Objective. As propolis is a highly valued bee product, we aimed to verify the quality of aged propolis, investigating their phenolic and flavonoid composition, levels of toxic metals, radical scavenging and antimicrobial activities. Material and Methods. Samples of fresh and aged propolis of six different beekeepers, from the same geographical location, were investigated in terms of their phenolic and flavonoid composition and levels of Pb, Cd, and Cr, as well as radical scavenging and antimicrobial activities. Results. The two groups of propolis had similar qualitative composition by HPLC-PDA and ESI(-)-MS. Fresh propolis and aged propolis show no differences when average values of extraction yield, flavonoids, EC50, or MIC were compared and both types of propolis showed good antimicrobial activity at low concentrations. Only levels of phenolic compounds were higher in fresh propolis. Conclusion. The propolis samples considered in this study, aged or fresh, had similar qualitative composition, although they were collected in different periods. Samples only differed in their levels of total phenolic content. Moreover, aged propolis conserves significant radical scavenging and antimicrobial properties. We suggest that aged propolis should not be discarded but explored for alternative applications. Propolis is a resinous hive substance containing beeswax, plant exudates, and salivary secretions from bees. Extracts of propolis are used as phytochemical ingredient in functional foods at levels that may confer health benefits , 2. The Flavonoids and phenolic acids or their esters often form up to 50% of all propolis constituents . SeveralBrazilian propolis is appreciated worldwide. From 2010 to 2012 the price of a kilogram of raw Brazilian propolis increased more than 50% in the international market. According to data from the Japan Trade Organization, 92% of raw propolis consumed in Japan is from Brazil. Propolis which remains long periods in hives (aged propolis) has a dry consistency and is usually discarded by beekeepers. In this study we aimed to verify the quality of aged propolis. Therefore, samples of fresh and aged propolis from six different beekeepers but from the same geographical location were investigated in terms of their phenolic and flavonoid composition and levels of metals, as well as radical scavenging and antimicrobial activities.Apis mellifera) were collected from hives of six different beekeepers from Prudentópolis, Paraná State, Brazil. On April 7th, 2009, the propolis samples considered as aged (M) (because they had remained in hives for at least 180 days) were collected (1st collection). By the occasion of this collection, wooden collectors were placed in hives to promote the production of fresh propolis (F) (Propolis samples from honeybees (olis (F) . The setGround propolis (5 g) from both the 1st and 2nd collection was extracted with 50 mL of a 70% v/v hydroalcoholic solution. After stirring in shaker at room temperature for 100 h, these solutions were filtered. The ethanolic solvent was removed under reduced pressure yielding ethanolic extracts of propolis identified as aged propolis M (1st collection) and fresh propolis F (2nd collection).μBondapak C-18 analytical column  was used. A binary mobile phase of acetonitrile and 0.5% of aqueous formic acid was employed at an elution rate of 1 mL min−1. Linear gradient was performed starting with 30% of acetonitrile to 100% for 30 min. All propolis extract solutions were prepared in acetonitrile at 1000 μg mL−1 and 5 μL and were injected through a loop system.The chromatographic profiles of the different propolis extracts were determined by HPLC (Waters 600) with photodiode array detector operating in a 1D detection mode at 292 nm. A thermostatized (30 ± 1°C) -MS to obtain representative fingerprints and compare their qualitative profiles − ions of p-coumaric acid, 4-hydroxy-3-prenylcinnamic acid, and betuletol, respectively. Ions of m/z 299 were detected at two different retention times (17.2 and 21.6) and were attributed to kaempferide and 3,5-diprenyl-4-hydroxycinnamic acid (artepillin C). Similarly, ions of m/z 301 (at 12.8 min and 25.5 min) were attributed to dihydrokaempferide and E/Z communic acid, respectively.ESI(-) samples . LC-MS a(Paraná) , 16. DicWhen extraction yield, total phenolic and flavonoid levels for aged and fresh propolis were compared in pairs for the same beekeeper, a trend toward higher amounts of extraction yield and total phenolic acids in fresh propolis is noticed . Convers50 value) than the extracts obtained from aged propolis from the same beekeeper, except for extracts 6M and 6F.The DPPH assay was performed for aged and fresh propolis obtained from different beekeepers in order to verify the effect that long periods in hive have on propolis radical scavenging activity . All ext in vitro antibacterial activity was assessed through the values of MIC and MBC of six aged (M) and six fresh (F) propolis samples against strains of Gram-positive and Gram-negative bacteria.  S. aureus and M. luteus, at low concentrations showing a good antimicrobial activity. These propolis extracts were however not effective against Gram-negative bacteria, with MIC values higher than 4000 μg mL−1 (data not shown).The−1, fresh propolis: 12 ± 6 mg g−1), and (iii) EC50 in DPPH test , considering the six beekeepers altogether. The same result was obtained when MIC and MBC mean values for each bacterium were statistically compared: (i) Staphylococcus aureus ; (ii) Enterococcus faecalis ; Micrococcus luteus . For Gram-negative strains, the antibacterial activity was weak for both fresh and aged propolis. Extracts of propolis are extensively described as being effective against Gram-positive bacteria but very weak against Gram-negative ones [−1) than the aged samples (107 ± 10 mg g−1).To statistically compare aged and fresh propolis samples, they were considered as two experimental groups and the quantitative data obtained were analyzed by a one-way ANOVA. No statistical difference at the 95% confidence level was observed between average values of (i) extraction yield ; (ii) flavonoids levels , popularly known as “alecrim-do-campo,” which is largely distributed in South America from southeastern Brazil to Argentina and Uruguay. Propolis from B. dracunculifolia is rich in phenolic acids, particularly prenylated derivatives of p-coumaric acid, as shown in our data.The qualitative chemical profile observed by HPLC-PDA, ESI(-)-MS and LC-MS for all samples was alike despite aged and fresh propolis having been collected in different periods and from different beekeepers Figures  and 3. C sources , 26, 27. sources  reported Staphylococcus aureus and Escherichia coli [ Candida albicans [Among tropical countries, Brazil has the widest chemical diversity of propolis types; however variations in qualitative chemical composition of Brazilian propolis due to seasonal effect are not always observed. In this regard, Simões-Ambrosio et al.  evaluatehia coli  and agaialbicans , 18 and albicans  of propo Candida albicans [The amounts of phenolic and flavonoid constituents vary widely according to propolis types and seasonal factors , 32, 33.albicans . Recentlalbicans .μg/mL). In this regard, Banskota et al. [The potent antioxidant activity of Brazilian propolis observed in our study was also shown by Guimarães et al.  who testa et al.  reported S. aureus. Nevertheless, when these activities are compared considering all the fresh samples and all the aged samples, no statistical differences between average values were observed. Others studies also reported that propolis is active mainly against Gram-positive bacteria but shows a limited activity against Gram-negative bacteria [Concerning the antimicrobial activity, when fresh and aged propolis are pair-compared for the same beekeeper slightly better radical scavenging and antimicrobial activities were found for fresh propolis which showed a good antimicrobial activity mainly againstbacteria –43. Thesbacteria . Though,bacteria , 45–47. bacteria  showed t−1, respectively [−1 [Finally, it is also important to highlight that toxic elements such as Pb, Cd, and Cr were not detected in fresh or aged propolis extracts. The presence of these metals in propolis has been attributed to environmental contamination of air, plants, soil, or waters around the hives due to anthropic activities . Pb, Cd,ectively  and for vely [−1 . We can This study showed that despite being collected in different periods, propolis samples had a very similar qualitative composition. Samples differed from each other only in relation to their levels of total phenolic content (93.67–149.30) mg/g. Even though aged propolis generally has a different appearance and a drier consistency and, according to beekeepers, this type of propolis is depreciated, data collected in this study indicates that aged propolis still has significant radical scavenging and good antimicrobial activities. These results therefore suggest that aged propolis should not be discarded. Toxic metals  were not detected in propolis extracts.


