To investigate whether mitochondrial DNA haplogroups M or N are related to occurrence or manifestations of systemic lupus erythematosus (SLE), we collected M/N haplogrouping and clinical characteristics from 868 Han Chinese women with SLE, as well as for 870 age-matched healthy Han Chinese control women. M/N haplogroups were determined in all subjects using allele-specific amplification. The frequency of M haplogroup in all patients was 429 (49.4%) and the frequency of N haplogroup, 439 (50.6%). The corresponding frequencies in controls were 456 (52.4%) and 414 (47.6%) (P = 0.213). Among women older than 50 years at onset age, the N haplogroup was significantly higher in patients than in healthy controls . The N haplogroup was associated with significantly higher risk for certain SLE characteristics: hematological system damage , skin impairment , neurological disturbance  and alopecia . Our results suggest that in Han Chinese women, the mtDNA N haplogroup is associated with higher risk of late-onset SLE, skin impairment, neurological disturbance, hematological system damage and alopecia. Polymorphisms in mitochondrial DNA (mtDNA), a 16.6-kbp double-stranded molecule encoding several rRNAs, tRNAs and proteins3DNA damage and T cell pathology due to excessive ROS production occur in systemic lupus erythematosus (SLE)56910Ethnic and regional genetic heterogeneity may reduce the reliability of SLE studies based on SNPs, including GWAS. This is similar to the case in diabetes research, where GWAS have reported conflicting results for the same disease susceptibility gene candidates12Genetic studies of disease susceptibility may be more reproducible and accurate when based on not only individual SNPs but also haplotypes, since human genetic studies focusing on haplotypes indicate that several SNPs can show linkage, giving rise to synthetic genetic effects. Given a US study suggesting that specific mtDNA SNP haplogroups may affect risk of breast cancer in womenThe control group included 870 healthy women with an average age of 34.01 ± 10.28 years; the patient group, 868 women with an average age of 35.42 ± 11.66 years. The age distribution was similar between the two groups (P > 0.05).Age of SLE onset in patients averaged 31.85 ± 10.69 years. Most frequent clinical manifestations at onset included hematological system damage, renal impairment, arthritis, skin lesion and positivity for anti-dsDNA antibodies .Haplogroup was determined in all patients and controls, the way to determine haplogroup M and N are showed in To control for possible confounding effects due to differences in estrogen, which is important in SLE pathogenesis observed by many observations, we defined subgroups of patients and controls according to whether they were younger or older than 50 years at onset age, which corresponds to the average age of menopause in Han Chinese. The results suggest that among women younger than 50 years, haplogroup frequencies were not significantly different between patients and controls  and with some complications of SLE15We analyzed our data for possible associations between M/N haplogroup and SLE activity based on SLEDAI score. The results showed that the N haplogroup was associated with significantly higher SLE activity than the M haplogroup .We show here for the first time that there is an association between mtDNA M/N haplogroups and risk of SLE in Han Chinese women. While neither haplogroup is associated with increased risk in women younger than 50 years at onset age, the N haplogroup is associated with significantly higher SLE risk in women older than 50 at onset age. This is surprising given the previously proposed rule that nuclear genes contribute more to risk of SLE in younger individuals, whereas environmental factors contribute more to risk in older individualsSNPs in mtDNA have already been shown to interact with the environment. Mutation rates of mtDNA are higher in skin tissue exposed to ultraviolet irradiation and lung tissue exposed to cigarette smoke than in unexposed tissues. This makes mtDNA mutations and resulting changes in ROS production a sensor of exposure to environmental changesThe association between the N haplogroup and risk of SLE in older women likely reflects the combined action of several mtDNA SNPs. The SNP 8701 A/G is a mutation-prone site in the gene encoding the ATP6 subunit of ATP synthaseOur data suggest that, the mtDNA haplogroup is associated with significantly increased risk of several clinical manifestations of SLE: neurological disturbance, hematological system damage and alopecia. Associations of mtDNA SNPs with neurological disturbances and hematological system damage have also been reported in other diseases including stroke and epilepsyWe found the mtDNA N haplogroup to be associated with higher SLE activity. We speculate that at least one reason for this association is that the decrease in ATP production and increase in ROS production in mitochondria of