other chemicals, no significant difference at the 5 % significance level was found with lipid adjustment.There were significant differences by education level for nearly all contaminants. Generally, mothers with higher education had higher contaminant concentrations. Sampling year showed a significant effect for nearly all POPs. Further to this, women who participated in the study in 2011 had significantly lower concentrations of nearly all POPs compared to women who participated in 2008.Fish consumers had significantly higher levels of nearly all POPs excluding the PFASs. Bacon, pork, poultry and steak consumption showed little effect on contaminant concentrations however participants who ate hamburger 2 or more times per week showed significantly lower levels of the OC pesticides β-HCH, DDE, and the PCBs . Only PFOA levels were higher for participants who cooked with non-stick cooking vessels. However no association was observed if they microwaved with non-stick cooking vessels.No significant differences were detected when grouping by infant gender, maternal smoking status or season of collection for PFOA in cord plasma , 48 (PFOS) and 64 (PFOS) percent. In a small subsample we found maternal plasma PFASs concentrations to be highly correlated and to have good reproducibility between the 1st and 3rd trimester (ICCs 064-0.83). The cord:maternal plasma concentration ratios ranged from 0.14 (PFOS) to 0.87 . Similar to other studies, we found parity, maternal age, income, education, smoking status, pre-pregnancy BMI and fish consumption to be significant predictors for most chemicals. Those participants who were foreign-born had significantly higher concentrations of organochlorinated pesticides and PCBs.In comparison to other national surveys, the participants of the MIREC cohort had lower geometric mean concentrations of DDE. MIREC’s DDE concentrations were lower than those measured in females from the population-based Canadian Health Measures Survey (CHMS)  as well r = 0.88) and PFOS (r = 0.87), with mean levels being higher in the 1st trimester. The MIREC maternal PFASs concentrations were similar to those found in females aged 20–39 in the population based study CHMS and another Canadian pregnancy cohort study based in Vancouver (CHiRP) [p < 0.0001), as seen in other studies [In the MIREC study cord plasma, the PFASs were detected more frequently than any other POPs. PFOA and PFOS had detection rates of 64 and 48 %, which is in contrast to the OC pesticides and PCBs that had only a 7–15 % detection rate. The 1st and 3rd trimester PFASs were found to be strongly correlated and moderately to strongly reproducible, with 1st trimester levels consistently higher, and ICCs ranging from 0.64 (PFOA) to 0.83 (PFHxS). Fei et al. [ (CHiRP)  both con (CHiRP) , Norway  (CHiRP) , Denmark (CHiRP)  and the  (CHiRP)  from Ham studies .PBDE 47 was the most frequently detected PBDE in the MIREC study. However, the MIREC PBDE 47 concentrations are notably lower than those found in earlier studies of pregnant women  and femaParity, maternal age, income, education, smoking status, pre-pregnancy BMI, year of collection and fish consumption were found to be significant predictors for most chemicals. Foreign Born participants had higher levels of PCBs and organochlorinated pesticides.Generally, as parity increases contaminant concentrations tended to decrease. The organochlorinated concentrations were significantly higher in older age groups, except the PFASs where both PFHxS and PFOA had lower concentrations in older mothers. Fei et al. , 51 alsoMost contaminants were found to be higher in non-smokers and former smokers than current smokers, except for PFOS. This is in contrast to the findings in other studies which have shown a positive association with smoking and POP concentrations –61 or shn = 39) had significantly lower OC concentrations  with and without lipid adjustment, suggesting that lipid adjustment alone may be insufficient to account for postprandial changes in these contaminant concentrations in pregnant women and other confounding measures must be considered.OCs are highly lipophilic and partition among various tissues depending on their lipid content. Phillips et al.  showed iForeign-born participants had significantly higher concentrations of organochlorinated pesticides and PCBs. Foreign born participants were mainly from Europe (35 %) and Asia (19 %), followed by the Caribbean, South America and the U.S , Africa (8 %), Middle East (7 %) and Oceania (1 %). A Trinational Biomonitoring study  also shoBoth fish and hamburger consumption showed significant effects on contaminant concentrations albeit in different directions. Fish consumers had significantly higher levels of most POPs (excluding the PFASs). Fish consumption is known to affect the levels of POPs –68. HambWe found that the year of the sample collection had a significant effect on POP concentrations. Lower levels were seen in 2011 compared to 2008 for nearly all POPs except PFHxS. This could reflect the fact that POP levels are decreasing over time as a result of bans. It may also reflect a difference in recruitment methods over time (e.g. older moms) and should be noted as an important consideration in cohort studies collecting data over a number of years.ow) and concentration ratios. Further exploration into the predictors of the maternal:fetal concentration ratio is warranted.The lipid adjusted cord:maternal serum concentration ratios for the OC pesticides, PBDEs and PCBs are all less than 1 and similar to a recent study from Spain  which anAn examination of the chemicals measured in the serum of the pregnant women shows that most women had multiple PCBs, PBDEs and PFASs detected in their plasma. This result is supported by the work by Woodruff et al.  who showOne limitation and possible reason for lower concentrations in pregnant women compared to non-pregnant females in the CHMS is that we did not adjust for albumin. Albumin measurements can be used as a surrogate for plasma volume expansion in pregnancy. This plasma volume expansion may dilute environmental chemical concentrations in the plasma , 72. WooMultiple chemicals were detectable in the serum of the pregnant women in our study cohort, however the concentrations were much lower than those seen in other studies. We found parity, maternal age, income, education, smoking status, pre-pregnancy BMI, fish consumption and year of collection to be significant predictors for most chemicals. Foreign-born participants had significantly higher concentrations of OCs and PCBs. In maternal plasma the highest detection rates were seen for the PFOS, PFOA, DDE, and PCB153. However in cord plasma, PFOA had the highest detection rate. In a small subset of participants we found the PFASs maternal plasma concentrations to be highly correlated and to have moderate to high reproducibility between the 1st and 3rd trimester.