Transient elastography (TE) is widely used for non-invasive assessment of liver fibrosis in HIV-HCV co-infected patients. TE, however, cannot determine liver morphology. Acoustic radiation force impulse (ARFI) imaging is a novel procedure enabling assessment of liver fibrosis during a conventional ultrasonographic examination. This study evaluated the correlation between liver fibrosis measurements by TE and ARFI.Each of 46 HIV-HCV patients underwent both ARFI and TE within 6 months. Patients were evaluated by the “equivalent METAVIR” scoring system, using previously established cut-off values. Agreements between the ARFI and TE scores were estimated by Kappa coefficients, with Kappa values ≥0.40, ≥0.60, and ≥0.80 defined as moderate, good and very good agreement, respectively.ARFI and TE yielded "Equivalent Metavir" fibrosis scores of F1 in 26 and 31 patients, respectively; F2 in nine and seven, respectively; F3 in three and two, respectively; and F4 in eight and six, respectively. The two methods showed very good agreement in predicting overall stages [Kappa = 0.82] and for F ≥3 [Kappa = 0.80] and moderate agreement in predicting significant fibrosis F ≥2 [Kappa = 0.50]. Morphologic ultrasound analysis concomitant to ARFI detected two hepatocarcinomas.ARFI showed promising results in the non-invasive assessment of liver fibrosis in HIV-HCV patients, with liver fibrosis staging similar to that of TE. Moreover, ARFI can assess morphology and fibrosis during the same session. Since the introduction of highly active antiretroviral therapy (HAART), chronic liver disease has emerged as one of the leading causes of morbidity and mortality in HIV-HCV co-infected patients,2. ThereEvaluation of liver fibrosis is considered essential to the management of patients with chronic hepatitis C, providing prognostic information and assisting in making therapeutic decisions-7. To daTE (FibroScan®) is currently the most extensively used elastography technique in clinical practice-19. ManyTE, however, has several drawbacks, including the requirement for specific equipment that performs only elastography, its ability to assess only the right liver, and its inability to visually determine the site of measurement. Moreover, ascites and obesity are limiting factors. ARFI imaging has many advantages over TE. The technology used for ARFI has been incorporated into a conventional ultrasound system, allowing ultrasound analysis of liver morphology at the same time. In addition, ARFI allows elastography to be performed with a flexible metering box at variable depths, enabling the exact localization of the measurement site, and the examination can also be performed in patients with ascites. ARFI measurements have been found to correlate well with the stage of hepatic fibrosis-36. HoweThe study was approved by the institutional review board of our University hospital. Between July 2009 and May 2012, HIV-HCV co-infected patients regularly followed up at the HIV out-patient Clinic of the Department of Internal Medicine and Infectious Disease of our hospital were prospectively enrolled. All patients provided written informed consent before inclusion.3), plasma HCV- RNA concentrations (IU/ml), and HCV genotype. Men and women were considered heavy drinkers if they consumed >3 and >2 glasses of alcohol per day, respectively. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), and bilirubin concentrations were also measured.The demographic, clinical and biological information of all patients was obtained from their medical records. Factors included patient age, body mass index (BMI), alcohol consumption, medications, specific antiretroviral drugs, plasma HIV-RNA levels (IU/ml), complete blood counts, absolute number of CD4+ T-cells  and TE were performed by two different observers, with each subject undergoing the two examinations no more than 6 months apart. The physician performing the second procedure was blinded to the results of the first procedure.TE was performed in the right lobe of the liver through the intercostal space as described,40. LiveARFI imaging is a method of quantifying the mechanical properties of tissues, without manual compression, by measuring the shear wave velocity induced by acoustic radiation and propagating in the tissue. This quantitative technique provides a single unidimensional measurement of tissue elasticity like TE, although the measurement area can be positioned on a two-dimensional B mode image. The region is a 1 × 0.5-cm rectangle, which can be freely moved in the two-dimensional B mode image to a maximum depth of 8 cm from the skin plane. Measurements are expressed as m/s, indicating shear wave speed traveling perpendicular to the shear wave source. The principle of ARFI is described in Figure All patients underwent quantitative elastographic examinations in fasting conditions with a Siemens Acuson S2000TM ultrasound system  and an abdominal curved transducer (4C1), using the virtual touch tissue quantification system.The first step assessed liver morphology and echo-structure, looking for cirrhosis, signs of portal hypertension and tumor lesions. In the second step, 10 ARFI measurements of the right liver through the intercostal space were performed by a single examination. To standardize these examinations, an area of liver tissue free of large blood vessels was chosen at a minimal distance of 2 cm below the liver capsule. ARFI measurements were expressed in m/s as median (M), mean and standard deviation, and interquartile range (IQR), with the IQR/M ratio used to evaluate the variability of the measurements. An IQR/M < 0.3 was considered a homogeneous set of measurements. Median ARFI value was expressed according to the ‘equivalent Metavir’ score using previously determined cutt-off values for HCV mono-infected patients of 1.34, 1.55, 1.80 m/s for liver fibrosis scores of F ≥ 2, F ≥3, and F = 4, respectively.Quantitative characteristics were expressed as mean (standard deviation), median (first-third quartile) and range, and compared using non-parametric Wilcoxon statistics. Qualitative characteristics were expressed as frequencies and proportions, and compared using Fisher’s exact test.Correlations between TE and ARFI measurements were estimated using Spearman’s correlation coefficient and its two-sided 95% confidence interval (CI), as calculated with Fisher's z transformation.Agreements between “equivalent Metavir” fibrosis stages on ARFI and TE were assessed by simple Kappa coefficients for predictions of F ≥ 3 or F ≥2, and F = 4, and weighted Kappa coefficients for predictions of F1 to F4 Metavir scores; two-sided 95% CIs were also estimated. Kappa values ≥0.60 and ≥0.80 were considered indicative of good and very good agreement, respectively. Additional analysis was performed in patients infected with HCV genotypes 1 and 4, who usually have a poorer response than patients infected with genotypes 2 and 3.Quantitative and qualitative variables associated with discordances between ARFI and TE staging were assessed by the Wilcoxon test and Fisher’s exact test, respectively. Only univariate analyses were performed because of the small number of subjects.Of the 86 examinations, 40 were excluded because of missing data (n = 8), too long delay (>6 months) between ARFI and TE (n = 24) or repeated examinations for the same patient (n = 8). When liver stiffness was measured repeatedly in the same patient, only the measurement with minimal delay between ARFI and TE was included in the analysis. Valid results of both ARFI and TE were obtained for 46 patients; their demographic and clinical and biological characteristics are summarized in Table Of the 46 patients, 28 did not receive any treatment for HCV, whereas the other 18 were treated with peginterferon and ribavirin (SOC). At the time of ARFI, eight had achieved a sustained virologic response, whereas the other 10 had failed treatment. No patient received any direct-acting antiviral agents (DAAs). Moreover, all 46 patients had received highly active antiretroviral therapy (HAART), including 28 who received third-generation protease inhibitors (PIs), three who received integrase inhibitors (INIs), eight who received non-nucleoside reverse transcriptase inhibitors (NNRTIs), and seven who received other treatment combinations  and three with benign liver lesions. The two HCCs presented with arterial hypervascularity and wash-out in venous and delayed phase on dynamic enhanced MRI. Their dThe median delay between ARFI and TE in the 46 evaluated patients was 10 days range, 0–25 days). Median liver stiffness on TE was 6.1 kPa (range 3.4–35.3 kPa) and median shear wave velocity (SWV) on ARFI was 1.29 m/s (range 0.93–2.86 m/s). Median liver stiffnesses on TE in patients with F1, F2, F3, and F4 fibrosis stages were 5.4 kPa (range 4.1–6.1 kPa), 7.2 kPa , 11.6 kPa  and 24.6 kPa , respectively. Median SWVs on ARFI in patients with F1, F2, F3 and F4 fibrosis stages were 1.15 m/s , 1.44 m/s , 1.60 m/s , and 2.25 m/s , respectively. Figure 5 days. MSpearman correlation analysis showed that the correlation coefficient between ARFI and TE measurements was 0.76 . Table 3) cells; HCV genotype; increased serum AST, ALT, ALP, and GGT (>2N) and bilirubin concentrations; steatosis; cirrhosis; HIV and HCV treatment; and response to HCV treatment. Only age was associated with the discordance between the two methods (p = 0.047) with older patients in the discordant group. Discordance also tended to be associated directly with hepatomegaly and inversely with overweight, cytolysis, and cholestasis. No significant difference was found with HIV (p=0.22) and HCV(p=0.55) treatment.Of the 46 patients, 14 (30%) showed discordant Metavir scores between the two methods, 11 in predicting F ≥ 2 and three in predicting F ≥ 3. ARFI yielded a higher fibrosis score than TE for 11 of these patients. As the discordance was more important for predicting F ≥ 2, factors possibly associated with these discordances were analyzed, including age; sex; BMI; alcohol consumption; plasma HCV-RNA levels; absolute CD4+  and for the diagnosis of liver cirrhosis (AUROC = 0.93). MoreoveWe observed a moderate agreement between the two techniques for predicting F ≥ 2. Earlier studies also showed that TE or ARFI was more accurate for predicting extreme stages (F1 or F4) than moderate (F2) fibrosis. For example, the AUROCs for ARFI were higher for predicting F=4  than F≥ 2 ,33,49.In addition, agreement between the TE and ARFI scores was determined in patients infected by genotype 1 and 4 who usually have poorer response than patients infected with genotype 2 and 3. Similarly, there was no difference in concordance level between patients with genotypes 1 and 4 and those with other genotypes. The TE and ARFI methods showed very good agreement in predicting overall fibrosis stage and F ≥ 3 in patients with genotypes 1 and 4.This study included a small number of patients (n = 46). However, it was designed as a pilot study, with intraclass correlation coefficients precise enough to draw conclusions by comparing the lower limit of the 95% CIs with 0.6, deemed to represent the lower limit of good agreement.ARFI has several advantages compared with TE. TE has been reported to have an average failure rate of 3.1% and to be highly dependent on BMI. Moreover, TE measurements were estimated to be unreliable in 15.8% of patients in a very large study. We founTE and ARFI showed a discrepancy in predicting fibrosis score in 30% of patients, especially in predicting