T lymphocytes induces apoptosis and necrosis6The central role of mtDNA in vital metabolic processes and physiological reactions means that its polymorphisms are unlikely to cause grave effects for carriers, since such mutations would likely be lethal. Consistent with this idea, our data suggest that the mtDNA N haplogroup acts as a minor predisposing genetic factor in SLE.The study protocol was approved by the Ethics Committee of the third Xiangya Hospital, Central South University, Changsha, China. All participants provided written informed consent according to the Declaration of Helsinki prior to the study. All methods were carried out in accordance with the approved guidelines.Han Chinese women with SLE were recruited between October 2012 and June 2013 from among outpatients and inpatients in the rheumatology departments of Xiangya Hospital, the Second Xiangya Hospital and the Third Xiangya Hospital, all affiliated with Central South University. All patients satisfied the criteria for SLE published by the American College of Rheumatology in 1997. Patients who also had other connective tissue diseases, severe infection or a family history of autoimmune diseases were excluded.During patient recruitment, healthy Han Chinese women were also recruited as controls. Women were recruited after coming in for voluntary health check-ups at the community health center of the Third Xiangya Hospital. Controls had no history of SLE on their first, second or third-degree relatives.The following clinical information was collected: patient ethnicity, gender, current age, age at disease onset, disease stage, family history, clinical manifestations, antinuclear antibody spectrum, anti-dsDNA antibody status, white blood cell count, platelet count, urine protein profile, 24-hr urine protein quantitation, urine red blood cell count and renal cast status. SLE activity was evaluated using the scoring system of the SLE disease activity index (SLEDAI). The following ratings were used: 1, severe (SLEDAI ≧ 15 points); 2, moderate (SLEDAI = 10–14); 3, mild (SLEDAI = 5–9); 4, mildly inactive (SLEDAI = 0–4).oC.DNA was extracted from peripheral white blood cells using conventional methods. The quality of genomic DNA was verified using agarose gel electrophoresis, and concentration was determined from the absorbance at 260 nm. Genomic DNA was stored at −80 ATP6), 10398 (ND3), 10400 (ND3), 9540 (COII), 14783 (cytb), 15043 (cytb) and 15301 (cytb). Of these SNPs, 8701 (A/G) and 10398 (A/G) are missense mutations, and both are in tight linkage with 10400 (C/T).The following SNPs were initially considered for M/N haplotyping of mtDNA from our patients and controls (www.mitomap.org): 8701 (Therefore the mtDNA SNPs 8701 (A/G), 10398 (A/G) and 10400 (C/T) were screened to determine M/N haplogroup. Individuals with the sequence GGT for these loci were haplogrouped as M, while those with AAC or AGC were haplogrouped as N. Cycling conditions were 94 °C for 5 min and 35 cycles of 94 °C for 30 s, 57.5 °C for 28 s, and 72 °C for 30 s.Amplifications (10 μL) contained 60 ng DNA, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4) 2 test was used to determine whether the relative proportions of mtDNA M/N haplogroups were in Hardy-Weinberg equilibrium in SLE patients and healthy controls. Possible associations between M/N haplogroup and age of SLE onset or occurrence of specific SLE symptoms and ages were assessed using the χ2 test or Fisher’s exact test. Possible associations between M/N haplogroup and SLE activity were assessed using the Mann-Whitney U rank sum test. All statistical tests were performed in SPSS 18.0 , and P < 0.05 was defined as the threshold of significance. In The χHow to cite this article: Tang, Y. et al. Association of mtDNA M/N haplogroups with systemic lupus erythematosus: a case-control study of Han Chinese women. Sci. Rep.5, 10817; doi: 10.1038/srep10817 (2015).


Luoyutong (LYT) capsule has been used to treat cerebrovascular diseases clinically in China and is now patented and approved by the State Food and Drug Administration. In this retrospective validation study we investigated the ability of LYT to protect against cerebral ischemia-reperfusion injury in rats. Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion followed by reperfusion. Capsule containing LYT (high dose and medium dose) as treatment group and Citicoline Sodium as positive control treatment group were administered daily to rats 30 min after reperfusion. Treatment was continued for either 3 days or 14 days. A saline solution was administered to control animals. Behavior tests were performed after 3 and 14 days of treatment. Our findings revealed that LYT treatment improved the neurological outcome, decreased cerebral infarction volume, and reduced apoptosis. Additionally, LYT improved neural plasticity, as the expression of synaptophysin, microtubule associated protein, and myelin basic protein was upregulated by LYT treatment, while