Integrating HIV/AIDS and methadone maintenance treatment (MMT) services with existing health care delivery system is critical in sustaining efforts to fight HIV/AIDS in large injection-driven epidemics. However, efficiency of different integrative service models is unknown. This study assessed behavioral and health-related quality-of-life (HRQOL) outcomes of MMT in four service delivery models and explored factors associated with these outcomes of interest.A cross-sectional survey was conducted in two HIV epicenters in Vietnam: Hanoi and Nam Dinh Province. All patients in five selected MMT clinics were invited to participate, and 1016 were interviewed (80–90 % response rate).Respondents had a mean age of 35.8, taken MMT for average 16.5 months and 3.3 % on MMT for 36–60 months. The MMT integrated with rural district health center (DHC) has the highest prevalence of concurrent drug use (11.3 %). The percentage of condom use  with primary and casual partners was lowest in the MMT at urban DHCs. Patients at the rural DHC reported very high proportions of pain/discomfort (37.8 %), anxiety/depression (43.1 %), and mobility (13.3 %). In regression models, poorer HRQOL outcomes were found in MMT models in the rural areas or without general health care, and among those patients who were HIV positive, reported concurrent drug use, and had higher numbers of previous drug rehabilitation episodes. Mobility and anxiety/depression are factors that increased the likelihood of concurrent drug use among MMT patients.Outcomes of MMT were diverse across different integrative service models. Policies on rapid expansion of the MMT program in Vietnam should also emphasize on the integration with comprehensive health care services including psychological supports for patients. In Asia, since injecting illicit drugs is recognized as a major risk factor for acquiring HIV, opioid substitution treatments have been considered an important component of HIV/AIDS prevention strategies –4. MethaAlong with HIV/AIDS, opioid illicit drug use was linked to other physical and psychological problems as well as high risk of mortality , 13–18. In Vietnam, since the first MMT program was piloted in 2008, 156 MMT clinics have been established and operated with 28,000 DUs enrolling by April 2015 , 34. WitMMT + HIV voluntary testing and counseling services (VCT) at Nam Dinh PAC;MMT + rural DHC in Xuan Truong District,MMT + urban DHC in Tu Liem and Long Bien districts, andMMT + urban Ha Dong RPC.From January to August 2013, a cross-sectional survey was conducted in two HIV epicenters in Vietnam: Hanoi and Nam Dinh, with 20,717 and 3685 HIV-positive reported cases, respectively. Five clinics were purposively selected in Hanoi and Nam Dinh Provinces. These settings were selected based on some criteria: (1) providing MMT services; (2) covering a wide range of health care levels such as provincial, regional, and district levels; and (3) having adequate patients for the study. These sites were classified into four delivery models:Both rural and urban DHCs in this study provide MMT along with antiretroviral treatment (ART) and general health care. Meanwhile, the Ha Dong RPC only provides general health care. During the period of this study, those services were co-located in one site with different health workers. Eligible subjects were 18 years or above, participating or having demand to enroll into the program. Patients meeting the criteria and presenting at the clinic during the whole study period were invited; and if they agreed to participate, an informed consent was given to them for signature. A total of 1016 respondents were recruited in the study. Since patients in our sample were receiving MMT free-of-charge, they did not receive any extra incentive for answering the survey. The response rate was 80–90 % across different sites  with intimate, casual, and commercial sex partners that were self-reported by respondents. HRQOL was measured by using EuroQol - five dimensions - five levels EQ-5D-5L) instrument. The descriptive system includes five domains: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression with five levels of response: no problems, slight problems, moderate problems, severe problems, and extreme problems, giving 3125 health states with respective single indexes. To compute those indexes, the EQ-5D-5L value set of Thailand was used in the absence of such values for Vietnam . AdditioD-5L instp values of log-likelihood ratio test <0.1 included. Statistical significance was set at p value <0.05.ANOVA and chi-squared test were used to assess the difference of characteristics and behavioral and HRQOL outcomes between different MMT models. Multivariate linear regression and logistic regression were performed to determine the factors related to outcomes of interest. Predictors of outcomes included sociodemographic characteristics, history of drug use and rehabilitation, health status and HIV infection, current drug-related behaviors, and service delivery models. Backward stepwise selection strategy was used to reduce the models, with variables having The research was approved by the Scientific Committee of the Authority of HIV/AIDS Control, Ministry of Health, Vietnam.A total of 1016 patients enrolled into the study, 997 taking MMT daily for average 16.5 months; 19 other patients were not included in this analysis since they had registered but not yet taken methadone. There were 19.7 % of the patients taking MMT for over 24 months; and 3.3 % were treated for 36–60 months. The mean age of patients was 36.7 (SD = 7.6) years. Among those, only 1.28 % were female, 67.7 % lived with spouse or partners. The majority had less than high school education (55.3 %) and cult of ancestors  (88.2 %). The percentage of participants reporting HIV-positive status was 8.1 %. Compared to other sites, the rural MMT clinics integrated with Xuan Truong DHC have a large proportion of patients who were Catholic (25.8 %) and who were manual workers or farmers (35.8 %)  were employed to allow for the comparability of measurements. However, the study has limitations. First, the cross-sectional design may not allow the causal relations between MMT delivery models and the changes of HRQOL as well as risk behaviors of respondents. Second, the collected data was based on self-reported information, which was subject to desirability bias due to respondents’ recall. In this study, concurrent drug use was self-reported that might underestimate the actual prevalence among MMT patients. In addition, we only interviewed the patients who remained at the MMT clinics while missing those who dropped out of the program. In addition, we did not have information regarding MMT doses and patient responses. Finally, the generalization of study was limited due to convenience sampling technique.In conclusion, the study supports the effectiveness of MMT for drug users in Vietnam and preferable outcomes of integrating MMT with existing health services. Heterogeneities in behavioral and HRQOL outcomes across different integrative MMT service models suggest a need not only to provide HIV-related interventions but also the value of comprehensive health care including psychological supports for MMT patients especially in rural areas. Future research should examine the costs and efficiency of satellite models for dispensing MMT where distance to the clinic is a barrier to service access and utilization.


Buckwheat honey possesses super DPPH radical scavenging activity and strong ferric reducing antioxidant power. Administration of buckwheat honey for 10 weeks significantly inhibited serum lipoprotein oxidation and increased serum oxygen radical absorbance capacity. Moreover, buckwheat honey significantly inhibited aspartate aminotransferase and alanine aminotransferase activities, which are enhanced by carbon tetrachloride. Hepatic malondialdehyde decreased and hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) increased in the presence of buckwheat honey. In a comet assay, lymphocyte DNA damage induced by carbon tetrachloride was significantly inhibited by buckwheat honey. Therefore, buckwheat honey has a hepatoprotective effect and inhibits DNA damage, activities that are primarily attributable to its high antioxidant capacity.Buckwheat honey, which is widely consumed in China, has a characteristic dark color. The objective of this study was to investigate the protective effects of buckwheat honey on liver and DNA damage induced by carbon tetrachloride in mice. The results revealed that buckwheat honey had high total phenolic content, and rutin, hesperetin, and  The liver plays important roles in metabolism, secretion, excretion, and biotransformation. In China, where liver disease is common, there are approximately 130 million individuals with hepatitis B virus, which may contribute to chronic hepatitis, cirrhosis, or liver cancer. Therefore, there has been an increasing interest in the treatment and prevention of liver disease. Oxidative stress, which is involved in the pathogenesis of liver diseases, leads to hepatic damage –4.4) is one of the most widely used toxins for the experimental induction of liver damage in laboratory animals. The hepatotoxicity of CCl4 stems from reductive dehalogenation products, such as trichloromethyl (CCl3∙) and trichloromethyl peroxyl (CCl3O2∙) radicals [4-induced liver damage [4-induced liver damage [Carbon tetrachloride , which is cultivated in several Asian and European counties, is an important source of nectar and pollen for bees. Buckwheat honey has a characteristic dark color and its antioxidant activity has been studied for more than 10 years. Pasini et al. [ p-hydroxybenzoic and p-coumaric acids. Phenolic antioxidants from buckwheat honey are bioavailable and increase the antioxidant activity of plasma. Gheldof et al. [ in vitro serum lipoprotein oxidation and thiobarbituric acid reactive substances (TBARS) were not significantly affected following a single consumption of buckwheat honey. Therefore, long-term