F ≥ 2. Apart from age, no factor was clearly associated with these discrepancies. However, the study size was not powered for this aim. Liver biopsy should have been performed in patients with discordance, but was not except in one patient, in whom ARFI, but not TE, agreed with biopsy results. Moreover, when a discrepancy was observed between the two techniques, it was not possible to conclude whether TE or ARFI was more correct. These discrepancies may be linked to the cut-off values used, as these are not the same in all published studies. The TE cut-off values we used yielded Finally, although liver biopsy has been considered the gold standard for the assessment of liver fibrosis, we elected to use non-invasive methods for comparison. FibrosiThis pilot study showed good agreement between TE and ARFI in evaluating liver fibrosis in HIV-HCV co-infected patients, suggesting that ARFI may replace TE in this population. In addition to being a new and promising sonography-based method for non-invasive assessment of liver fibrosis, ARFI is cost effective, allowing routine ultrasound examination and fibrosis assessment during the same session.Written informed consent was obtained from all patients for publication of this paper; copies are available for review by the Editor of this journal.This study and its publication were approved by our local ethics committee: CPP Sud-Ouest et Outre Mer III Bordeaux.AASLD: American Association for the Study of Liver Diseases; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; ARFI: Acoustic radiation force impulse; AST: Aspartate aminotransferase; AUROC: Area under the receiver operating characteristic curve; BMI: Body mass index; CT: Computed tomography; DAA: Direct-acting antiviral agents; GGT: γ-glutamyl transferase; HAART: Highly active antiretroviral therapy; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; INIs: Integrase inhibitors; IQR: Interquartile range; LB: Liver biopsy; LSE: Liver stiffness elastography; MRI: Magnetic resonance imaging; ND: Not done; NNRTIs: Non-nucleoside reverse transcriptase inhibitors; PIs: Protease inhibitors; RNA: Ribonucleic acid; ROI: Region of interest; RT: Reverse transcriptase; RTE: Real-time elastography; SOC: Standard of care; SWE: Shear wave elastography; SWV: Shear wave velocity; TE: Transient elastography; US: Ultrasound.The authors state that they have no competing interests.NF, HT, and PM designed and supervised the study. NF supervised all ARFI measurements. NF, JD, and FM, extracted the data. PP and JA analyzed the data. NF, PM, PP, and JA interpreted the data. NF wrote the manuscript. PM, JA, PP, and HT drafted and finalized the manuscript. All authors revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript. All authors are permanent employees of the Bordeaux University Hospital, which funded this study and the preparation of this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/405/prepub


Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes . pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the  Oryza sativa L.), which is one of the most important cereal crops in the world, is a fundamental food source that provides nutrition for nearly half of the global population [Rice  investigation of the status of transgenic rice in China, (II) construction and validation of a reference plasmid, and (III) application to practical sample analysis. Additionally, the novel standard reference molecule developed in this study, which includes rice endogenous reference genes and sequences of GM elements from rice, was demonstrated to be a valid substitute for certified positive reference materials in GM rice detection.μL of nuclease-free water. DNA quantification was performed using a PicoGreen assay , and the samples were diluted to a 50 ng/μL working stock, which was stored in aliquots at −80°C. Restriction enzymes were purchased from New England Biolabs . DNA synthesis, primer synthesis, and DNA sequencing were performed by Genewiz, Inc. .To prepare samples containing the exogenous and endogenous target sequences, genuine seeds from the GM rice varieties TT51-1, Kangyou97, Kefeng6, Kefeng8, KMD1, Bar68-1, and Bar66-1 as well as nontransgenic rice seeds were collected by members of our laboratory. Plant genomic DNA was isolated from rice flour using the Wizard Magnetic DNA Purification System for Food according to the manufacturer's instructions . The DNA pellets were dissolved in 100  EcoRV site of a pUC57 vector. We used validated PCR-based methods to amplify exogenous and endogenous genes using Chinese National Standard PCR primers (μL reaction volume containing GoTaq Green Master Mix (2x) 12.5 μL, each primer 0.5 μL (10 μM), DNA template (25 ng/μL) 2.0 μL, and ddH2O 9.5 μL), and amplification cycling parameters . The ampicillin-resistant plasmid was transformed into Escherichia coli, and the transformed strain was stored at −80°C. Five liters of Luria-Bertani (LB) medium containing 100 μg/mL ampicillin was inoculated with 5 mL of a preculture of E. coli containing the plasmid pBJGMM001 and was shaken vigorously overnight at 37°C. The culture was then centrifuged at 4°C, and the plasmid DNA was isolated and purified using the PureYield Plasmid Midiprep System  according to the manufacturer's protocol. Plasmid samples were stored in Axygen tubes ; 106 copies/μL).To efficiently construct the plasmid, the exogenous and endogenous fragments were cloned into the primers , amplifi E. coli cells was sequenced completely to verify that all target DNAs were present and correctly cloned.Plasmid DNA isolated from the transformed A260/A280 ratio of approximately 1.8 and an A260/A230 ratio above 2.0 [The concentration and purity of the plasmid DNA preparation were measured via UV spectrophotometry. The purity of the nucleic acid was assessed spectrophotometrically by comparing the UV absorbance of the sample at 260 nm to that at 280 nm. Taking into account the generally accepted mean extinction coefficients for double-stranded DNA at 260 nm and 280 nm, pure nucleic acid samples are expected to have anbove 2.0 .The purity of plasmid pBJGMM001 was analyzed by agarose gel electrophoresis.As a reference molecule, pBJGMM001 was processed as follows.http://www.cnki.net/), the Wanfang database (http://www.wanfangdata.com.cn/), scientific papers , the GMO Detection Method database (http://gmdd.shgmo.org/), and Detection and Monitoring of GM Crops of China (http://www.gmcrop.cn/). We summarized the available information on exogenous genes, regulatory elements, and screening markers in GM rice. Based on this information, a statistical analysis was performed to select the genes that appear most frequently in GM rice.GM rice in China was investigated using the following tools: the Chinese National Knowledge Infrastructure database (http://dx.doi.org/10.1155/2015/948297). These varieties include TT51-1, which received a biosafety certificate, and other GM lines undergoing field trials and lines that are currently in the development stage in China. The investigation revealed a large number of commonly occurring transgenic elements, including the following: the cauliflower mosaic virus 35S promoter (CaMV35S), the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the hygromycin phosphotransferase gene (Hpt), the ubiquitin gene (Ubi), the β-glucuronidase gene (GUS), the bar gene (Bar), the actin promoter (P-Act), the Agrobacterium tumefaciens nopaline synthase promoter (P-NOS), the neomycin phosphotransferase II gene (NPTII), and the cauliflower mosaic virus 35S terminator (T-35S).When designing the novel plasmid, we aimed to use fewer elements to achieve greater detection coverage. As a first step toward this goal, we investigated the status of GM rice development in China from 1989 to 2014. Based on a database search, we determined that the total number of papers published on GM rice varieties is approximately 250 . The use of initial screens that target elements common to multiple GM events can facilitate rapid and cost-effective discrimination of GMO and GMO-free samples . Based o EcoRV site of the pUC57 vector. In vitro DNA synthesis of the entire target fragment saves time and is easier compared to traditional overlapping PCR. Using gene synthesis, researchers save money on the reagents needed for plasmid construction, cloning, and sequencing and can also save time by outsourcing the synthesis of gene sequences that are difficult to clone, thereby avoiding repeated cloning failures. Moreover, ~10 bp was added to both amplicons to increase the amplification efficiency.The pBJGMM001 plasmid contained DNA sequences from part of the rice taxon-specific SPS gene, the PEPC gene, and the exogenous elements CaMV35S, T-NOS, Ubi, Bar, and Hpt . The exo A260/A280 and A260/A230 ratios measured for the plasmid solution were 1.90 ± 0.04 and 2.12 ± 0.03, respectively, indicating sufficient DNA purity; however, such values do not exclude the possibility that traces of contaminating protein may be present. Because no smear was visible in the plasmid preparation and no RNA band was visible, it can be concluded that the plasmid preparation was not contaminated with external genomic DNA or a large amount of RNA (data not shown). However, traces of genomic DNA or RNA from host bacterial cells cannot be excluded in the final plasmid preparation. Such traces do not influence the detection of target sequences.Plasmid pBJGMM001 was fully sequenced by three independent laboratories. The results were consistent with expectations (data not shown). The sequence analysis did not reveal the presence of a mixed population of plasmids. TheAmplified fragments of SPS (287 bp), PEPC (271 bp), T-NOS (180 bp), CaMV35S (195 bp), Ubi (314 bp), Bar (430 bp), and Hpt (472 bp) were observed when pBJGMM001 DNA was used as a template for amplification . The priIn this study, to investigate the utility of pBJGMM001 in practical samples, we tested samples from all potential commercial GM rice lines, including TT51-1, Kangyou97, Kefeng6, Kefeng8, KMD1, Bar68-1, and Bar66-1. Only the positive samples contained the target elements, and amplification signals were not observed for the non-GM rice .The GM rice materials used in the application assay are currently undergoing testing and are therefore not yet available for human consumption. Many other GM rice varieties are also in development. However, trace amounts of these GM varieties have been found in the food supplies in Europe and China . Our tesRice is one of the most important food crops worldwide, and most people in developing countries depend on rice as a staple food for nutrition and caloric intake. Due to the importance of rice to humans, major efforts have been made to improve its properties through genetic alterations . The newpBJGMM001 was designed after considerable investigation and will be used as a certified reference material. The detection coverage of pBJGMM001 reached 96% in all GM rice lines in China. Furthermore, the developed assays were successfully used to test six practical samples from different GM rice lines. All of the results indicated that the established plasmid is a convenient, rapid, and low-cost method for the routine detection of GM rice. The sequence of pBJGMM001 is available from NCBI under accession number KR493382.Supplementary Table S1: The investigation results of the status of genetically modified rice in China from 1989 to 2014. We show the total list of papers published on development of GM rice varieties is approximately 250.