neurofilament 200 expression was reduced. Moreover, levels of brain derived neurotrophic factor and basic fibroblast growth factor were increased. Our results suggest that LYT treatment may protect against ischemic injury and improve neural plasticity. Stroke is one of the leading causes of death worldwide and causes long-term disability. It has a significant impact upon health, well-being, and social interactions. Despite this, the available treatments remain limited and unsatisfactory and there is a considerable demand for novel therapies –3. The uNeuronal plasticity involves changes in intracerebral structure and function in both gray matter and white matter. Gray matter consists mainly of neuronal cell bodies and unmyelinated axons. Within the gray matter, synaptic distribution and density can be measured by expression of the synaptic vesicle protein synaptophysin, while dendrites can be visualized using microtubule associated protein (MAP-2) as a marker. White matter is composed mainly of myelinated axons and myelin-producing oligodendrocytes as well as other glial cells. Myelin basic protein (MBP) is a marker of myelin and neurofilament 200 (NF200) is expressed in myelinated axons. The effect of stroke on gray matter has been well studied in the past, but more attention has been paid recently to white matter injury following stroke .Luoyutong (LYT) capsule contains eight active TCM ingredients  and is pμm) composed of eight ingredients  was in the form of a dried superfine powder . Animals were housed in an environmentally controlled room at 22 ± 2°C, with a 12 h/12 h light/dark cycle and were allowed free access to food and water throughout the entire study. The study was approved by the Institutional Animal Care and Use Committee of Capital Medical University and was in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Focal cerebral ischemia was induced in rats as previously described . BrieflyIn total, 90 rats were randomly divided into five groups: sham group, MCAO group, medium dose (0.4 g/kg) of LYT (LYTM), high dose (0.8 g/kg) of LYT (LYTH), and CS (0.1 g/kg). Each group was divided into two subgroups, one treated for 3 days and the other for 14 days. Each subgroup contained nine animals; six were used for 2,3,5-triphenyltetrazolium chloride (TTC) staining to evaluate brain infarct and swelling volume and three rats were allocated for histological and Western blot analyses. A drug solution (2 mL) was administered by gavage and was calculated according to the body surface area based on the daily clinical dosage recommended for humans. Treatment started 30 min after reperfusion and was administered once a day for 3 or 14 days. Animals in the sham and MCAO groups received 2 mL 0.9% NaCl in the same manner. The experimental design is illustrated in Behavioral tests were performed blindly by a trained investigator to eliminate bias. Three tests were carried out to evaluate various aspects of neurological function. (1) The first one is Longa's score test , where aThe animals were sacrificed 3 or 14 days after reperfusion in a carbon dioxide chamber and the brains were quickly removed and sectioned into six consecutive coronal slices of 2 mm thickness. The slices were stained by immersing in 2% TTC for 30 min at 37°C, followed by fixation in 8% formalin. The border between infarcted and noninfarcted tissues was outlined with an image analysis system. The area of infarction was measured by subtracting the area of the nonlesioned ipsilateral hemisphere from that of the contralateral hemisphere based on Swanson's method . The infRats were euthanized 14 days after reperfusion with intraperitoneal injections of chloral hydrate (300 mg/kg) and were perfused transcardially with 4% w/v paraformaldehyde in phosphate-buffered saline (PBS). The brains were dehydrated in 30% sucrose in 4% formaldehyde in PBS. Frozen brains were sectioned coronally. Apoptotic cell death was detected using the In Situ Cell Death Detection Kit, POD , according to the manufacturer's instructions.μg total protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by electrophoretic transfer to polyvinylidene difluoride membranes. Membranes were incubated overnight at 4°C in a 1 : 1000 dilution of primary antibodies against caspase-3 , synaptophysin (Abcam), MAP-2 , MBP (Abcam), brain derived neurotrophic factor (BDNF) (Abcam), or basic fibroblast growth factor (b-FGF) (Abcam). Chemiluminescent detection of antigens was performed following incubation with horseradish peroxidase-conjugated secondary antibodies  for 60 min at room temperature using an enhanced luminescence kit .The forward fontanelle, cut at a coronal slice thickness of 2 mm from the optic chiasma, was harvested 3 or 14 days after reperfusion. Samples were homogenized in lysis buffer  containing protease inhibitors  and phosphatase inhibitors . For each sample, 100 Rats were euthanized 14 days after reperfusion with intraperitoneal injections of chloral hydrate (300 mg/kg) and perfusion with cold saline. The brains were dehydrated in 30% sucrose and 4% formaldehyde in PBS. Frozen brains were sectioned coronally for immunohistochemistry. Following incubation for 2 h in a blocking solution containing 1% bovine serum albumin, 2% normal goat serum, 0.3% Triton X-100, and 5% nonfat dry milk in PBS, sections were labelled using primary antibodies against synaptophysin (Abcam), MAP-2 , MBP (Abcam), NF200 (Abcam), BDNF (Abcam), b-FGF (Abcam), or neuron-specific nuclear protein (NeuN) (Millipore) at a dilution of 1 : 50. Sections were then incubated in fluorescently conjugated secondary antibodies (Alexa 488/Alexa 594-conjugated anti-mouse/anti-rabbit IgG) and fluorescence was detected using a fluorescence microscope .P values less than 0.05 were considered statistically significant.Statistical analysis was performed using SPSS 11.0 . Data were expressed as means ± SEM and were statistically analyzed by one-way analysis of variance (ANOVA) followed by LSD post hoc test. P < 0.01; 0–12 neurological score test, P < 0.05), but the effect was not obvious in LYTM group and CS group, indicating improved neurological function in the LYTH group. Scores in the modified foot fault test were lower following all treatments . LYTM did not impact infarct volume as significantly as LYTH. CS treatment only decreased infarct volume after 14 days of treatment (P < 0.05).Cerebral infarct volume was measured by TTC staining Figures . The infP < 0.05). The expression of activated caspase-3 was reduced by LYTH, LTYM, and CS treatment, but a significant reduction was only observed in the LYTH group (P < 0.05).To measure apoptosis, TUNEL staining was performed in the cortex of ipsilateral cerebral tissue 14 days after reperfusion . The numTo investigate the effect of LYT on neural plasticity following ischemia-reperfusion injury in rats, we measured the levels of synaptophysin, MAP-2, and MBP by Western blotting. We also analyzed the colocalization of synaptophysin and MAP-2 with NeuN in the cortex and MBP with NF 200 in the corpus callosum by immunofluorescence.P < 0.05). Both of LYT and CS had no obvious effect on it (P < 0.05). After 14 days, there were no significant differences between sham, MCAO, and treatment groups (Synaptophysin expression was decreased in the MCAO group compared with the sham group 3 days after reperfusion (t groups . Immunoft groups .P < 0.05). MAP-2 levels were increased by 3 days of LYTH treatment after reperfusion (P < 0.05), whereas administration of LYTM and CA did not rescue MAP-2 expression. After 14 days, there was no difference between MCAO and treatment groups . We observed an increased MBP expression after 3 and 14 days of treatment with LYT and CS . MBP immunoreactivity was affected mostly by myelin sheaths in the sham group. However, low immunoreactivity was detected for MBP after MCAO, while NF200 staining increased in intensity in the MCAO group. These effects were reversed by all treatments . LYT and CS treatment dramatically increased BDNF expression 3 days after reperfusion and it was even higher than that in the sham group (P < 0.05). After 14 days, no significant differences were observed in BDNF expression between the groups  and only treatment with LYTH could reverse this effect. No difference in b-FGF expression was observed 14 days after reperfusion (b-FGF expression was also lower in the MCAO group compared to the sham group 3 days after reperfusion (erfusion . Immunoferfusion .Clinical research showed that LYT has a therapeutic effect of stroke , 13; howIn order to make clear whether the therapeutic effect of LYT depends on antiapoptosis function, we measured apoptosis by TUNEL and quantified the expression of activated caspase-3 in cerebral tissue 14 days after reperfusion. Both CS and LYTH have pronounced influence on apoptosis, but only LYTH could reduce the expression of activated caspase-3. Since caspase-3 is not only a known regulator of apoptosis, it can also influence neural plasticity . TherefoNeural plasticity is important for neurological function recovery after reperfusion, and neural plasticity has different performance at different time points. Neural plasticity plays a vital role in ischemic injury , depressIn addition, LYT treatment upregulates the expression of BDNF and b-FGF following ischemia-reperfusion injury, both of which are critical mediators of neural plasticity and survival , 29–35. In summary, we have provided evidence that LYT has a protective effect after ischemia-reperfusion injury in rats by suppressing neuronal apoptosis and repairing neural plasticity of both gray matter and white matter. These effects may be mediated by regulating caspase-3, BDNF, and b-FGF expressions. Further elucidation of the signaling pathways involved is required. It is likely that the herbal ingredients of LYT act synergistically, but the relative contribution of each herb to the beneficial outcome has not yet been defined.