studies on oxidative stress-induced illnesses are necessary to investigate whether buckwheat honey has antioxidant-related health benefits. In this study, we assessed the antioxidant capacity of buckwheat honey in mice and evaluated its protection potential for attenuating CCl4-induced liver and DNA damage.Buckwheat  was approximately 61%. Buckwheat honey samples were stored at 4°C.Buckwheat honey was obtained from Shaanxi Bee Master Co., Ltd. . The pollen frequency .Fluorescein disodium (FL), 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-hydrazyl (DPPH), 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH), dimethyl sulfoxide (DMSO), Trolox, and silymarin were obtained from Sigma-Aldrich . Agarose was purchased from BioRad . Diagnostic kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and protein were obtained from Nanjing Jiancheng Bioengineering Institute . Lymphocyte separation medium was purchased from Tianjin Hao Yang Biological Manufacture Co., Ltd. CCl2CO3. An hour later, absorbance was measured at 760 nm. TPC was expressed as the gallic acid equivalents per gram of buckwheat honey (mg GA/g).We used a modified Folin-Ciocalteu method to determine TPC in buckwheat honey . Brieflyμm). The mobile phase adopted was methanol (A) and 0.15% aqueous acetic acid solution (B) (v/v) using a linear gradient elution of 5–15% A at 0–10 min, 15–35% A at 10–15 min, 35–55% A at 15–20 min, 55–65% A at 20–25 min, 65–80% A at 25–30 min, and 80% A at 30–35 min. The injected volume was 5 μL, and flow rate was 1.0 mL/min. The column was operated at 30°C. The diode-array detector was performed at 360 nm.The contents of individual phenols in buckwheat honey were estimated by HPLC-DAD analysis as proposed by Liang et al. . An AgilDPPH radical scavenging activity of buckwheat honey was assessed according to the method proposed by Wang et al. . BrieflyμL of buckwheat honey (0.2 g/mL) was mixed with 50 μL of 1 mM iron vitriol and 20 μL of 1 mM ferrozine. The total volume was adjusted to 1 mL with methanol and incubated at room temperature for 10 min. The absorbance of the ferrozine-Fe2+ complex was measured at 562 nm. Ferrous ion-chelating activity was expressed as Na2EDTA equivalents per gram of buckwheat honey (mg Na2EDTA/g).The ferrous ion-chelating activity of buckwheat honey was measured by the method reported by Singh and Rajini with some modifications . In this3; 25 mL of 0.3 M acetate buffer, pH 3.6), mixed well and incubated at 37°C for 4 min. Absorbance was measured at 593 nm. FRAP was expressed as Trolox equivalents per gram of buckwheat honey (mg Trolox/g).FRAP of buckwheat honey was assessed by the method reported by Benzie and Strain . Buckwhe(1) Animals. Male Kunming mice (18–22 g) were obtained from Xi'an Jiaotong University and housed in cages with six mice per cage. The animal ethical approval communication number is SCXK 2012-003. The animal experiments followed the guidelines and regulations of the State Committee of Science and Technology of the People's Republic of China.4-treated mice were administered distilled water via gavage at 0.22 mL/10 g BW, twice daily for 10 weeks. According to the doses of honey and silymarin reported by Cheng et al. [After acclimatization to laboratory conditions for 7 d, the mice were randomly divided into four groups (12 mice/group). Control mice and CClg et al. , the mic4-induced liver damage, all mice were continuously intragastrically administered distilled water, buckwheat honey, and silymarin for the next week. Two hours following the last administration, all mice (except control mice) were administered a CCl4/peanut oil mixture ; control mice received only peanut oil. Subsequently, the animals were fasted for 16 h and bled by cardiac puncture. Half of the blood samples were transferred to anticoagulant tubes for separating lymphocytes, and the other half were transferred to ordinary centrifuge tubes for serum collection.To investigate the protective effects of buckwheat honey on CCl(2) Serum Lipoprotein Oxidation. Serum lipoprotein oxidation was assessed by the method reported by Regnström et al. [2HPO4, 1.8 Mm KH2PO4, 27 mM KCl, and 138 mM NaCl) to 0.5%. Copper ions at 12 μmol/L were added to the diluted serum samples. Oxidation kinetics was determined at 234 nm every 20 minutes at 37°C. Diluted serum samples without copper were used as blanks. The area under the oxidation curve (AUC) was plotted and the percentage inhibition of serum lipoprotein oxidation was calculated according to the following equation:control is the area under the oxidation curve for the control group serum samples and AUChoney is the area under the oxidation curve for the honey group serum samples.m et al. . Serum s(3) ORAC Assay. The ORAC assay was performed in 96-well plates and measured in a multifunctional plate reader  [μL of 78 nM FL and 50 μL of 1% serum were transferred to 96-well plates. The blank consisted of 50 μL of phosphate buffer instead of serum. The mixture was preincubated at 37°C for 30 min before rapidly adding 25 μL of 221 mM AAPH solution. The plate was automatically shaken prior to each reading. Fluorescence was measured every 5 minutes. The assay was performed in triplicate, and the results were expressed as inhibition of the area under the curve (AUC) according to the following equation: control = AUCcontrol − AUCblank; net AUChoney = AUChoney − AUCblank.(4) Comet Assay. The comet assay is the preferred technique for detecting DNA damage in single cells. In this study, lymphocytes isolated from control mice, CCl4-treated mice, and honey mice were analyzed by the comet assay to assess the protective effects of buckwheat honey on CCl4-induced DNA damage. Lymphocytes from silymarin mice were set as the positive reference. Following the methods proposed by Singh et al. [5/mL. After fixing the lymphocytes on slides, the slides were immersed in lysis buffer ) for 2 h. Subsequently, the slides were immersed in electrophoresis buffer  for DNA unwinding. After 30 min, electrophoresis was run at 25 V (300 mA) for 20 min in the dark. All slides were treated with ethidium bromide and observed under a fluorescence microscope . The results were scored and analyzed using an automated analysis system of the Comet Assay Software Project (CASP). At least 50 cells were scored from each slide. The degree of DNA damage was scored by determining the percentage of DNA in the tail (tail DNA %) and olive tail moment (OTM), defined as the fraction of tail DNA multiplied by the distance between the means of the head and tail:h et al.  with sli(5) Assessment of Liver Function. Serum was obtained following the centrifugation of blood samples at room temperature for 20 min at 3,000 rpm. Serum ALT and AST values were measured using commercially available diagnostic kits.(6) Determination of MDA, SOD, and GSH-Px Activities. After the animals were sacrificed, livers were immediately excised. With the exception of a portion of the left lobe to be used for histopathological examination, the livers were homogenized in phosphate buffer  and centrifuged at 2,500 rpm for 20 min at 4°C. The MDA content, SOD, and GSH-Px activities along with protein levels in the supernatant were estimated according to commercially available diagnostic kits.(7) Histopathological Examinations. A left lobe portion of the liver was incubated for 24 h in 10% neutral formalin solution. Based on standard procedures, we obtained 5 μm sections for histopathological studies using hematoxylin and eosin (H&E) staining.P value < 0.05).We analyzed the data in triplicate using SAS software version 8.1 . Tukey's posttest was used to assess statistical significance ( p-coumaric acid (12.52 mg/kg).To study the antioxidant activity, the TPC and individual phenolic compounds of buckwheat honey were determined, and the results are shown in  in vitro are shown in 2EDTA/g. The FRAP assay is often used to determine the antioxidant properties of foods based on their electron-donating capacity [The results of antioxidant activities of buckwheat honeycapacity . As showcapacity .The administration of buckwheat honey  for 10 weeks resulted in the inhibition of serum lipoprotein oxidation. Buckwheat honey inhibited serum lipoprotein oxidation by 65.71% . Serum O4-induced damage is shown in 4-treated group, a significant increase in the tail length of comet was observed. However, the lymphocyte from the mice administered honey and silymarin showed a similar decrease in the tail length of comet. As shown in Figures 4-treated group were 30.91% and 53.03%, respectively, whereas the mean tail DNA and OTM in the control group were 11.76% and 5.21%, respectively. Therefore, significant increases in the mean tail DNA and OTM of lymphocytes were associated with CCl4 exposure (P < 0.05). Interestingly, pretreatment with buckwheat honey  for 11 weeks decreased lymphocyte damage significantly (P < 0.05). Silymarin, as a positive reference, had super protective effect on DNA damage induced by CCl4.The protective effect of buckwheat honey on CCl4-treated group, serum ALT and AST activities were 170.68 and 55.01 U/L, which were 15x and 1.52x higher than those of the control group, respectively (P < 0.05). In the honey and silymarin groups, serum ALT and AST activities were 11.12 and 27.77 U/L and 12.43 and 25.96 U/L, respectively. There were no significant differences in the hepatic enzyme activities between the control, honey, and silymarin groups. Therefore, buckwheat honey treatment  for 11 weeks inhibited an increase in serum ALT and AST activity.Serum ALT and AST activities were determined in this study and the results are shown in 4-treated group relative to the control mice. Pretreatment with buckwheat honey  and silymarin  for 11 weeks significantly decreased hepatic MDA levels in the CCl4-treated mice (P < 0.05) .Hepatic MDA levels and GSH-Px and SOD activities were monitored in this study and the results are shown in  < 0.05) . The act4-treated mice revealed extensive liver damage characterized by severe hepatocellular