Several studies have investigated the association between polymorphisms in protamine 1 and 2 genes and male infertility risk, with inconsistent results to date. This meta-analysis based on the 13 published case-control studies, including 7350 cases and 6167 controls, was performed to further establish the potential association between the 6 common single nucleotide polymorphisms  in protamines 1 and 2 and male infertility. The -190C > A (rs2301365) polymorphism was identified as a risk factor for male infertility under all models. Interestingly, rs1646022 and rs737008 polymorphisms exerted protective effects against male sterility in Asian and population-based under some models. No associations between the remaining SNPs and male sterility were observed. Infertility is a widespread reproductive health problem affecting family well-being and social stability worldwide. Globally, around 15% of heterosexual couples are unable to conceive children without assistance3456Protamines, which were first isolated from spermatozoa a century ago, play vital roles in spermatogenesis. The nucleoprotein gene products, protamine 1  and protamine 2 , are closely linked in a stretch of DNA 13–15 kb long on human chromosome 16p13.3, along with the gene encoding transition protein 2 (TNP2), categorized as members of the protamine gene family. Protamine is the major DNA-binding protein in sperm nucleus that promotes DNA condensation and packaging in spermatozoa by histone replacement during spermatogenesis. The structure of chromatin undergoes constant remodeling involving complex morphologic, physiologic and biochemical modifications810A number of molecular epidemiological studies have been conducted to examine the association between PRM1 and PRM2 polymorphisms and male infertility in diverse populations. Among these, rs2301365 of PRM1 is in the 5′- untranslated regions (5′-UTR), rs2070923 and rs1646022 of PRM2 are located in the intron, while rs35262993 and rs737008 are two synonymous single nucleotide polymorphisms (SNPs) involving nucleotide exchange at positions 54 (exon 1) and 230 (exon 2). In addition, a non-synonymous SNP at position 102 (exon 1) resulting in the amino acid substitution Arg34Ser (A34R) has been extensively investigated12131415161718192021Through literature search and selection based on inclusion criteria, 13 articles were identified after reviewing potentially relevant articles . The chaIn total, 9 studies on the rs737008 polymorphism, including 1447 cases and 1284 controls, met the inclusion criteria and were selected for meta-analysis. The number of cases included varied from 53 to 304, with a mean  of 160.78 (±70.26) and controls varied from 50 to 376, with a mean (±SD) of 142.67 (±104.11). The characteristics of all nine studies are summarized in vs. CC: OR = 0.75, 95% CI = 0.57–0.97, P = 0.030), while no such correlation was observed for the other subgroups, such as the Asian group, under the heterozygous model  and the Caucasian group under the dominant model . Specific data are presented in Data on the association between rs737008 and male infertility risk are summarized in vs. CC: OR = 1.32, 95% CI = 1.08–1.60, P = 0.006; for CA vs. CC: OR = 1.27, 95% CI = 1.04–1.56, P = 0.022; for AA vs. CC: OR = 1.66, 95% CI = 1.07–2.58, P = 0.024; for AA + CA vs. CC: OR = 1.53, 95% CI = 1.00–2.36, P = 0.052; for A vs. C: OR = 1.28, 95% CI = 1.09–1.51, P = 0.003). Subgroup analyses revealed significant associations within the Caucasian subgroup , the polymerase chain reaction (PCR) sequence subgroup  and the PB subgroup , but not the remaining subgroups. Specific data are summarized in Data on the association between the rs2301365 polymorphism and male infertility risk are summarized in vs. GG: OR = 0.68, 95% CI = 0.48–0.97, P = 0.032) and PB . This correlation was not observed in other subgroups under all models. Specific data are described in Data on the association between rs1646022 and male infertility risk are summarized in No significant association was observed in overall analysis between the SNPs and male infertility under all models. To clarify the effects of potential ethnic, methodological and control source differences, subgroup analyses were performed. No significant association was evident between the polymorphisms examined and estimated risk of male infertility in all subgroups under all models.Begg’s funnel plot and Egger’s test were performed to assess publication bias. For all SNPs, funnel plot shapes did not reveal any evidence of obvious asymmetry. Egger’s test was used to provide statistical evidence of funnel plot symmetry. The results suggest no publication bias or small-study effects.We additionally conducted sensitivity analyses on SNPs under all models by omitting one study at a time in the calculation of a summary outcome. Although the sample sizes for cases and controls in all eligible studies varied, corresponding pooled ORs and 95% CIs were not qualitatively altered with or without studies on small samples. No other single study influenced pooled OR and 95% CI qualitatively.Spermatogenesis is a complex process involving mitotic and meiotic division of germ cells resulting in the formation of haploid spermatozoa. Highly coordinated expression of genes and subtle post-transcriptional regulation are therefore crucial for normal germ cell development. During the process of male reproduction, at least 150 different genes are involved in spermatogenesis. Changes in a cohort of genes and expression patterns affect spermatogenesis and its products, leading to spermatogenesis dysfunction and consequently, male sterilityTo date, two protamine types have been identified in mammals, designated PRM1 and PRM2. The nucleoprotein PRM1 is present in all species of vertebrates while PRM2 exists in some mammalian species, including human and mouseet al.et al.Variants of PRM1 and PRM2 have been shown to be related to male infertility in humans and animals192210111617http://www.gwascentral.org/) and National Human Genome Research Institute GWAS Catalog (http://www.genome.gov/26525384), but failed to identify a relevant genome-wide association (GWAS) study on all SNPs.The present meta-analysis including 7350 cases and 6167 controls from 13 case-control studies explored the association between PRM1 and PRM2 polymorphisms and male infertility. We searched available databases, such as GWAS Central (vs. GG (heterozygous model) and T vs. G  among the case and control groups. No association of SNPs of PRM1 with male infertility was observed  and publication bias  did not exist in the meta-analysis. He et al.Initially, we focused on the relationship between PRM1 and PRM2 polymorphisms and male infertility risk. 6 studies dating from 2006 to 2012 were included. In rs35576928, no mutant homozygotes were identified. We only compared the distribution of GT vs. CC: OR = 0.75, 95% CI = 0.57–0.97, P = 0.03). In addition, an edge effect may exist in the PB subgroup under the heterozygous model . One of the most important goals of the meta-analysis was to identify the source of heterogeneity. Accordingly, studies were stratified according to ethnicity, control source and method. However, we failed to observe heterogeneity in all subgroups.For rs737008, 9 studies that met the inclusion criteria were selected, including 1447 cases and 1284 controls dating from 2003 to 2012. In overall analysis, no significant association was observed between the polymorphism and male infertility under all models. However, a strongly protective effect was specifically observed between rs737008 and male infertility in the PB subgroup under the dominant model  has clearly been shown to be associated with male infertility14The available literature on the relationship between the PRM1 -190C > A (rs2301365) polymorphism and male infertility risk documents controversial findings. We selected six studies dating from 2008 to 2012 that met the inclusion criteria in the meta-analysis, including 1025 cases and 819 controls. The study focused on whether the SNP is a risk for male infertility under all models. Data from our meta-analysis showed that mutation increases the risk of male sterility under all models. A potential explanation for the association between PRM1 -190C > A polymorphism and infertility is that the region encompassing this polymorphism resulted in clear DNase I footprint protection P = 0.032) and the PB subgroup under the dominant model  and allele model . After considering a comprehensive list of factors, the following possible causes were proposed: SNP markedly decreases the risk of male infertility in Asian and PB, since this mutation may lead to some key enzymes failing to cut and joint PRM gene during the protamine translation process. Inconsistent findings with Caucasian and other populations indicate that ethnicity may contribute to differences in male infertility susceptibility. Moreover, progression of male sterility is known to be the outcome of interactions between genes and environment. Caucasians are used to a meat-based diet and bear considerably more stress in terms of lifestyle, which may affect the development of male germ cells. Finally, the joint effects of SNPs on PRM haplotypes should also be considered. An individual with a clinical disorder is not the product of the single disrupted gene. Genetic disruption is embedded within the context of the entire genome of the individual. After applying the nonparametric trim and fill method, we observed no publication bias in SNPs under all models in the meta-analysis.For rs1646022, 6 studies were selected from 2008 to 2012 that met inclusion criteria, including 898 cases and 641 controls. No significant correlation of this SNP, located in the intron area of the PRM2 gene in chromosome 16p13.2, was observed with male sterility in overall analyses under all models. However, a strong protective effect of rs1646022 against male infertility was observed in the Asian subgroup under the dominant model  and varied dietary habits during the lengthy evolution process, which may affect the mode of action and potency of the two SNPs, leading to differences in results among the populations from distinct regions.A number of limitations of the current meta-analysis need to be addressed. Firstly, only 13 studies were enrolled, and the relatively small total sample size had limited power for exploring the real association. Secondly, subgroup analyses by method, ethnicity and control source involved relatively small groups, which may not impart sufficient statistical power to explore the real association and are more likely to reveal greater beneficial effects than large-scale trialsIn conclusion, data from our meta-analysis support further exploration of the true association between these SNPs and male infertility. The -190C > A (rs2301365) polymorphism is associated with risk for male infertility under all models. Interestingly, the rs1646022 polymorphism has a protective effect against male infertility in Asian and PB populations under dominant and allele models, while the rs737008 polymorphism exerts a protective effect in PB under dominant and heterozygous models. However, no associations were observed between the other SNPs examined and male sterility. In terms of male infertility with multifactorial etiology, further studies with larger sample sizes and stratified by different ethnic backgrounds, environmental exposure or other risk factors are warranted to clarify the potential roles of PRM1 and 2 polymorphisms in the pathogenesis of male infertility.Data from single reports were extracted . We systInclusion criteria were as follows: (1) research focus on rs35576928, rs737008, rs35262993, rs2301365, rs1646022, rs2070923 polymorphisms and male infertility, (2) studies on human beings, (3) case-control study design, (4) contained details of genotype frequency of cases and controls, (5) papers where the full text was available. Two reviewers assessed the full text of eligible studies from the above databases. Additional studies were identified from a manual search of references of original or review articles on this topic.Information on the enrolled studies is listed in P-values < 0.05 considered significant. Heterogeneity across the studies was evaluated with the Chi-square-based Q testP < 0.05. A random-effects model using the DerSimonian and Laird method and fixed-effects model using the Mantel–Haenszel method were used to pool data from the meta-analysisP-value for heterogeneity was >0.10 and I2 < 50%, indicating absence of heterogeneity between studies, the fixed-effect model was employed to evaluate the summary OR. Conversely, if the P-value for heterogeneity was ≤0.10 or I2 ≥ 50%, indicating a high extent of heterogeneity between studies, the random-effects model was employed to evaluate the summary OR.The strength of association between polymorphisms and male infertility risk was assessed based on OR with 95% CI. The combined ORs were respectively calculated for five genetic models . The statistical significance of pooled OR was determined with the Z-test, with vs. W). Stratified analysis was further performed based on ethnicity (Asian and Caucasian), method  and others) and control source . A sensitivity analysis was conducted in which a single study within the meta-analysis was deleted each time. To determine the influence of individual data sets on overall pooled OR, forest plot analysis was performed to assess result stability. Funnel plots and Egger’s liner regression test were applied to test publication bias,The six polymorphisms were evaluated for the associations with male infertility susceptibility based on four genetic models. Compared to the wild-type homozygote (WW), we first estimated the effects of the rare homozygous (RR) and heterozygous (WR) genotypes on risk of infertility, and subsequently evaluated the risk of infertility under four genetic models. In addition, allele model associations were estimated .