Rhynchospora pubera and R. tenuis reveals that during meiosis I sister chromatids are bi-oriented, display amphitelic attachment to the spindle and are subsequently separated. During prophase II, chromatids are connected by thin chromatin threads that appear instrumental for the regular disjunction of homologous non-sister chromatids in meiosis II.Meiosis is a specialized cell division in sexually reproducing organisms before gamete formation. Following DNA replication, the canonical sequence in species with monocentric chromosomes is characterized by reductional segregation of homologous chromosomes during the first and equational segregation of sister chromatids during the second meiotic division. Species with holocentric chromosomes employ specific adaptations to ensure regular disjunction during meiosis. Here we present the analysis of two closely related plant species with holocentric chromosomes that display an inversion of the canonical meiotic sequence, with the equational division preceding the reductional. In-depth analysis of the meiotic divisions of  The absence of a defined centromere in organisms with holocentric chromosomes presents particular problems for the control of chromosome segregation during meiosis. Cabral et al. present evidence that two plant species overcome this challenge by inverting the conventional sequence of meiotic divisions. Meiosis is a special type of cell division that occurs in sexually reproducing organisms and results in the formation of gametes. DNA replication is followed by two rounds of chromosome segregation, thereby halving the genomic content. The canonical sequence of events is characterized by sister centromeres segregating together during the first  division and then separating during the second meiotic  division. Regular chromosome disjunction during meiosis I depends on physical connections between homologous non-sister chromatids. These connections, termed chiasmata, correspond to regions that have undergone interhomologue recombination. Recombined DNA strands together with sister chromatid cohesion hold the homologous chromosomes together until the transition from metaphase I to anaphase I. At anaphase I onset, cohesion of chromosome arms is released but maintained in proximity of sister centromeres. The sister centromeres are co-oriented and attached to the same spindle. In contrast, in metaphase II, sister centromeres are oriented in a bipolar manner and are attached to different spindles resulting in sister centromere separation1Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs) catalysed by the conserved Spo11 protein 35Arabidopsis. In contrast, pairing and synapsis in the worm Caenorhabidits elegans and the fly Drosophila melanogaster are independent of DSB formation10C. elegans these processes rely on specific chromosomal regions that associate with chromosome-specific zinc-finger proteins12Drosophila males, chromosomes segregate reductionally even without the formation of chiasmata , which has been explained by interactions of heterochromatic regions, at least in the case of sex chromosomes and the autosome 4 , may circumvent the problem of meiosis by a different strategy. They were reported to display a diploid number of individualized chromatids at prophase II, indicating complete loss of sister chromatid cohesion in meiosis I. Accordingly, several authors have suggested that sister chromatids may segregate at anaphase I leading to the concept of inverted meiosis in which the order of reductional and equational division is inverted and separation of homologous non-sister chromatids follows sister chromatid segregation2332333435363738Hemiptera) in which a diploid individual with a heteromorphic chromosome pair was analysed33Some other organisms with holocentric chromosomes, including plants  and R. tenuis (n=2). Our data support the occurrence of inverted meiosis in plants based on the observation that sister chromatids display amphitelic attachment to the spindle, that sister chromatids are subsequently separated during meiosis I and that homologous non-sister chromatids display mostly regular disjunction in anaphase II. Furthermore, the availability of a R. pubera individual with a heteromorphic chromosome pair allows the non-ambiguous reconstruction of the inverted meiotic sequence. The analyses of both species are complementary, since R. pubera displays chiasmatic meiosis, which is most commonly found among plants with holocentric chromosomes, while R. tenuis represents an exceptional case with achiasmatic meiosis.Here we present an in-depth analysis on the meiotic behaviour of two Cyperaceae species with holocentric chromosomes, R. pubera meiosis has been described previouslyn=1,379 diakinesis and metaphase bivalents). Bivalents with two chiasmata and univalents were also observed, but with lower frequencies . We infer that several aspects of R. pubera are similar to meiotic progression of other plants . First, we see deposition of the axial element protein ASY1 . We were interested whether chromatid association would be directed by 45S rDNA repeat regions (to be found on 3 of the 5 chromosomes of R. pubera)D. melanogaster. We performed chromomycin A3 (CMA) staining that labels the 45S rDNA clusters in many plant species4344R. pubera  the parts of the chromatids that face each other or the chromatin threads are associated with CMA staining , 20% had four to six isolated chromatids (class II), 45.5% had only two isolated chromatids (class III) and 25.5% had no isolated chromatids  of all meiosis II products