hydropic degeneration and necrosis around the central vein, dilated sinusoidal spaces, inflammatory cell infiltration, and ballooning degeneration  [Phenolic compounds are present in plants and food products, including honey. Phenolic compounds possess powerful antioxidant capacity by acting as hydrogen donors to free radicals and as electron donors to metal ions . AccordiEDTA/kg) . The metEDTA/kg) . Flavono2+-induced oxidation of serum lipoprotein was determined. This method provides an indication of diene formation in lipoprotein fatty acids when exposed to Cu2+. Diene formation is assessed by measuring changes in absorbance at 234 nm. High absorbance values correspond to diene formation as a result of serum lipoprotein oxidation, and low absorbance values correspond to inhibition of serum lipoprotein oxidation and, consequently, to high antioxidant activity [2+-induced oxidation of serum lipoprotein and increased the antioxidant capacity of mice. Serum ORAC is another method for measuring serum antioxidant capacity. This method, which incorporates FL as the fluorescent probe, is commonly used in biological samples and foods. The ORAC method is based on the inhibition of peroxyl-radical-induced oxidation initiated by the thermal decomposition of AAPH [To investigate whether buckwheat honey could increase the antioxidant capacity of the mice, Cuactivity . The admactivity . Antioxiactivity , 29. The of AAPH . FL bloc of AAPH . Accordi4 is a classic method used to induce oxidation and liver damage [4 include highly reactive free radicals, which initiate the chain reaction of lipid peroxidation, thereby affecting polyunsaturated fatty acids and phospholipids [4-induced liver damage. Therefore, serum ALT and AST activities in the CCl4-treated group were significantly higher than those of the control group. Interestingly, pretreatment with buckwheat honey inhibited the increase of serum ALT and AST activities induced by CCl4. With increasing serum ALT and AST activities as a result of CCl4-induced damage, lipid peroxidation products  accumulate in hepatic cells. Therefore, hepatic MDA levels were monitored in this study. As shown in 4-treated group relative to the control mice. Pretreatment with buckwheat honey and silymarin significantly decreased hepatic MDA levels in the CCl4-treated mice. To delineate the mechanisms underlying the protective effects of buckwheat honey, the activities of hepatic antioxidant enzymes  were determined. In this study, the activities of GSH-Px and SOD in the CCl4-treated mice decreased significantly compared to those in the control mice . Meanwhile, histological observations further affirmed that administration with buckwheat honey significantly attenuates CCl4-induced liver damage.Intraperitoneal administration of CClr damage , 8. Metaholipids . Lipid p Murraya koenigii L., yam peel, and bee pollen have been studied for their hepatoprotective effects [4-induced DNA damage can be inhibited by the administration of buckwheat honey. Therefore, buckwheat honey, which has a free radical scavenging ability, reduces lipid peroxidation and increases antioxidant capacity, thereby attenuating CCl4-induced liver damage in mice.In the present study, eight phenolic compounds were identified in buckwheat honey, of which rutin and hesperetin are the majority. Rutin has been verified to exert renal-protective effects by inhibiting ROS and antioxidant activities . Hespere effects , 9, 10.  effects  were con4-induced DNA damage. Lymphocyte DNA damage induced by carbon tetrachloride was assessed by alkaline single cell gel electrophoresis, that is, the comet assay. This method is a rapid and sensitive technique for measuring and analyzing DNA damage in individual cells [4 are shown in Figures 4 caused a significant rise in DNA damage in peripheral lymphocytes. This result could be attributed to the in vivo action of carbon tetrachloride metabolites, that is, trichloromethyl and/or trichloromethyl peroxy radicals, which pass from the liver to the circulatory system, or to the appearance of stimulated nuclear cells in circulation. According to Kujawska et al., oxidative damage to DNA increases by 33% in mice following intraperitoneal administration of CCl4 [4 did not increase DNA damage in rat blood leukocytes [4 poisoning. In this study, a marked rise in tail DNA and OTM was obtained in mice treated with CCl4. More importantly, pretreatment with antioxidants can inhibit the increase of mean tail DNA and OTM. This protection should be attributed to the phenolic compound existing in buckwheat honey, which could scavenge the free radicals produced in the metabolism of CCl4 and thus attenuate the damage.Additionally, another aim of this study was to assess whether buckwheat honey can attenuate CClal cells . The moral cells . In the  of CCl4 . On the ukocytes , which m4-induced liver and DNA damage in mice. Buckwheat honey demonstrated high TPC, free radical scavenging capability, and ferric-reducing antioxidant properties. Pretreatment with buckwheat honey for 10 weeks in mice significantly increased serum antioxidant activities. Therefore, buckwheat honey exhibited hepatoprotective effects in mice. Additionally, pretreatment with buckwheat honey protected DNA from CCl4-induced oxidation.In conclusion, the results of this study demonstrated that buckwheat honey increased the antioxidant capacity and attenuated CCl


We expanded upon our previous experience using involved-field fractionated radiotherapy (IFRT) as an alternative to whole brain radiotherapy or stereotactic radiosurgery for patients with surgically resected brain metastases (BM).All patients with single BM who underwent surgical resection followed by IFRT at our institution from 2006 to 2013 were evaluated. Local recurrence (LR)-free survival, distant failure (DF)-free survival, and overall survival (OS) were determined. Analyses were performed associating clinical variables with LR and DF. Salvage approaches and toxicity of treatment for each patient were also assessed.Median follow-up was 19.1 months. Fifty-six patients were treated with a median dose of 40.05 Gy/15 fractions with IFRT to the resection cavity. LR-free survival was 91.4%, DF-free survival was 68.4%, and OS was 77.7% at 12 months. No variables were associated with increased LR; however, melanoma histopathology and infratentorial location were associated with DF on multivariate analysis. LRs were salvaged in 5/8 patients, and DFs were salvaged in 24/29 patients. Two patients developed radionecrosis.Adjuvant IFRT is feasible and safe for well-selected patients with surgically resected single BM. Acceptable rates of local control and salvage of distal intracranial recurrences continue to be achieved with continued follow-up. Brain metastases (BM) are the most common central nervous system (CNS) tumors in adults , 2. ProgTherapeutic options for BM include corticosteroids, surgery, WBRT, and stereotactic radiosurgery (SRS). Two phase three randomized trials demonstrated decreased LR and improved OS and quality of life for patients with single BM treated with surgery combined with WBRT compared to WBRT alone , 11. TheThese outcomes justify use of WBRT following surgical resection of single BM; however, these benefits come at the cost of late neurocognitive side effects. Due to improvements in systemic therapy, patients can live to experience the late effects of WBRT, prompting an interest in improving control of intracranial disease while minimizing late neurocognitive effects –15. TreaSince recurrences often occur at the surgical cavity following metastasectomy, another approach is to treat the surgical cavity with adjuvant radiotherapy to decrease LR while sparing the majority of the brain. Focal approaches including SRS have been used to improve control of intracranial disease, while decreasing late neurocognitive toxicity following surgical resection of BM –22. AnotFrom 2006 to 2013, 56 consecutive patients with single BM underwent gadolinium-enhanced pre-operative magnetic resonance imaging (MRI) or computed tomography (CT) of the brain if MRI was contraindicated followed by surgical removal of BM followed by IFRT. Patients who were selected for this treatment approach had good performance status with either controlled primary or extracranial metastatic disease. In addition to good performance status and well-controlled metastatic disease, patients were selected for this treatment approach to improve pre-treatment neurological symptoms, relieve mass effect, or provide histopathological confirmation of metastatic disease. Patients underwent post-operative MRI within 48 h to confirm extent of surgery. This study was approved by our institutional review board.Prior to radiotherapy, CT simulation was performed. Patients were immobilized using a thermoplastic head mask, and 1.25 mm axial CT images were obtained using a LightSpeed RT 16 CT Simulator . The images were transferred to the Eclipse treatment planning system  and registered with the pre-operative T1-weighted pre and post-contrast and T2-weighted MRI to aid in target delineation. In patients who were unable to undergo MRI examination due to contraindications , the CT simulation images were registered with the pre-operative pre- and post-contrast CT of the brain.The surgical resection cavity was identified on planning CT, and was defined as the clinical target volume (CTV). Substantial collapse of the surgical cavity identified on post-operative MRI or CT brain was treated with SRS. The planning target volume (PTV) was defined as a 10 mm isometric margin around the CTV . RadiothPatients had clinical and radiologic follow-up with neurological examination and MRI scans 1 month after completion of IFRT and every 3 months thereafter.All patients with single BM who underwent surgery followed by IFRT from 2006 to 2013 were identified by reviewing the clinical charts of patients treated in our department, with patient characteristics collected and recorded in a database.en bloc versus piecemeal