Background: In many Western populations, blood pressure varies moderately with season and outdoor temperature. Relatively little is known about effects of seasonal changes in blood pressure on the detection and control of hypertension in general populations, especially in low- and middle-income countries.Methods: We analysed cross-sectional data of 57 375 (42% men) participants aged 30–79 (mean 52.3) years who were enrolled during 2004–08, as part of the China Kadoorie Biobank, from a rural county in the south-east costal Zhejiang Province. Analyses related daily mean outdoor temperature, obtained from local Meteorological Bureau, to mean systolic (SBP) and diastolic blood pressure (DBP), rate of newly detected hypertension and, among those with self-reported physician-diagnosed hypertension, rate of adequate blood pressure control, using multiple linear and logistic regression models.Results: The overall mean blood pressure was 135.9 mmHg for SBP and 80.5 mmHg for DBP. Daily outdoor temperature ranged between −2.9 and 33.7°C, with July being the hottest month (mean 29.4°C) and January the coldest (mean 4.0°C). Comparing January (the coldest month) with July (the warmest), the differences in the adjusted SBP/DBP were 19.2/7.7 mmHg. Each 10°C lower ambient temperature was associated with 6.9/2.9 mmHg higher SBP/DBP,14.1% higher prevalence of newly detected hypertension and, among those with pre-diagnosed hypertension, 13.0% lower hypertension control rate.Conclusion: In rural China, lower outdoor temperature is strongly associated with higher mean blood pressure and hypertension prevalence as well as poorer hypertension control, and should be considered when conducting population-based hypertension surveys and providing treatment for hypertensive patients. The significant seasonal variations in blood pressure were primarily driven by changes in outdoor temperature.When outdoor temperature dropped, blood pressure increase dramatically and a higher prevalence of hypertension could be detected. This should be taken into account when conducting and evaluating population-based hypertension survey.Lower outdoor temperature could attenuate the blood pressure control rate in people with known hypertension. This should be considered by hypertensive patients and their care/treatment providers.Hypertension, or raised blood pressure, is a common, dangerous and treatable condition, but its detection and control remain a major health challenge in many parts of the world, particularly in low- and middle-income countries such as China. Several environmental and lifestyle factors are known to be associated with raised blood pressure, including cold temperature, alcohol drinking, adiposity and salt intake. In Western populations where most people have good access to adequate housing and health care, mean blood pressure in adults tends to vary only moderately with outdoor temperature.To fill in this knowledge gap, we report a detailed analysis of cross-sectional data of over 50 000 adults from a rural coastal area in the south of Yangzi River, from the China Kadoorie Biobank (CKB) study.Detailed information about the CKB study design and recruitment strategy has been reported previously.The study was approved centrally by the ethics committees of the University of Oxford and the Chinese Center for Disease Control and Prevention (CDC). In addition, ethics approval was also obtained from the institutional research board at the Zhejiang Provincial CDC and Tongxiang CDC. All participants provided written informed consent.Information about socio-demographic, lifestyle and medical history was collected using an interviewer-administered laptop-based electronic questionnaire.In the present analysis, hypertension detection rate was defined as proportion of people who had mean SBP ≥140 mmHg or mean diastolic blood pressure (DBP) ≥90 mmHg among those without self-reported physician diagnosed hypertension. Hypertension control rate was defined as proportion of people with prior hypertension whose measured SBP <140 mmHg and DBP <90 mmHg.Local daily outdoor temperature data collected at four time points  during the day for whole of the study period was provided by Tongxiang Meteorological Bureau. Mean monthly, spring , summer , autumn , and winter  outdoor temperature was calculated as the average of outdoor temperatures during the days when participants were surveyed in that month or season.Sedentary leisure time was defined as time spent in sitting-down activities outside work, such as watching TV, reading a newspaper and using a computer. Height and weight were measured following standard protocols and BMI was calculated as weight (in kg) divided by the square of standing height (in m).Least squared means of SBP and DBP were calculated for each season (regardless of the year) and calendar month during the 3.5 years’ study period, with adjustment for age, sex and education level. In additional analyses, outdoor temperature was also adjusted for.The least square means of SBP in each calendar month were also analysed separately in certain subgroups, including by age , BMI categories, smoking status , alcohol drinking status , hypertensive status and fruit intake. Analyses were adjusted for mean age, sex and education level, where appropriate.Multiple linear regression models were used to estimate changes in blood pressure per 10°C lower outdoor temperature, adjusting for age, sex and education level. In additional analyses, season, physical activity (daily hours spent on sedentary leisure activities), dietary factors (i.e. fruit and pickled vegetable intake) and BMI were adjusted for, first individually and then jointly.Using logistic regression models, age-, sex- and education-adjusted hypertension detection and control rates were calculated for each calendar month and season (regardless of the year). To estimate changes in detection and control rates per 10°C temperature, parameters of linear regression lines were obtained from plots of age-, sex- and education- adjusted rates in each month against monthly outdoor temperature. All analyses were performed using SAS 9.3 .2 and 6.4%had BMI ≥28 kg/m2 (cut-off points often used to define overweight and obesity in Chinan = 8216) reported having physician-diagnosed hypertension, and of these 90.7% reported taking antihypertensive drugs. A further 26.9% (29.5% in men and 25% in women) had measured SBP ≥140 and/or DBP ≥90 mmHg (i.e. newly detected hypertension) at the baseline survey. The overall mean (SD) SBP and DBP were 135.9 (21.4) and 80.5 (10.7) mmHg, respectively, with SBP and DBP both being about 2 mmHg higher in men than in women ] age was 52.3 (9.9) years, 41.6% were men and 55.9% had formal education, which was higher in men than in women (73% vs 43.7%) . Among min women .Table 1eTable 1, available as Supplementary data at IJE online).Participants recruited in different seasons were very similar in terms of age and BMI. For both men and women, those recruited in spring had the highest prevalence of physician-diagnosed hypertension and antihypertensive treatment, and those in autumn had the lowest . In general, SBP reached a peak (146.1 mmHg) in January and dropped to the lowest level (126.9 mmHg) in July. This variation was slightly larger in men than in women (20.3 vs 18.1 mmHg), increased with age (from 16.1 mmHg in those aged 30–39 years to 22.9 mmHg in those aged 70–79 years), and was more extreme among those with BMI <18.5 kg/m2 (24.5 vs 18.5–19.8 mmHg in the other three groups), but not much affected by other factors (Average daily outdoor temperature during the study period ranged from −2.9 (°C) to 33.7°C (data not shown), with the highest mean monthly temperature observed in July (29.4°C) and the lowest in January (4.0°C) . When ou factors . The seaeTable 1, available as Supplementary data at IJE online).eFigure 2, available as Supplementary data at IJE online). Using individual data in the model, each 10°C lower temperature was associated with 6.9/2.9 mmHg higher SBP/DBP. These associations hardly changed after additional adjustment for season, physical activity, fruit intake, pickled vegetable intake and BMI (data not shown).There was an almost perfect linear inverse association between mean monthly outdoor temperature and mean monthly BP, regardless of study year (The highest hypertension detection rate was seen in January (56.0%) and the lowest in July (16.7%) . By contIn this large study of rural farmers from the south-east coastal region of China, we observed large seasonal variations in blood pressure, driven primarily by changes in outdoor temperature. Each 10°C lower outdoor temperature was associated with 6.9/2.9 mmHg higher SBP/DBP, a 14.1% absolute increase in hypertension detection rate and, among those with self-reported physician-diagnosed hypertension, a 13.0% absolute decrease in hypertension control rate. Comparing with the hottest month (July), participants recruited in the coldest month (January) were three times more likely to be diagnosed with hypertension (56.0% vs 16.7%), and six times less likely to have appropriate control of blood pressure if they had had hypertension (7.9% vs 45.5%).,Several studies have previously reported on the seasonal variation of blood pressure, using either spot clinic blood pressure,,A few mostly hospital-based studies have also reported on the association of season or ambient temperature with detection and control of hypertension. In a small study of 275 women aged 18–40 years in Delhi, India, where the mean outdoor temperature differed by about 19°C between winter and summer, mean SBP increased by 11 mmHg from summer (114 mmHg) to winter (125 mmHg) and at the same time the detection rate of hypertension almost doubled, from 16% to 31%.,Seasonal variation in blood pressure is not a new phenomenon and is probably mediated mainly by ambient temperature.In this study, we used the average of two blood pressure measurements made at the study clinics during daytime, which may not provide a good estimate of an individual’s usual level of blood pressure in a given condition, including season. Moreover, we used daily temperatures measured at the nearest meteorological offices from survey sites, which may not be a perfect measure of the ambient temperature to which the study participants had recently been exposed. Furthermore, no information was available on indoor temperature at home, workplace or survey clinics, nor about the time each participant spent indoors, so their effects could not be assessed directly. If, however, the measured outdoor temperatures were only partially correlated with the usual temperature to which participants were exposed during the day, our study findings may greatly underestimate the real association between ambient temperature and blood pressure.,Our study findings have implications for assessing the population burden and evaluating the clinical management of hypertension, even though the study population is not intended to be representative of the general population in China or in Zhejiang Province.Supplementary data are available at IJE online.Kadoorie Charitable Foundation; Wellcome Trust; UK Medical Research Council; British Heart Foundation and Cancer Research UK. The baseline survey and first re-survey in China were supported by a research grant from the Kadoorie Charitable Foundation in Hong Kong. Follow-up of the project during 2009–14 is being supported by the Wellcome Trust in the UK (grant 088158/Z/09/Z). The project is also supported by core funding to the Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU) at Oxford University from the UK Medical Research Council; the British Heart Foundation and Cancer Research UK.The China Kadoorie Biobank was supported by the:


Wolbachia into Aedes aegypti mosquitoes. This primarily has been proposed as a tool to control dengue virus (DENV) transmission; however, evidence suggests Wolbachia infections confer protection for Ae. aegypti against other arboviruses. At present, it is unknown whether or not ZIKV can infect, disseminate, and be transmitted by Wolbachia-infected Ae. aegypti. Using Ae. aegypti infected with the wMel strain of Wolbachia that are being released in Medellin, Colombia, we report that these mosquitoes have reduced vector competence for ZIKV. These results support the use of Wolbachia biocontrol as a multivalent strategy against Ae. aegypti-transmitted viruses.Zika virus (ZIKV) is causing an explosive outbreak of febrile disease in the Americas. There are no effective antiviral therapies or licensed vaccines for this virus, and mosquito control strategies have not been adequate to contain the virus. A promising candidate for arbovirus control and prevention relies on the introduction of the intracellular bacterium  Flaviviridae. It currently is causing an explosive outbreak of febrile disease in the Americas. In humans, ZIKV infection typically causes a mild and self-limiting illness known as Zika fever, which often is accompanied by maculopapular rash, headache, and myalgia1345Aedes mosquitoes89Aedes aegyptiAedes species, e.g., Aedes albopictus and Aedes hensilli1213Zika virus (ZIKV) is an arbovirus that belongs to the family Wolbachia into Ae. aegypti mosquitoes. Wolbachia is a maternally-inherited intracellular bacterium that is present in numerous insect species worldwide, including mosquitoes, butterflies, beetles, ants, and bees15Ae. aegypti has no native Wolbachia symbionts. The wMelPop strain of the bacterium therefore was introduced into Ae. aegypti with the intent to control dengue virus (DENV) transmission by shortening the lifespan of female mosquitoesWolbachia no longer is being considered for biocontrol because infected mosquitoes displayed reduced fitness in small-scale field releasesWolbachia interfere with viruses and other microbes in the same host. For example, certain Wolbachia variants  partially block DENV transmission without greatly impacting Ae. aegypti fitness2021wMel strain conferred protection against chikungunya virus (CHIKV) and yellow fever virus (YFV) in Ae. aegypti as well2324Wolbachia biocontrol represents a significant new development in the fight against arbovirus transmission. Indeed, because DENV, CHIKV, YFV, and now ZIKV2627Wolbachia biocontrol could potentially be used as a multivalent strategy against all four of these Ae. aegypti-transmitted viruses. But the ability of ZIKV to infect, disseminate, and be transmitted by Wolbachia-infected Ae. aegypti has not been evaluated.Despite the continued spread of the virus, there remain no effective antiviral therapies or licensed vaccines. Thus, with its continued invasion of the new world, the only tools presently available to combat Zika target mosquito populations, mostly with insecticides and larval source reduction. To date, these strategies have not prevented invasion of this virus into new locales and have not been adequate to control the virus upon arrival. A promising candidate for arbovirus control and prevention relies on the introduction of the intracellular bacterium wMel-infected and wMel-free Ae. aegypti from Medellin, Colombia. This was done because medium-scale deployments of wMel-infected Ae. aegypti began in the DENV metropolitan area of Medellin in the spring of last year (2015) [see www.eliminatedengue.com/colombia], and now ZIKV co-circulates with DENV in Colombia. The data presented herein provide information on the effectiveness of Colombian Wolbachia-infected Ae. aegypti in blocking the transmission of ZIKV, as well as describe a biologically relevant model for studying ZIKV transmission dynamics  that does not rely on animal blood spiked with cultured virus. These data argue for the expansion of this technology to ZIKV in South and Central America and are useful in the broader context of ZIKV-mosquito interactions in the Americas.Accordingly, we assessed vector competence for ZIKV in Wolbachia-infected laboratory colonies of Colombian Ae. aegypti for ZIKV. Adult, female, mosquitoes were exposed to infectious bloodmeals containing ZIKV and mosquitoes that ingested blood containing virus were assayed for infection, dissemination, and transmission potential at 4, 7, 10, 14, and 17 days (d) post feeding (PF). ZIKV infection status was confirmed by plaque assay to differentiate infectious from non-infectious virus. As expected, infection, dissemination, and transmission rates were high for WT exposed to blood containing ZIKV. In contrast, (COL)wMel that ingested blood containing ZIKV displayed poor peroral vector competence as compared to WT. In fact, there was a significant reduction  in ZIKV infection status as compared to WT at all timepoints assayed and across replicates, and (COL)wMel remained non-infective over the duration of experimentation . After feeding on ZIKV-infected mice with moderate viremia (6.02 log10 PFU/ml), 100% of WT mosquitoes had established infections at 4 d PF, but none of the mosquitoes disseminated virus nor was infectious virus detected in the saliva (10 PFU/ml) did not have detectable virus in the saliva until 14 d PF  via water-jacketed membrane feeder maintained at 36.5 °CwMel and WT exposed to viremic mice as compared to mosquitoes exposed to a membrane feeder spiked to a higher viremia  of ZIKV was about 15 daysAe. aegypti as early as 4 d PF and the EIP of ZIKV ranged from 7–14 days depending on viremia of the host. This may indicate differences between the propensity of African versus Asian lineage ZIKV to infect and replicate in Ae. aegypti but will require further validation in the laboratory.There is a paucity of data available on vector competence for ZIKV. The few studies available have primarily focused on two urban vectors, Ae. aegypti were highly capable of transmitting ZIKVAe. aegypti strains from Kedougou and Dakar  were not competent to transmit ZIKVAe. aegypti strains from the Americas, which were susceptible to ZIKV infection but had unexpectedly low transmission potentialAe. aegypti from Medellin, Colombia was highly susceptible to ZIKV infection and were competent in their potential to transmit the virus to a new host. It should be noted that Ae. aegypti with poor competence but high population density, have been capable of sustaining arbovirus outbreaks3839Ae. aegypti-ZIKV combinations.More recently, it was demonstrated that Singapore’s Wolbachia can limit infection in Ae. aegypti with other arboviruses24Wolbachia-infected Ae. aegypti could potentially be used to simultaneously control DENV, CHIKV, YFV, and ZIKV. As a result, we evaluated whether Colombian mosquitoes infected with the wMel strain of Wolbachia reduced ZIKV transmission potential. Studies like this one, aimed at assessing the potential effectiveness of Wolbachia biocontrol against ZIKV, will help inform the viability of using this technology as a multivalent strategy for multiple Ae. aegypti-transmitted arboviruses. This novel approach is already being tested in five countries around the globe  against related arboviruses, so these results warrant further exploration, both in the laboratory and the field, on the feasibility of expanding this technology beyond DENV and informing whether Wolbachia biocontrol can be used to supplement or replace existing vector control strategies.The history of ZIKV has been reminiscent of CHIKV, which re-emerged out of Africa and now circulates on all inhabited continents and represents a major global health problem. Not surprisingly, ZIKV has emerged in ColombiaGuide for the Care and Use of Laboratory Animals. All animals and animal facilities were under the control of the School of Veterinary Medicine with oversight from the University of Wisconsin Research Animal Resource Center. The protocol was approved by the University of Wisconsin Animal Care and Use Committee .This study was carried out in strict accordance with recommendations set forth in the National Institutes of Health 2. Aedes albopictus mosquito cells,  were maintained in MEM supplemented with 10% FBS, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and incubated at 28 °C in 5% CO2. ZIKV strain PRVABC59 (GenBank:KU501215), originally isolated from a traveler to Puerto Rico with three rounds of amplification on Vero cells, was obtained from Brandy Russell . Virus stocks were prepared by inoculation onto a confluent monolayer of C6/36 mosquito cells.African Green Monkey kidney cells  were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and incubated at 37 °C in 5% COAe. aegypti used in this study were maintained at the University of Wisconsin-Madison as previously describedWolbachia) were established from several hundred eggs collected from 41 ovitraps placed around the municipality of Bello (commune 1), a northwest suburb of Medellin, Colombia. The Wolbachia-infected ((COL)wMel; infected with the wMel strain of Wolbachia pipientis) mosquito line was generated by crossing wild-caught Aedes aegypti with a wMel-infected laboratory strain of Ae. aegypti using a scheme of mating to field-collected males as developed by Yeap et al.Wolbachia-infected mosquito. The wMel-infected laboratory population of Ae. aegypti originated from eggs provided by Scott O’Neill . Briefly, females from the wMel-infected laboratory population were backcrossed to wild-caught males until generation five at which time the lines were closed (restricting matings to mosquitoes from within the line) with the population size maintained at several thousand adults. An outcrossed line then was established by continuous backcrossing laboratory mosquitoes to the progeny of wild-caught Ae. aegypti from the Bello region . The WT line was derived from material collected from the same 41 ovitraps distributed throughout the suburb of Bello, Colombia. This line has never had any previous contact with Wolbachia-infected mosquitoes. Wild-caught males from the Bello region were routinely collected and introduced into the (COL)wMel and WT colony cages after each generation to prevent inbreeding effects and to ensure the genetic homogeneity of these two lines. Wolbachia infection status was routinely verified using PCR with primers specific to the IS5 repeat elementIfnar−/− mice. These mice have abrogated type I interferon signaling and as a result develop lethal infection and a high viremia. Ifnar−/− mice on the C57BL/6 background were obtained from Eva Harris  and were bred in the pathogen-free animal facilities of the University of Wisconsin-Madison School of Veterinary Medicine. Groups of three-week-old mixed sex mice were used for mosquito exposures. Mice were infected in the left, hind foot pad with 106 plaque forming units (PFU) of ZIKV in 50 μl of animal diluent (AD: 1% heat-inactivated FBS in Dulbecco’s PBS). Uninfected mosquitoes (both WT and (COL)wMel) were allowed to feed on mice two or three days post infection at which time sub-mandibular blood draws were performed and serum was collected to verify viremia. Mice fed upon two days post infection  yielded an infectious bloodmeal concentration of 6.02 log10 PFU/ml ± 0.67  and mice fed upon three days post infection  yielded an infectious bloodmeal concentration of 4.74 log10 PFU/ml ± 0.06. A third biological replicate was performed, whereby mosquitoes were exposed to a ZIKV-infected bloodmeal via water-jacketed membrane feeder maintained at 36.5 °C10 PFU/ml .Mosquitoes were exposed to ZIKV by feeding on isoflurane anesthetized ZIKV-infected Infection, dissemination, and transmission rates were determined for individual mosquitoes and sample sizes were chosen using long established procedures25442 for four days for plaque development. Cell monolayers then were stained with 3 ml of overlay containing a 1:1 mixture of 1.2% oxoid agar and 2X DMEM with 2% (vol/vol) FBS, 2% (vol/vol) penicillin/streptomycin, and 0.33% neutral red (Gibco). Cells were incubated overnight at 37 °C and plaques were counted. Viral RNA from human serum samples from Colombia was quantified by qRT-PCR using the primers and probe designed by Lanciotti et al.TM Universal One-Step RT-qPCR kit  on an iCycler® instrument . Primers and probe were used at final concentrations of 600 nm and 100 nm respectively. Cycling conditions were as follows: 37 °C for 15 min, 50 °C for 30 min and 95 °C for 2 min, followed by 50 cycles of 95 °C for 15 sec and 60 °C for 1 min. Virus concentration was determined by interpolation onto an internal standard curve made up of a 7-point dilution series of in vitro transcribed RNA.All ZIKV screens from mosquito tissues and titrations for virus quantification from mouse serum or virus stocks were completed by plaque assay on Vero cell cultures. Duplicate wells were infected with 0.1 ml aliquots from serial 10-fold dilutions in growth media and virus was adsorbed for one hour. Following incubation, the inoculum was removed, and monolayers were overlaid with 3 ml containing a 1:1 mixture of 1.2% oxoid agar and 2X DMEM  with 10% (vol/vol) FBS and 2% (vol/vol) penicillin/streptomycin. Cells were incubated at 37 °C in 5% COInfection, dissemination, and transmission rates were analyzed using an Exact unconditional testHow to cite this article: Aliota, M. T. et al. The wMel strain of Wolbachia Reduces Transmission of Zika virus by Aedes aegypti. Sci. Rep.6, 28792; doi: 10.1038/srep28792 (2016).