had incorrect numbers of chromosomes (n=37). We assume that segregation of chromatids during meiosis II not only yielded ~80% of products with the correct number of chromatids but also with the correct set, since we only observed limited pollen abortion in anthers  or shows terminal associations to bivalents  at metaphase I, and, importantly, bivalents orient themselves with the long axis parallel to the spindleR. pubera, bivalents are oriented with the longer axis perpendicular to the spindle and they appear to have several microtubule attachment sites with kinetochores of sister chromatids attached independently to microtubules emanating from opposite poles (amphitelic attachment). At anaphase I, C. elegans homologues are separated, while in R. pubera sister chromatids are separated from each other and pulled to different poles. At this stage, sister chromatids of C. elegans are still held together by cohesins and, in contrast, the non-sister chromatids of R. pubera appear individualized, with some being connected with thin chromatin threads. Importantly, the diploid number of DAPI-stained bodies can clearly be visualized in each half of the dyad in prophase II, following the expectations of sister separation during meiosis I in R. pubera. In C. elegans at metaphase II/anaphase I, sister chromatids align and are subsequently separated. In R. pubera, chromatids associate with the help of an unknown mechanism and subsequently undergo disjunction during anaphase II. These observations are in strong favour of sister separation during meiosis I in R. pubera , each of the two cells of the resulting dyad should contain the same number of DAPI-stained bodies (chromatids and chromatid fragments). In contrast, if homologous chromosomes are separated then the number of DAPI-stained bodies (chromatids and chromatid fragments) is not expected to be the same, with one cell of the dyad receiving the intact chromosome 2 and the other one the two parts of the fragmented chromosome 2 , visible during diakinesis (n=24) clearly showed mirror images of the heteromorphic chromosome pair, demonstrating that meiosis I in R. pubera is indeed equational  is closely related to R. pubera, and its analysis provides further support for the occurrence of inverted meiosis in the Cyperaceae family. An advantage for analysis is that the two chromosomes differ in their size and can easily be distinguished. To analyse chromosome pairing and synapsis during R. tenuis early prophase, the small chromosome pair was specifically labelled with CMA  during early prophase I (n=33) suggesting the absence of pairing also for this chromosome  had an irregular genomic content  and anaphase II . In metaphase II, chromatids align and subsequently undergo disjunction during anaphase II. In the case of achiasmatic meiosis, following the canonical pathway, the second meiotic division is expected to resemble an equational, error-free division. In contrast, the second division of an inverted meiosis is expected to face the problem of distributing homologous non-sister chromatids. Depending on the accuracy of a hypothetical mechanism to promote regular disjunction, errors during chromatid disjunction are expected in the second meiotic division of an inverted meiosis. Indeed, about 30% of all meiotic products of R. tenuis show irregularities. These results strongly support the claim of inverted meiotic events in R. tenuis  in meiosis I. Furthermore, sister chromatid cohesion at the onset of anaphase I is lost and sister chromatids segregate to opposite poles. It should be noted that the haploid genome complement of R. tenuis comprises only two chromatidsR. tenuis prophase II, which connect chromatids with each other, could be instrumental to support regular disjunction. Although we do not have indication of their sequence composition, these threads could be of heterochromatic nature and resemble those found to connect achiasmatic chromosomes during meiosis I in D. melanogaster. It is important to note that in R. tenuis these connections cannot be the remnants of recombination, since this species displays achiasmatic meiosis. Pradillo et al.Arabidopsis mutants with impaired DSB formation or interhomologue bias. Certainly one can also envisage that in R. pubera and R. tenuis, SC-related proteins may support regular segregation of homologous non-sister chromatids during meiosis II.Does meiosis in R. tenuis are of very different size and would, therefore, according to their holocentric nature accommodate more or less microtubule attachment sites, we envisage a model of balanced spindle forces only in case one large and one small chromosome is connected to each spindle pole, thereby promoting regular chromatid disjunction  moved towards each pole. Together, this indicates loss of sister chromatid cohesion and equational division.Meiotic progression is more complex in R. pubera each bivalent has multiple microtubule attachment sites. These sites are in the central region of each half bivalent, with sister chromatids being attached to spindles from different poles  non-sister chromatids in preparation for the second meiotic division.In D. melanogaster, which employs in some instances heterochromatic 45S rDNA repeat containing regions to connect achiasmatic chromosomes during meiosis I  would eventually have one sister chromatid connected to two different non-sister chromatids that would subsequently interfere with chromosome segregation; however, anaphase bridges have not been observed (n=30). The idea of chromatin connections between non-sister