surgical resection, lesion location , and tumor depth . Statistical analysis was performed using SPSS software . The Kaplan–Meier method was used to calculate the rate of OS, LR-free survival, and DF-free survival. Univariate and multivariate analyses of significance were performed using Cox regression analysis. A p-value of 0.05 or less was considered statistically significant.Clinical endpoints include LR, defined as the presence of new enhancement along the resection cavity identified on serial MRI, and DF, defined as enhancement consistent with BM and/or leptomeningeal spread outside the IFRT volume. Time to local or distant progression was defined from the date of surgery to the first MRI demonstrating recurrence. OS was calculated from date of BM diagnosis until date of death. Parameters associated with LR and/or DF were assessed including active extracranial disease, histopathology of primary tumor , tumor maximal diameter (<3 cm or ≥3 cm), Fifty-six patients with single BM who underwent surgery followed by IFRT were included in this study. Detailed characteristics of the study group are shown in Table en bloc and 29 (52%) with piecemeal resection. The most common tumor histopathology was NSCLC in 20 (36%), breast cancer in 15 (27%), and melanoma in 11 patients (20%). Thirty-eight (68%) had supratentorial lesions, while 18 (32%) had infratentorial lesions. Seventeen (30%) had superficial lesions (involvement/abutment of dura) and 39 (70%) had deep lesions (surrounded by parenchyma). Mean tumor size  was 3.4 cm , with 35/56 having tumors ≥3 cm in size.All 56 patients underwent gross total resection (GTR), with 27 (48%) having their BM removed 3 . Patients were treated with radiotherapy to a median dose of 40.05 Gy/15 daily fractions over 3 weeks at 2.67 Gy per fraction  . Surgical cavity volume was contoured as a CTV using CT simulation image registered with post-operative MRI. The median volume of the post-operative cavity in all patients was 15 cm07 days. Thirty-nine of 56 (70%) received systemic anti-neoplastic therapy, with 22 starting treatment prior to their course of IFRT and 17 starting systemic therapy following completion of IFRT. None received systemic anti-neoplastic therapy concurrently with IFRT.p = 0.52), tumor maximal diameter ≥3 cm (p = 0.78), piecemeal resection of tumor (p = 0.41), and superficial tumor depth with involvement of dura mater (p = 0.73).Median follow-up was 19.1 months , with local control achieved in 48 (85.7%). Actuarial rates of LR-free survival at 12 and 24 months were 91.4 and 85.1%, respectively, 95% CI [not reached]  = 2.46, 95% CI (1.06–5.74)] and infratentorial location  were associated with increased incidence of DF. Melanoma histopathology  and infratentorial location  continued to be associated with DF on multivariate analyses. Thirteen of 29 (45%) who developed DF had infratentorial tumors, and 5/10 who presented with leptomeningeal spread had infratentorial tumors.Distant failure was observed in 29 (51.8%), and actuarial rates of DF-free survival at 12 and 24 months were 68.4 and 45.7%, respectively, 95% CI 10.1–31.1)  developed leptomeningeal spread. Five had single BM located in the posterior fossa, and three had BM superficial in location. Histopathology was not significantly associated with increased risk of leptomeningeal spread.Patterns of intracranial failure were recorded, with 3 (5%) having LR, 21 (38%) having DF, and 5 (9%) having both LR and DF.At most recent follow-up, 69.1% of patients were alive. Survival was not significantly associated with RPA class. Median survival was 19.1 months . Twelve and 24-month OS rates were 77.7 and 61.1%, respectively (figure not shown). Of all patients who died, three died of neurologic death from progression of intracranial disease, seven died of death due to progression of extracranial disease, one died of natural cause unrelated to malignancy, and six died of unknown cause.LR were salvaged in four patients – two with surgery and SRS, one with SRS, and one with surgery and WBRT. DF were salvaged in 24 patients – 16 patients with SRS, three with WBRT, two with a second course of IFRT, two with chemotherapy and IFRT, and one with chemotherapy alone.Two presented with ≥grade 2 adverse effects of treatment with CNS necrosis [CTCAE Version 4.0]. One patient with diagnosis of BRAF mutated metastatic melanoma, developed symptomatic radiation effect with headaches 2 years following her course of IFRT to 40.05 Gy/15 fractions as seen on brain MRI, with extensive confluent FLAIR and T2-weighted hyperintensity, but no evidence of disease progression in the area of prior radiation treatment. Her symptoms are presently being managed with steroids, at a dose of 40 mg daily. Another patient with NSCLC, initially treated with IFRT to 40.05 Gy/15 fractions, developed histologically confirmed radionecrosis that was initially thought to be a LR on surgical resection following salvage treatment of distant BM with SRS. Late neurocognitive effects of treatment were not systematically recorded in the medical record.We report the only series using IFRT following surgical resection of single BM in 56 patients with a median follow-up of 19.1 months, expanded from our previous series of 33 patients . A 12-mo3, patients were treated with a multi-dose SRS regimen of 27 Gy in three fractions, resulting in a 9% risk of radionecrosis or adverse radiation effect. In another series with 65 patients and a median surgical cavity volume of 8.1 cm3, treatment with multi-dose stereotactic radiotherapy (SRT) resulted in a local control rate of 70% at 24 months . Patients presenting with larger surgical cavity volumes will, therefore, be selected for treatment with IFRT instead of multi-dose SRT not only to avoid increased risk of toxicity from treating larger volume surgical cavities but also due to physician preference.With comparable rates of local control, SRS and brachytherapy have advantages compared to IFRT with respect to treatment duration. In our series, IFRT was delivered predominantly over 3 weeks, in contrast to SRS and brachytherapy, which can be delivered in a single day. The disadvantages of brachytherapy include risk of infection, limited availability, and operator experience . The pri4 months , 33. Theen bloc) did not adversely affect local control in our series on univariate analysis , the lack of information regarding targetable mutations or rearrangements to further characterize this selected population, and the inherent biases associated with a retrospective study. Yet, our motivation for our treatment approach is to decrease the risk of neurocognitive toxicity as one randomized trial demonstrated measurable decline at 4 months with SRS followed by WBRT compared to SRS alone using the Hopkins Verbal Learning Test-Revised , 15. DueIn conclusion, we demonstrate feasibility and safety of using IFRT following surgical resection of single BM for patients with good performance status and well-controlled primary disease. IFRT can be considered as an alternative therapeutic approach to SRS for patients with resected metastases and surgical resection cavities ≥3cm, and in lieu of WBRT for these select patients with close follow-up.All authors  have contributed to the conception, draft/revision, approval of final draft, and are accountable for all aspects of work in ensuring that questions related to accuracy or integrity of work are appropriately investigated and resolved.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.


Association between Cyclin D1 (CCND1) polymorphism and cervical cancer risk are conflicting with published articles. We performed a meta-analysis to investigate the association between CCND1 G870A polymorphism and cervical cancer risk.PubMed, Embase and CNKI data were researched to conduct a meta-analysis on the associations between CCND1 G870A polymorphism and cervical cancer risk. Ten published case–control studies including 2,864 patients with cervical cancer and 3,898 controls were collected in this meta-analysis. Odds ratio (OR) with 95% confidence interval (CI) were applied to assess the relationship; meta-regression, sensitivity analysis and cumulative analysis were also conducted to guarantee the strength of results.P = 0.76 I2 = 74.5%), codominant model , dominant model  and recessive model . Similarly, in the stratified analysis by ethnicity, study design and genotyping type, no significant association detected in all genetic models either.Overall, no significant association between CCND1 G870A polymorphism and cervical cancer risk were found in allele contrast  for this article can be found here:  Cervical cancer is one of the most common malignant diseases; it is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females with approximately 529,800 new cases and 275,100 deaths among females in 2008 worldwide [Cervical cancer is a multifactorial and multistep disease. Innate immune deficiency, environmental aggravation, and genetic mutation have been considered as important pathopoiesis factors. New molecular epidemical studies revealed that human papillomavirus (HPV), particularly HPV 16 and 18 infections may be the common and important factor contributing to the development of cervical cancer, which is known to cause approximately 70% of cervical cancers ,3.Abnormal cell proliferation is an important step in cancer development. Cyclins are a family of proteins that control cell progression through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes . Cyclin Recently, molecular epidemiologic studies have directed considerable attention toward the association between genetic mutation and cancer susceptibility. Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation among people. The change in a nucleotide may alter gene functions and may influence protein expression, which could inhibit or promote cell proliferation and increase susceptibility to cancer development. In 1995, Betticher et al. [Catarino et al. published the first research about the association between CCND1 G870A polymorphism and cervical cancer risk in 2005 . Since tThree electronic databases, including Pubmed, Embase, and China National Knowledge Infrastructure (CNKI), were searched with the terms “CCND1”, “Cyclin D1”, “cervical cancer”, and “polymorphism” for studies published from 1995 to June 2014. Additional studies were identified through manual searches of the references of original studies or review articles on the topic of interest. Only studies published in English or Chinese were included. All studies selected for our meta-analysis met the following criteria: (a) observational (case–control or prospective) studies on the association between CCND1 G870A polymorphism and cervical cancer risk, (b) sufficient published data for estimating an odds ratio (OR) and 95% confidence interval (CI), and (c) the largest or most recent samples were selected when they overlapped with other studies.Data from all included studies were extracted independently by two investigators (Niu and Zheng). The extracted data included the name of the first author, publication date, country of origin, sources of controls, racial descent of the study population , number of different genotypes, and Hardy-Weinberg equilibrium (HWE) in controls.P value of less than 0.05 was considered significant disequilibrium. Stratified analyses were used to evaluate ethnicity, study design, and genotyping type technique. Heterogeneity was explored with the use of a chi-squared test, and the quantity of heterogeneity was measured by the I2 statistic. I2 values of 25%, 50%, and 75% represent low, moderate, and high heterogeneity, respectively. The OR of each model was estimated by using the fixed-effects model (Mantel–Haenszel method) when I2 ≤ 50%; otherwise, the random-effects model (DerSimonian and Laird method) was used. Meta-regression analyses were performed to assess potential covariates that can predict intertribal heterogeneity. Publication bias was assessed on the basis of modified Egger’s bias test and Begg’s funnel plot. Statistical analysis was performed by using STATA versions 11.0 . Two-sided P value (P < 0.05) was considered statistically significant.The strength of the association between CCND1 G870A polymorphism and cervical cancer was evaluated by ORs with 95% CI, comprised with allele contrast (A vs. G), codominant model , dominant model (GA + AA vs. GG), and recessive model (AA vs. GG + GA). The HWE of the control group was assessed, and a n = 12), Embase (n = 9), and CNKI (n = 4) ; codominant model ; dominant model . In the succeeding analysis of HWE studies, similar associations were found. In the stratified analyses of ethnicity, study design, and genotype, no significant association was found between CCND1 G870A polymorphism and cervical risk in almost all models. Heterogeneity was observed in all five genotype models. Meta-regression and stratified analyses were conducted, but no critical factors were found to explain heterogeneity in the subgroup of ethnicity, design, and genotype either .Overall, no significant association was found between CCND1 G870A polymorphism and cervical cancer risk in this meta-analysis Table . Values Studies included in the meta-analysis were deleted one by one to reflect the influence of an individual dataset on the pooled ORs. The results were consistent Figure .Figure 3P = 0.643 for A vs. G; GA vs. GG: P = 0.427; AA vs. GG: P = 0.558; (GA + AA) vs. GG: P = 0.423; AA vs. (GG + GA): P = 0.884).Funnel plot and Egger’s test were used to estimate publication bias. The shapes of the funnel plots for all genetic models did not reveal any asymmetrical evidence. Figure Cervical cancer is one of the most dangerous causes of health deficiency and death worldwide. Epidemiological studies have revealed that HPV infection is an important factor contributing to cervical cancer. Furthermore, gene mutation and abnormal tumor cell proliferation may serve critical functions in cancer development.The CCND1 gene is located at chromosome 11q13 and encodes a key cell cycle regulatory protein with 295 amino acids. CCND1 is an activator of CDK, which can regulate cell division by accelerating/decelerating the transition from G1 to S phase. Over-expression of CCND1 could result in the aberrant proliferation of DNA damage and the accumulation of genetic errors. Some SNPs of CCND1 have been reported. The G-to-A mutation is found in a well-known locus in the boundary of exon 4 and intron 4. This mutation does not alter any amino acid in the protein sequence. CCND1 G870A mutation results in an alternatively spliced transcript with a longer half-life than the CCND1 G allele and thus enables abnormal cells to pass through the G1-S checkpoint easily. Previous studies have demonstrated that CCND1 G870A polymorphism is significantly associated with the development of various cancers, such as breast cancer, prostate cancer, colorectal cancer, and other cancer types -25.In 2005, Catarino et al. firstly find that CCND1 GG polymorphism is associated with a 3.45-fold higher risk for the development of cervical cancer in a Portuguese population  . The samIn 2011, Ni et al. reported the first meta-analysis on the association between CCND1 G870A polymorphism and cervical cancer risk . Only fiOur analysis has some limitations. First, some heterogeneity still exists despite stratified analysis, and the meta-regression could not be explained successfully. Second, some environmental factors, such as smoking, drinking, and HPV infection, were not included in our meta-analysis because of data deficiency. Third, the sample size was relatively small, which might have yielded false results and inaccurate conclusions.In conclusion, this meta-analysis indicated that CCND1 G870A polymorphism may not be a risk factor of cervical cancer development. Large and well-designed case–control studies are needed to validate our findings further.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


Arabidopsis thaliana wild type semisolid callus cultures were integrated into fully automated plant cultivation containers and exposed to spaceflight conditions within the Simbox hardware on board of the spacecraft Shenzhou 8. The related ground experiment was conducted under similar conditions. The use of an in-flight centrifuge provided a 1 g gravitational field in space. The cells were metabolically quenched after 5 days via RNAlater injection. The impact on the Arabidopsis transcriptome was investigated by means of whole-genome gene expression analysis. The results show a major impact of nonmicrogravity related spaceflight conditions. Genes that were significantly altered in transcript abundance are mainly involved in protein phosphorylation and MAPK cascade-related signaling processes, as well as in the cellular defense and stress responses. In contrast to short-term effects of microgravity , this mission identified only minor changes after 5 days of microgravity. These concerned genes coding for proteins involved in the plastid-associated translation machinery, mitochondrial electron transport, and energy production.The Simbox mission was the first joint space project between Germany and China in November 2011. Eleven-day-old  Ceratopteris richardii, [Arabidopsis thaliana) exhibit gravisensitivity [Gravitation biology is a field of research which has made considerable progress within the last years, involving prokaryotes, fungi, plants, and animals. Plants are especially interesting, because, as sessile organisms, they possess high versatility in responding to environmental challenges and abiotic as well as biotic ones. In order to investigate responses to altered gravitation, a large range of methods is available that allows for modification of the Earth's gravitational field. These involve centrifugation (hypergravity), clinorotation, magnetic levitation, and random positioning (simulated microgravity), or parabolic flights of planes and sounding rockets, as well as satellites and spacecrafts (deliver microgravity). Experiments with plants show that not only tissues and organelles , 2 but achardii, , 9) and chardii, –12 or hositivity –16. Expesitivity –20. They Arabidopsis  cell suspension cultures, the response to short-term microgravity was investigated intensely in our group by means of parabolic flights [Arabidopsis), Fabaceae, and Poaceae has recently been published [ Arabidopsis callus cultures and 18-day-old seedlings after a nearly 13-day-space mission. Furthermore, the set of altered genes detected in whole seedlings was different from that in callus cultures [ Arabidopsis seedlings [For flights . A combiublished , 22–31. ublished , for exaublished , 24 detecultures . Therebycultures , 16. Witeedlings  observed Arabidopsis were exposed to a 17-day spaceflight on board of the Chinese spacecraft Shenzhou 8. Due to reduced viability after longer periods of exposure within the flight hardware, the callus cultures were metabolically quenched after 5 days in space. Results of a whole-genome microarray screening  revealed major differences between both 1 g controls but a minor impact of microgravity.In this study, we report on results of a spaceflight experiment. This experiment was part of the Simbox (Science in Microgravity Box) mission, a joint project between the space agencies from Germany (Deutsches Zentrum für Luft- und Raumfahrt e. V.) and China (China Manned Space Engineering) in November 2011. As one out of 17 biological experiments, semisolid callus cultures of2, and O2 content as well as activation of the pump system were recorded and transmitted.The Simbox was a modification of the Biobox-6 , 34 whic Arabidopsis thaliana (cv. Columbia Col-0) plants were used for callus formation on 1.2a media [Sterile cuttings (about 50 mm long) of stems of wild type2a media  containi2a media . New medlater . This reagent is also used to stabilize nucleic acids. Twenty mL of this fixative was filled into the fixative/waste unit attached to the bottom of the EC. Between handover and integration into the Simbox