Thalassemia is the most common inherited disease in southern China. However, this disorder is usually ignored by Jiangxi provincial health system and government due to lack of epidemiological data.A total of 9489 samples from Hakka Han and Gan-speaking Han in three geographical areas of Jiangxi Province were analyzed for both complete blood cell (CBC) count and reverse dot blot (RDB) gene chip for thalassemia.1182 cases of suspected thalassemia carriers with microcytosis (MCV<82 fL) were found by CBC count, and were tested by RDB gene chip to reveal a total of 594 mutant chromosomes, including 433 α-thalassemia mutant chromosomes and 172 β-thalassemia mutant chromosomes. Our results indicated a higher prevalence of thalassemia with the heterozygote frequency of 9.49% in southern Jiangxi province, whereas the low frequency was found in middle (3.90%) and northern Jiangxi (2.63%).Based on the epidemiological data, the estimated numbers of pregnancies in Jiangxi province in which the fetus is at risk for β-thalassemia major or intermedia, Bart's hydrops fetalis and Hb H disease are 34 , 79  and 39  per year, respectively. We suggested that prevention network of thalassemia should be established, especially in high prevalent southern Jiangxi (Hakka Han), including establishment of thalassemia database collection, hematological analysis laboratories, genetic counselling clinics, prenatal diagnosis centers and neonatal screening centers. Thalassemia is inherited as an autosomal recessive disorder characterized by microcytic hypochromic anemia, and a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. It is probably the most common monogenic gene disorder in the world and is especially frequent in tropical and sub-tropical areas such as Mediterranean countries, the Middle East, the Indian subcontinent, southern Far East Africa and Southeast Asia http://www.gztj.gov.cn), Jiangxi province lies on the southern bank of the Yangtze River. It shares a border with Anhui to the north, Zhejiang to the northeast, Fujian to the east, Guangdong to the south, Hunan to the west, and Hubei to the northwest. 99.73% of inhabitants of Jiangxi province are Chinese Han, predominantly Gan-speaking Han and Hakka Han. Two decade have elapsed since a relative low incidence of thalassemia  was reported by a large scale survey of hemoglobin variants and thalassemia in Jiangxi province (1987), this disorders was usually ignored by health system and government Previous studies have indicated that there was a high population frequency of thalassemia in southern China, mainly in southern regions of the Yangtze River, particularly in the three most southerly provinces of China-Guangdong, Guangxi and Hainan Here, we perform a large-scale survey of thalassemia in 9489 healthy subjects, which firstly allows a sight into the prevalence and molecular characterization of thalassemia in Jiangxi province. It is a prerequisite for defining a specific policy for carrier screening, genetic counseling and prenatal diagnosis.The study population included 9489 unrelated subjects between August 2011 and November 2011. These subjects visited medical units for routine healthy examination including blood tests, and the discarded blood samples were used for further study. The ages of these subjects ranged from 18 to 70-year-old and about 99% were Jiangxi natives, i.e. Information sheets with nationality, sex, age, dialect, natives or not and written consent forms were available in Chinese to ensure comprehensive understanding of the study objectives, and informed consent was signed or thumb printed by the participants. All studies were approved by the Ethics Committee of First Affiliated Hospital of Gannan Medical University and Ethics Committee of Chaozhou Central Hospital Affiliated to Southern Medical University. 2, was obtained from each participant. RBC indices were determined according to standard laboratory procedures by Beckman Coulter-LH750 blood analyzer . Quality control of complete blood count (CBC) was monitored through participation in the National External Quality Assurance Scheme of China and Randox International Quality Assessment Scheme (RIQAS). Subjects with low red blood cell mean corpuscular volume (MCV) values (<82 fL) were considered possible thalassemia carriers.These subjects received hematological screening for the presence of thalassemia in Medical Laboratory of the First Affiliated Hospital of Gannan Medical University, Central Hospital of Iron Industry Limited Corporation of Xinyu and Jiangxi Provincial People's Hospital. 2 mL peripheral blood, which was anti-coagulated with 4.0 mg EDTA-K3.7, −α4.2, two α-thalassemia mutations [Hb Constant Spring (HBA2: c.427T>C) and Hb Quong Sze (HBA2: c.377T>C)], and 16 known β-thalassemia mutations most commonly seen in the Chinese population  were analyzed by a commercial thalassemia RDB gene chip . The kit of the gene chip was approved by Chinese SFDA (REG.NO: SFDA (P) 20123400399) Genomic DNA of subjects with MCV<82.0 fL were extracted from peripheral blood leukocytes by DNA blood mini kit . The DNA concentration was determined by UV spectrophotometer  at the wavelength of 260 nm. The three known α-thalassemia deletions –SEA, −α2 test. P<0.05 was considered statistically different.Statistical analysis was conducted with SPSS 16.0 statistical software. The prevalence of different thalassemia alleles was calculated from the standard Hardy-Weinberg formula. Data from the three regions in Jiangxi province and previous studies were analyzed by Pearson χA total of 9489 blood samples were obtained and analyzed from three regions of Jiangxi province. 1182 cases of microcytosis (MCV<82 fL) were found. The percentage of microcytosis in Ganzhou region, Xinyu region and Nanchang region was 20.15% (1006/4992), 4.69% (47/1002) and 3.69% (129/3495), respectively.3.7, −α4.2), two α-thalassemia mutations (Hb Constant Spring and Hb Quong Sze) and 16 known common Chinese β-thalassemia mutations. The results of the survey of population among 9489 blood samples were shown in P>0.05).All 1182 microcytosis samples were analyzed by a RDB gene chip for the three known α-thalassemia deletions , was also similar to average level of Guangdong province  P = 0.000) High prevalence of α/β-thalassemia in the Hakka population of southern Jiangxi province (9.49%) was similar to the situation in Hakka population of Meizhou region in Guangdong province . Children with β-thalassemia major need regular blood transfusions, iron chelation therapy with deferoxamine and even hematopoietic stem cell transplantation. The annual cost of blood transfusions and deferoxamine treatments for one patient with β-thalassemia major is estimated to be about ¥100,000/$15,690 in China Based on our epidemiological results, thalassemia is a large public health problem in Jiangxi province, especially in Ganzhou region. Ganzhou is a relative poverty-stricken mountainous region in Jiangxi province. The annual per capita disposable income of the urban residents and the annual per capita net income of rural residents are only ¥18,704.7/$3,069.4 and ¥5,301/$869.9 (2012), respectively (Data from the Webpage of the Ganzhou government (


Commentary on: KakuruA, JagannathanP, MuhindoMK, et alDihydroartemisinin-piperaquine for the prevention of malaria in pregnancy. N Engl J Med2016;374:928–39.26962728et al evaluated the artemisinin-based combination therapy (ACT), dihydroartemisinin–piperaquine, for IPTp. Dihydroartemisinin–piperaquine is already recommended by WHO for treatment of malaria in the second and third trimesters. A recent comparison of four ACTs for treatment of malaria in pregnancy in Africa showed that dihydroartemisinin–piperaquine had the best efficacy and a long post-treatment prophylactic effect, which supports its suitability for IPTp in high transmission areas.1Malaria in pregnancy has devastating consequences for mother and fetus. WHO recommends intermittent preventive treatment in pregnancy (IPTp) with treatment doses of an efficacious antimalarial during the second and third trimesters of pregnancy at predefined intervals. Sulfadoxine–pyrimethamine is currently recommended, but high-level parasite resistance threatens its efficacy. Recent trials showed that amodiaquine, mefloquine and chloroquine–azithromycin are not suitable alternatives due to poor tolerability. This trial by Kakuru et al was a double-blind, randomised, controlled, three-arm trial comparing three-course dihydroartemisinin–piperaquine and monthly dihydroartemisinin–piperaquine versus the standard three-course sulfadoxine–pyrimethamine regimen as IPTp among HIV-uninfected pregnant women between 12 and 20 weeks gestation, using a 1:1:1 allocation. The study was conducted in an area of high sulfadoxine–pyrimethamine resistance in Uganda. The primary outcome was the prevalence of histopathology-confirmed placental malaria. Secondary outcomes included symptomatic malaria, parasitaemia, anaemia and adverse birth outcomes. Women and newborns were followed up to 6 weeks post delivery.The trial by Kakuru Three hundred women were enrolled. The dihydroartemisinin–piperaquine regimens were well tolerated. Compared with sulfadoxine–pyrimethamine, three-course and monthly dihydroartemisinin–piperaquine regimens were associated with a 67% and 100% reduction in incidence of clinical malaria during pregnancy, respectively; a 32% and 46% reduction in malaria infection at delivery; and a 13% and 34% lower risk of anaemia at delivery. Compared with sulfadoxine-pyrimethamine, the relative risk of a composite adverse birth outcome was 0.49 (95% CI 0.23 to 1.04) in the monthly dihydroartemisinin–piperaquine arm, and slightly higher (RR 1.15 95% CI 0.65 to 2.02) in the three-course arm, compared with sulfadoxine–pyrimethamine. There were no differences in safety or adverse events by study arm.This exploratory trial showed that IPTp with dihydroartemisinin–piperaquine was superior to sulfadoxine–pyrimethamine in reducing malaria infections during pregnancy in an area of high sulfadoxine–pyrimethamine resistance, however the study was not powered to detect differences in adverse birth outcomes. The results are consistent with another trial in western Kenya, which showed that, relative to IPTp with sulfadoxine–pyrimethamine, three-to-four courses of dihydroartemisinin–piperaquine resulted in similarly high reductions in clinical malaria and malaria infection, and was also associated with up to a 75% lower risk of stillbirths and early infant mortality.3The WHO's Malaria Policy Advisory Committee reviewed the results from both Ugandan and Kenyan trials, and concluded that dihydroartemisinin–piperaquine is a promising regimen for IPTp, and recommended that larger trials be conducted, powered to look at efficacy on adverse pregnancy outcomes and to provide further reassurance on safety.4Furthermore, questions remain regarding pregnant women's adherence to dihydroartemisinin–piperaquine, a 3-day regimen, in non-controlled settings.