chromatids finds further support by the results presented above for the achiasmatic meiosis in the closely related plant R. tenuis and by a parallel study performed in the holocentric plant Luzula elegansR. pubera, we envisage a similar model as outlined for R. tenuis, with the addition that during prophase II chromatin connections may preferentially form between homologous non-sister chromatids since they originated from the same bivalent and were therefore in closer proximity.These threads could be heterochromatin; however, in contrast to s I ref. , the R. C. elegans the chiasma position defines the orientation of bivalents to the spindle and thus how they attach to spindle microtubules29C. elegans. It appears that R. pubera and R. tenuis have adapted a different mode to deal with the holocentric nature of chromosomes during meiosis characterized by (I) amphitelic sister chromatid attachment in metaphase I, by (II) equational sister chromatid separation in anaphase I and (III) by employing a chromatin thread-mediated mechanism in prophase II to associate (homologous) non-sister chromatids for regular disjunction in meiosis II. Altogether, these meiotic alterations can be framed under the term ‘inverted meiosis’.Nevertheless, alternative ways to ensure proper chromosome segregation during meiosis emerged in other organisms with holocentric chromosomes. For instance, in R. tenuis were collected in Porto de Galinhas  and individuals of R. pubera in Mata de Dois Irmãos . Both species were either cultivated in an open experimental garden or in a greenhouse. Vouchers are kept at the herbarium of the Federal University of Pernambuco.Individuals of −1, for 30 min and mounted in glycerol/McIlvaine . The CMA/DAPI banding was performed as previously described−1 CMA for 1 h and restained with 2 μg ml−1 DAPI for 30 min more. Before analysis, slides were aged for 3 more days.Anthers were fixed in ethanol-acetic acid (3:1 v/v) and stored in fixative at −20 °C. Anthers were digested in 4% cellulase, 4% pectolyase and 4% cytohelicase at 37 °C for 4 h, and meiocytes were squashed in a drop of 60% acetic acid. Coverslips were removed after freezing in liquid nitrogen and slides were kept at −20 °C before use. Slides were stained with DAPI, 1 μg ml−1) and denatured with 70% formamide for 10 min at 85 °C. Slides were then denatured once more together with the hybridization mix (50% (v/v) formamide, 10% (w/v) dextran sulfate, 2 × SSC, 10% (w/v) SDS, 2–5 ng μl−1 probe) for 10 min at 90 °C. After the stringency washes, slides were mounted with DAPI/Vectashield H-1000 (2 μg ml−1). The 45S rDNA was detected using the probe R2, a 6.5-kb fragment of an 18S–5.8S–25S rDNA repeat unit from Arabidopsis thalianaR. tenuis as a probeFISH was performed as described in ref. 2HPO4, 30 mM NaH2PO4) for 40 min at room temperature (RT) and squashed in a drop of the same buffer. After coverslips were removed, slides were stained with DAPI/PBS (2 μg ml−1) for selection of appropriate meiotic stages. Slides were then washed with PBS and blocked with 3% bovine serum albumin (BSA) for 10 min at RT. The antibodies used were rabbit anti-AtASY1 (ref. −1).The immunostaining for ASY1 and H2AThr120ph ref.  was perfSY1 ref. , dilutedImmunostaining for RAD51 was performed as described in ref. 4, 5 mM EGTA, 0.1% Triton X-100, pH 6.9) for 1 h at RT. Afterwards, anthers were squashed in the same buffer on a gelatin-coated slide. After coverslips were removed, slides were stained with DAPI/PEM (2 μg ml−1) and selected under a fluorescence microscope. The best slides were washed in PEM, digested with 1.4% β-glucuronidase for 30 min at RT and blocked with 3% BSA for 1 h at RT. The antibodies used were mouse anti-β-tubulin , diluted 1:40, and rabbit anti-mouse IgG TRITC conjugated (DAKO), diluted 1:25, or goat anti-mouse IgG Alexa568 conjugated , diluted 1:300.Immunolocalization of tubulin was performed as previously describedFor anti-grassCENH3 ref.  immunostPictures of cells stained with DAPI or CMA/DAPI were taken with a Leica DMRB fluorescent microscope equipped with a Cohu digital camera and Leica Q-FISH programme. FISH pictures, together with RAD51 and ASY1 immunostaining pictures, were taken on a Carl Zeiss Axioplan 2 equipped with Photometrics Quantix camera and MetaMorph 7.1.4.0 software (Molecular Dynamics). Pictures of microtubule immunostaining were taken with the confocal microscope Carl Zeiss LSM 510 Meta using the LSM 510 programme. Image deconvolution was performed with the Auto Deblur 9.2.1 software (AutoQuant Imaging). Alternatively, pictures were taken with a Leica DM5500B microscope equipped with a deconvolution system and a Leica DFC345 FX camera. Further processing of images was made with HeliconFocus 5.0 and Adobe Photoshop CS4 softwares.To analyse the substructures of immunosignals and chromatin beyond the classical Abbe/Raleigh limit at an optical resolution of ~120 nm (super resolution), structured illumination microscopy (SIM) was applied using a C-Apo × 63/1.2 W Korr objective of an Elyra microscope system and the software ZEN . Image stacks were captured separately for each fluorochrome using appropriate excitation and emission filters. Maximum intensity projections were generated from the stacks of optical SIM sections through the specimens by the ZEN software .G.C. and A.M. performed the experiments shown. P.S. and A.P.-H. conceived the experiments and G.C., A.M., A.P.-H. and P.S. analysed the data. P.S. wrote the manuscript with extensive contribution of all authors.How to cite this article: Cabral, G. et al. Chiasmatic and achiasmatic inverted meiosis of plants with holocentric chromosomes. Nat. Commun. 5:5070 doi: 10.1038/ncomms6070 (2014).Supplementary Figures 1-6Rhynchospora pubera, corresponding to Fig. 3a. Chromosomes in red, tubulin in green.Confocal planes of mitotic metaphase of Rhynchospora pubera,corresponding to Fig. 3c. Chromosomes in red, tubulin in green.Confocal planes of meiotic metaphase I of Rhynchospora pubera, corresponding to Fig. 3c. Chromosomes in red, tubulin in green3D view of meiotic metaphase I chromosomes and spindle of Rhynchospora pubera,corresponding to Fig. 3e. Chromosomes in red, tubulin in green.Confocal planes of meiotic metaphase II of