flight/ground incubator, the ECs were stored at nominal laboratory temperature conditions (22–24°C). The Simbox incubator was unpowered for about 3 hours during transport to the spacecraft. During this time, the lowest temperature was 21°C . Experiment zero time (EZT) was set when the spacecraft reached the orbit. At EZT, the centrifuge was activated to run with 74.40 rpm. Within the spacecraft, the oxygen partial pressure ranged from 18.04 to 27.32 kPa, and the carbon dioxide partial pressure was between −0.03 and 0.46 kPa. Radiation measurements yielded a total dose of 5.93 to 8.1 mSv and an equivalent dose of 0.37 to 0.51 mSv/d near the Simbox incubator . The pump system was activated after 5 days in space and injected the fixative solution from the fixative/waste unit into the CC's of FMs. This yielded a final RNAlater concentration of about 90% (v/v) after mixing. Temperature in CCs was kept at a nominal range of 22 to 24°C before, during, and after fixation  with a Long March 2F rocket from the cosmodrome in JSLC. The precise mission timings including sample fixation time points are illustrated in fixation . After 1Immediately after the launch, the laboratory equipment and cell cultures were brought back to the PITC by Chinese scientists. The ground experiment started with a one-day delay on November 2, 2011 . The EUEDuring the Simbox mission, the samples were exposed to different experimental conditions. In the experiment in orbit, FM 16002 was attached to the static platform of the Simbox incubator and experienced 5 days of microgravity . FM 16001 was centrifuged, resulting in a 1 g control . In the ground experiment, the same experimental design was used. FM 16003 was fixed to the static platform . In summary, we obtained one biological sample per CC, resulting in two replicates for each FM (front and rear CC) and for each experimental condition, respectively.http://www.affymetrix.com/support/technical/manuals.affx). The Affymetrix protocol EukGE-WS2_V4 was used for washing and staining procedures.Total RNA was extracted using the RNeasy Plus kit  according to the manufacturer's instructions. Quantity and quality controls were performed and samples were processed using the MessageAmp II-Biotin Enhanced, Single Round aRNA Amplification Kit  as described earlier , 37. Frahttp://www.ebi.ac.uk/arrayexpress, [2 expression values [t-test (P ≤ 0.1) with combined false discovery rate (FDR) correction to identify significantly altered transcripts (P < 0.1) between the sample groups FS and FC, FS, and GS, and FC and GS, respectively. Differentially expressed genes were determined by fold change (fc) calculation of log2 transformed expression data. Thereby, the threshold was set at −1 ≥ log2 (fold change) ≥ 1 for at least 2-fold altered transcripts [Expression data were calculated from raw values of the detected signal intensity of hybridization events of all spotted probe sets and saved as  .CEL data files. Microarray data are available in the ArrayExpress database  under aexpress, . Normalin values –42. Hiern values  in ordernscripts , 41, 44.nscripts ) for enrnscripts  within M Arabidopsis semisolid callus cultures after 5 days in space. Due to the availability of an in-flight centrifuge, it was possible to compare expression data with (a) real microgravity samples  and with (b) those from the ground controls (which should deliver effects of nonmicrogravity related spaceflight conditions). This was achieved with high-density oligonucleotide arrays.The aim of this experiment was to characterize the transcriptome ofThe hardware was thoroughly tested in order to retain viability of the callus cultures for as long as possible. These tests were focused on the biocompatibility of the used materials, gas-exchange properties of membranes, and viability of the cell cultures under the cultivation conditions within the EC. We also recorded the oxygen content within the CC . As thisn = 2) showed a high degree of similarity  and comparative analysis showed a relatively low response of semisolid callus cultures (P < 0.1) at the gene expression level, although 298 genes were at least 2-fold differentially expressed (275 up- and 23 downregulated) within flight space (FS) samples. In contrast, nonmicrogravity related spaceflight conditions interfered with gene expression, considerably. Eight hundred ninety-seven genes were significantly and differentially expressed  when 1 g ground and μg exposed flight samples were compared. Among them, 463 were upregulated and 434 genes were downregulated within FS (P < 0.1) differentially altered genes  were identical in both comparisons  with clear bands representing the 28S and 18S rRNA. Whole-genome microarray screening was performed for each sample. Due to the limited amount of total RNA, the confirmation of expression data by quantitative real-time PCR was not possible. The data analysis revealed experiment-specific properties of biological replicates which were visualized by hierarchical clustering on the basis of the calculation of the Pearson Correlation coefficient . In thismilarity . This familarity . The Peay n = 2, . Statistcultures . Interesithin FS . Compariparisons .μg exposure, we compared data generated out of the sample groups flight space (FS) and in-flight centrifugation (FC). Two hundred seventy-five genes were at least 2-fold differentially upregulated and 23 downregulated (P < 0.1) alterations at the expression level after 5 days in space. By means of a Gene Ontology [For detection of gene expression changes due to egulated . The appOntology  based GeP < 0.1) altered within spaceflight samples (FS and FC) compared to the 1 g ground control and were identical between FS and FC compared to GS. This overlap yielded 573 significantly altered (P < 0.1) DEGs (P < 0.1) impact on general stress-responsive (gene set number 20) and defense-related genes (3), especially those involved in the response to oxidative stress and respiratory burst responses (21). These are peroxidases 21, 4, 52, and 25 , catalase 3 (AT1G20620), and receptor-like kinases . The latter can be induced upon contact with the bacterial protein flagellin which is an important elicitor of the plant defense response. These kinases are also important members of the MAP kinase signaling cascade. Furthermore, general metabolic processes (gene set number 7), protein targeting (13), and rRNA processing (21) were overrepresented due to nonmicrogravity related conditions in space.One aim of this investigation was to separate responses to microgravity from those of nonmicrogravity related spaceflight conditions. Until today, only marginal data exist about these effects on plants in space. Thus, we screened for genes that were significantly (.1) DEGs . The GSE.1) DEGs . Further.1) DEGs . Additio Arabidopsis semisolid callus cultures show alterations in differential gene expression in response to microgravity. However, the influence of the spaceflight environment, in addition to microgravity, is significant.The expression data of Arabidopsis plants exhibited only 27 transcripts which were at least 2-fold altered at their expression level [Comparison between microgravity and 1 g space controls revealed about 298 differentially (but not significantly) expressed genes. This number is low in comparison to short-term exposures to microgravity within a range of minutes . This fon level  could al Arabidopsis in vitro callus cultures under simulated microgravity conditions  [ Ceratopteris richardii [Heat shock proteins (HSPs) dominate the group of transcripts which are reduced in amount (not shown). These proteins are involved in many forms of stress response. They enable the folding and membrane translocation of proteins and are thought to reconstitute the tertiary structure of proteins affected by stress events. This way they can increase the stress tolerance. A decreased expression (our study) should thus indicate a lower number of proteins affected in their structure and was also reported for1 Tesla)  as well ichardii . There aichardii , 21, 24.A group of plant genes which are always affected by altered gravity are those involved in cell wall modification , 49–51. μg exposed and 1 g space with 1 g ground samples. This resulted in a considerable number of identical genes altered in mRNA abundance (573 genes)  affect different biological processes  . We thusg et al.  reportedrocesses . Overreprocesses . These crocesses . These pbundance 3 genes  were identified . In cons et al. , in this Arabidopsis wild type semisolid callus cultures were investigated after a 5-day spaceflight and compared to on-board and ground controls. Faced with limited HW capacities (only 3 EUEs) and small amounts of biological material (n = 2 for each sample group), high-density oligonucleotide arrays were used to screen for changes at the gene expression level. For future investigations, it would thus be desirable to have flight repetitions and an adequate amount of samples for additional analysis . Unexpectedly, the response of callus cultures to long-term microgravity was less prominent compared to nonmicrogravity related spaceflight conditions. The latter, including space radiation, induced differential and significant expression changes of transcripts that are involved in the stress-induced antioxidant system, signalling chains, and defense-/resistance-related genes. These findings clearly highlight that the use of an in-flight reference centrifuge (1 g in-flight control) should be mandatory during space flight missions.In this study, gene expression changes withinSupplementary material S1 shows the accelerometer-recorded gravity level profile (x-/y-/z-axis) as measured during the Simbox mission from EZT (Experiment Zero Time) until landing on November 17, 2011 (data: China Manned Space Engineering). Data was provided by Chinese authorities to DLR/Astrium.Supplementary material S2 shows the formaldehyde agarose gel analysis of extracted RNA from flight (FC) and ground samples (GS), front and rear CC (Culture Chamber).