Introduction. The purpose of this observational cross-sectional study was to assess left ventricular mass (LVM) in prehypertensive individuals in comparison to normotensives and to determine if central blood pressure (BP) correlates better with LVM index (LVMI) than brachial BP. Methods and Result. Brachial and central BP measurements were completed at first visit and at 4 weeks in 65 healthy volunteers who were at least 40 years old and not on medication. Subjects were divided into two groups of normotensives and prehypertensives based on JNC-7 criteria and LVM was obtained using cardiac magnetic resonance imaging. Prehypertensives had significantly higher LVMI compared to normotensives (P < 0.01). Brachial and central BP also both positively correlate with LVMI  in both groups and neither method was superior to the other. After multivariate regression analysis and adjusting for cardiovascular risk factors, prehypertension remained an independent determinant of LVM. Conclusion. Prehypertension is associated with cardiovascular target organ damage, and central BP was not superior to brachial BP or vice versa for association with LVMI. Prehypertension (PHT) was first introduced by the Seventh Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) in 2003, replacing former categories of “high-normal” and “above-optimal” blood pressure (BP); and it was defined as systolic BP of 120–139 mmHg or diastolic BP of 80–90 mmHg based on 2 or more properly measured seated BP readings on each of 2 or more office visits [PHT has been associated with significant abnormality of left ventricular (LV) geometry . AlthougStudies have shown that brachial and central BPs may differ, especially the systolic component , 10, butThe aim of this study was (1) to assess the strength of association of brachial and central pressure with LVMI and (2) to compare these variables in prehypertensive and normotensive study participants. Cardiac magnetic resonance imaging (MRI) was used to diagnose LVM as it is more precise and reliable compared to other diagnostic modalities .We conducted an observational cross-sectional study of healthy volunteers from September 2008 to September 2009 at Cardiology Division of New York Hospital Medical Center of Queens/Weill Medical College of Cornell University. We recruited our participants using flyers distributed throughout our institution. We enrolled healthy volunteers who were at least 40 years old and not on medication. Exclusion criteria for the study were previous diagnosis of hypertension (HTN), diabetes mellitus (DM), renal disease, CVD, valvular heart disease, and atrial fibrillation.2. The second visit, which was 4 weeks after the first, included the second measurements of brachial and central BP and performing of a cardiac MRI. To ensure a more representative BP value for each patient, the average of the BP measurement at the first and second visit was used for analysis.Detailed history, physical examination, and brachial and central BP measurement were completed at the first visit. Height (m) and weight (kg) were measured and body mass index (BMI) was calculated as weight (kg)/(height (m) × height (m)) and unit was recorded as kg/mStudy population was divided into two groups of normotensives and prehypertensives based on their BP measurement, and LVMI was compared in both groups. PHT as defined by JNC-7 criteria is systolic blood pressure (SBP) between 120 and 139 mmHg and diastolic blood pressure (DBP) between 80 and 89 mmHg. Brachial and central BP measurements with their correlation to LVMI were also studied.The study was approved by Institutional Review Board of New York Hospital Medical Center of Queens.Brachial BP was measured with a BPM-300 noninvasive BP monitor  after the subject had been in a recumbent position for a minimum of 10 minutes. The device took 6 consecutive BP readings, excluded the first measurement, and derived an average. It uses an oscillometric technique to calculate systolic and diastolic BP. The device meets the Association for the Advancement of Medical Instrumentation Standards.SphygmoCor monitor was used to measure central BP. The tonometer, gently pressed against the radial artery pulse, acquires the radial pulse wave and the SphygmoCor's proprietary algorithm derives the pulse wave as it exists in the ascending aorta producing the central BP measurements noninvasively. k-space inversion-recovery with steady-state free precession imaging. LVMI was calculated using LV measurements in diastole, divided by height, squared, and expressed as g/m2.We used Siemens 1.5T cardiac MRI scanner to obtain LVM. We acquired ECG-gating and breath-holding during contrast-enhanced segmentedr was used for correlations for each variable of interest . Means of continuous variables were compared with independent t-test statistics.Continuous variables were summarized by the mean ± SD. Categorical variables were summarized by frequencies with 95% confidence interval (CI). Pearson P values ≤ 0.05 were considered statistically significant. Analyses were performed using SPSS statistical software (PASW statistics 18).Multivariate linear regression models were constructed using PHT as the primary risk factor and effect size was adjusted for typical potential confounders ; n = 65) consists of healthy volunteers (2) was 27 ± 4 (range of 19–42). 29 (45%) of volunteers were prehypertensive and 36 (55%) were normotensive. 58.5% of volunteers were Caucasian, 27.7% were African Americans, 6.2% were Hispanic, and the rest were Asians. There was no statistically significant difference between prehypertensive and normotensive group in all of the following parameters: age, gender, race, BMI, augmentation pressure, augmentation pressure index, and central SBP.The study population (lunteers . The aveP < 0.01 (P = 0.014 (standardized beta coefficient = 0.314).The main dependent variable we used in our statistical analysis was end diastolic LVMI . Prehypertensives had higher LVMI that was statistically significant as compared to normotensives, P < 0.01 . Simple There was no difference between brachial and central DBP  ; howeverr = 0.318, P = 0.012)   and central DBP and LVMI  was similar.Pearson's correlation showed statistically significant correlation between central SBP and LVMI (= 0.012)  and brac < 0.01) . The corMultivariate linear regression analysis, when adjusted for age, gender, race, and BMI, showed positive statistically significant association between PHT and end diastolic LVMI.Prehypertensive individuals have been shown to be at an increased risk of developing hypertension . AlthougOur study demonstrated a strong relationship between PHT and LVMI when compared to normal BP, even after adjustment for age, gender, race, and BMI. Another principal new finding in present study was that, in both prehypertensives and normotensives, brachial and central BP correlated positively with LVMI, and central BP was not superior to brachial BP or vice versa for association with LVMI.P = 0.03) compared to normotensive patients after adjustment for baseline characteristics. Our study supports this finding. We were able to establish the importance of PHT category to the increased risk of developing future CVD.Manios et al.  analyzedP = 0.011) and increased by 88% for each increase of 0.1 in BP index .Left ventricular hypertrophy (LVH), measured by LVMI, has been identified as the most powerful risk factor for future cardiovascular events causing morbidity and mortality . In factn = 65) of our study compared to the Bogalusa Heart Study (n = 1379). The gender differences might be explained by the age difference in both study populations. The average age of present study population was 54 ± 8 (43–77 years) as compared to the Bogalusa Heart Study with age range of 20–44 years. Arterial stiffening increases in both genders with age [PHT is associated with an increased prevalence of LVH , 17. In with age . This moRecent study on 1,940 young participants found higher LVM values in prehypertensives compared to normotensives even after adjustment for covariates , 17. In Elevated LVM is a well-defined independent modifiable risk factor for adverse cardiovascular event , 15, 22 Our study showed no difference between the diastolic components of both brachial and central BP but a slightly higher brachial SBP than central SBP Figures  and 3. Tβ coefficient = 0.311, P = 0.001 versus β coefficient 0.281, P = 0.003 resp.). Pini et al. [There is an on-going debate on the BP approach that correlates better with LVMI and CVD. Roman et al.  reportedi et al.  also sugi et al.  found thIn conclusion, this data provides evidence of increased LVMI in prehypertensive patients. An increased LVM has been shown to be an independent modifiable risk factor for adverse cardiovascular events and progression of PHT to sustained hypertension. Current guidelines recommend lifestyle modification for the management of PHT, but this has had no demonstrable effect on public health to date . The TriIn addition, both central and brachial BP positively correlate with LVMI in normotensive and prehypertensive patients and central BP was not superior to brachial BP or vice versa for association with LVMI.Our study population consists of relatively small sample of 65 volunteers which may limit the generalizability of the results. There is need for further studies with larger sample populations to replicate these findings. Furthermore, the average age of our study population was 54 ± 8, with older age individuals underrepresented; however, PHT remained an independent predictor of increased LVMI even in multivariable models where age was entered as covariate.2) in our study; however, there is on-going controversy on the best method to index LVM so as to account for body size. Indexing LVM to BSA is said to underestimate LVM in obese and overweight hypertensive patients when compared to height2.7 indexed LVM [2.7 as we might have underestimated the prevalence of increased LVM in our study population by using LVM indexed to BSA.BMI was indexed to body surface area (BSA) (kg/mexed LVM . Indexin