Alström syndrome is characterized by increased risk of cardiovascular disease from childhood.To explore the association between risk factors for cardiovascular disease, aortic pulse wave velocity, and vascular events in Alström syndrome.Cross-sectional analyses with 5-year follow-up.The UK NHS nationally commissioned specialist clinics for Alström syndrome.Thirty-one Alström patients undertook vascular risk assessment, cardiac studies, and aortic pulse wave velocity measurement. Subsequent clinical outcomes were recorded.Insulin resistance was treated with lifestyle intervention and metformin, and diabetes with the addition of glitazones, glucagon-like peptide 1 agonists, and/or insulin. Thyroid and T deficiencies were corrected. Dyslipidemia was treated with statins and nicotinic acid derivatives. Cardiomyopathy was treated with standard therapy as required.The associations of age, gender, and risk factors for cardiovascular disease with aortic pulse wave velocity were assessed and correlated with the effects of reduction in left ventricular function. Vascular events were monitored for 5 years.P = .001) and inversely with left ventricular ejection fraction (P = .036). Five of the cohort with cardiovascular events had higher aortic pulse wave velocity (P = .0247), and all had long duration of diabetes.Aortic pulse wave velocity was positively associated with the duration of diabetes (Duration of diabetes predicted aortic pulse wave velocity in Alström syndrome, which in turn predicted cardiovascular events. This offers hope of secondary prevention because type 2 diabetes can be delayed or reversed by lifestyle interventions. ALMS1, has been identified  autosomal recessive condition characterized by childhood-onset cone rod retinal dystrophy, neuronal hearing loss, obesity, and insulin resistance 27. Subtleentified , 10, andentified –13. Alstntified –, 15. Twontified –, 17. A fntified –. Coronarntified –. GreaterElevated central pulse pressure measured as aortic pulse wave velocity (PWV) is indicative of large artery stiffening. Meta-analysis of outcome studies has confirmed that there is a 2-fold increase in all-cause and cardiovascular mortality in those with the highest tertile compared with the lowest . This efThe present cross-sectional study has included a larger number and wider age range of Alström syndrome subjects. This report also explores the relationships between risk factors for cardiovascular disease including duration of diabetes as well as left ventricular function and aortic PWV in the syndrome. Cardiovascular events occurring during the poststudy period have been recorded.All subjects gave written informed consent and were included in the DAS study (Defining the phenotype in Alström Syndrome)  funded by a UK Science Lottery Grant in association with Alström UK.Twenty-two Alström subjects attended UK National Health Service (NHS) nationally commissioned specialist clinics for Alström syndrome at Torbay Hospital between 2005 and 2010. The other nine were reviewed and tested by the Torbay team at Alström clinics in Leeds, Nottingham, Cardiff, and Truro. Eleven of the 31 subjects were female, and ages ranged from 11–44 years at the time of the baseline aortic PWV measurement.All subjects in the study had cone rod retinal dystrophy, acanthosis nigricans, obesity, and insulin resistance. Dyslipidemia characteristic of insulin resistance was present at the time of aortic PWV measurement in 28 of the 31 subjects, serum high-density lipoprotein (HDL) cholesterol was < 1.1 mmol/L, and/or serum triglycerides were > 2.0 mmol/L. All but one patient had preserved intelligence. Patients were screened annually to identify secondary hypothyroidism and, in males, T deficiency. Hormone deficiencies were corrected for at least 1 year before the vascular investigations . Long-teALMS1  . Left vewww.scmr.org protocols). CMR was used to monitor myocardial fibrosis and to detect the occurrence of changes consistent with infarction. It was not considered ethically acceptable to perform CMR routinely on younger deaf blind Alström subjects, and therefore left ventricular ejection fraction was measured from echocardiography as above in all subjects.Cardiac magnetic resonance imaging (CMR) was performed on 17 of the adult subjects . Left and right ventricular volumes, mitral annular plane systolic excursion, ejection fraction, and left ventricular mass index were acquired in line with standard CMR protocols. Left atrial volumes were calculated using the biplane area-length method. The four-chamber and short axis steady-state free precession cine images  at the mid left ventricle (papillary muscle) were used for myocardial deformation analysis. Late gadolinium enhancement imaging was performed 7–10 minutes after 0.15 mmol/kg of gadolinium contrast bolus . All patients consented to receive iv gadolinium 0.15 mmol/kg . Samples were processed and deep-frozen for later assay within 2 hours of venesection. Serum lipids, renal function, blood glucose (fluoride), and HbA1c levels were measured by standard laboratory procedures. Insulin resistance was measured as postprandial serum C-peptide/glucose ratio or fasting serum insulin level , 28.Clinically significant events were confirmed by angina-like symptoms leading to confirmatory coronary angiography (two cases), autopsy after cardiac-related death (two cases), duplex arterial scans and serial CMR (one case), and carotid plaque (one case). The other patients had no symptoms suggestive of angina or evidence of myocardial infarction on serial electrocardiography or CMR. Patients over 20 years of age designated as free from cardiovascular disease had normal lower limb arterial duplex scans and an absence of carotid artery plaques.Supplemental Table 1). All statistics were carried out in the software program “R” (http://www.r-project.org/).Associations between aortic PWV and the following variables was sought: age, gender, body mass index (BMI), duration of diabetes mellitus, HbA1c, log serum creatinine, mean systemic arterial blood pressure, serum triglycerides, serum cholesterol, serum HDL cholesterol, non-HDL cholesterol, and left ventricular ejection fraction. The variables were then interacted against aortic PWV in a general linear model, with log transformation where appropriate to achieve conformity of Gaussian distribution. Insignificant terms were removed in a stepwise fashion using an F test , and left ventricular ejection fraction was negatively and independently associated (P = .036) (R2 = 0.61). Demographic, genetic, anthropometric, and clinical data are shown in ed P = .06 , all of whom had long-standing type 2 diabetes, developed clinically significant cardiovascular disease. Logistic regression in the general linear model was also applied to the occurrence of cardiovascular disease, which showed that only aortic PWV was independently associated with cardiovascular disease  was treated with hemodialysis for end-stage renal failure. Another subject (ID no. 31) received renal and whole pancreas transplantation 10 and 9 years before the baseline study and remains well 5 years later. One subject (ID no. 22) with long-standing type 2 diabetes had concurrent severe cirrhosis of the liver due to nonalcoholic liver disease in association with the syndrome. He had maintained low systemic arterial blood pressure, serum cholesterol < 3 mmol/L, intermediate aortic PWV 8.2 m/s, but relative freedom from cardiovascular disease verified by normal coronary arteriogram. He also had normal carotid ultrasound scans and minor disease on lower limb arterial duplex scans (15% stenosis of the right iliac artery).The very early occurrence of severe insulin resistance, dyslipidemia, hypertension, and progression to type 2 diabetes implies high risk for cardiovascular disease. The current case series demonstrates that aortic PWV, a highly significant determinant of future vascular events, is increased in Alström patients in direct relation to the duration of diabetes, and that this relationship is independent of age. It is well established that aortic PWV is strongly linked with advancing age in the general population , 29, 30.Four other subjects with severe insulin resistance but without progression to diabetes  have also remained free from overt cardiovascular disease . A detaiOne limitation of this study is the small number involved because of the rarity of Alström syndrome. The widespread organ fibrosis in Alström syndrome may have effects on both aortic PWV and cardiovascular disease not present in nonsyndromic metabolic syndrome and type 2 diabetes. With larger numbers of associations between aortic PWV and cardiovascular disease with renal failure, serial HbA1c levels, and non-HDL cholesterol levels, gender and age might have emerged. Its strengths lie in the coherence of the patient group, consistency of methodology, and completeness of follow-up. The insulin resistance of Alström syndrome does mirror the propensity for development of diabetes, arterial stiffness, and cardiovascular disease that is seen in the wider population , 38. As 39–In summary, we have studied vascular risk factors associated with aortic PWV and cardiovascular disease in a well-characterized group of subjects with a rare genetic insulin-resistant syndrome. The data are consistent with the hypothesis that arterial stiffness and cardiovascular disease are predominantly determined by the duration of diabetes in older subjects with the syndrome. Because the onset of diabetes can be delayed in Alström syndrome by lifestyle modification, there is a clear indication that every Alström family should be afforded the most appropriate access to nutritional advice and empowerment to exercise despite the dual sensory loss. It is also recommended that dyslipidemia in the syndrome should be treated from adolescence. Whether these tentative conclusions can be generalized to the growing number of obese insulin-resistant adolescents at high risk of type 2 diabetes is not known but is worthy of study.