P < 0.001) and locoregional recurrence-free survival (LRFS) rate  compared with non-drinkers. Drinking ≥14 drinks/week, and drinking ≥20 years were both independent unfavorable prognostic factors for OS . Stratified analyses further revealed that the negative impacts of alcohol were manifested mainly among older patients and among smokers. In conclusion, alcohol drinking is a useful predictor of prognosis in male NPC patients; drinkers, especially heavy drinkers have poorer prognosis.The relationship between alcohol drinking and the prognosis of nasopharyngeal carcinoma (NPC) is unknown. To investigate the prognostic value of alcohol drinking on NPC, this retrospective study was conducted on 1923 male NPC patients. Patients were classified as current, former and non-drinkers according to their drinking status. Furthermore, they were categorized as heavy drinkers and mild/none drinkers based on the intensity and duration of alcohol drinking. Survival outcomes were compared using Kaplan–Meier analysis and Cox proportional hazards model. We found that current drinkers had significantly lower overall survival (OS) rate (5-year OS: 70.2% vs. 76.4%,  Nasopharyngeal carcinoma (NPC) is a unique head and neck cancer (HNC) endemic in southeast Asia, and is among the most common causes of cancer mortality in ChinaLifestyle factors, such as tobacco smoking and alcohol drinking, are established risk factors for the development of NPC4et al.16There have been a number of studies on the prognostic value of alcohol intake on HNC7891011121314In order to provide additional evidence of the impact of alcohol intake on NPC, we reviewed a large database of NPC patients in our hospital. Besides comparing drinkers (former and current) with non-drinkers, we also categorized patients according to intensity and duration of alcohol consumption to further analyze the associations between drinking and prognosis of NPC. Since female drinkers were few in our database, only male NPC patients were taken into analysis to avoid the confounding effect of gender.A retrospective review was conducted of NPC patients treated at Sun Yat-sen University Cancer Center (SYSUCC) between January 2001 and December 2004. Inclusion criteria were: (1) newly diagnosed, histologically proven NPC; (2) without distant metastasis; (3) receiving radical radiotherapy; (4) male patients. A total of 2008 patients were identified. Exclusion criteria were: (1) lack of the record of alcohol intake habits (n = 72); (2) patients younger than 18 years old (n = 13). Thus, the remaining 1923 male NPC patients were enrolled in this study. Computed tomography (CT) and/or magnetic resonance imaging (MRI) was essential for disease staging before treatment, and all patients were restaged according to the 7th edition of the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) staging system for NPCThis study was approved by the Institutional Review Board of SYSUCC, and was conducted in accordance with the guidelines of our institution. As this was a retrospective analysis of routine data, we were granted a waiver of written consent, and verbal informed consent was obtained from the patients. The information of patients’ alcohol intake, including drinking status, amount of alcoholic beverages intake per month, duration and quitting time, was collected by physicians at entry and by nurses during hospitalization at SYSUCC. Only the records consistent between both physician and nurse were considered credible. The amount of alcoholic beverages intake was initially recorded in jin, a traditional Chinese unit of weight which equals to 500 grams, and was converted into ounces  later. In our institution, the information on types of alcoholic beverages intake is not necessarily registered on admission; not all medical records collected this information. For the 2008 patients identified initially in our study, there were 210 cases that we needed to collected information on types of alcoholic beverages through telephone follow-up. The telephone follow-up was conducted in June 2014. 72 patients couldn’t be reached for inquiry. These patients were excluded (as mentioned above in the Exclusion criteria). If patients did not drink certain type of alcoholic beverages regularly, it was defined as random type. A medium serving size (one drink) was defined as 12 ounces of beer, 6 ounces of wine, 1.5 ounces of liquor, and an average amount, 6.5 ounces, of random typePatients who had consumed alcoholic beverages at least once a week for a minimun of 1 year were categorized as drinkers and the rest as non-drinkers. Drinkers were further categorized as former drinkers who quitted drinking for more than six months and the rest as current drinkers. Quantitative analysis of alcohol intake was evaluated by the intensity and duration of alcohol consumption. The intensity of alcohol intake was defined in terms of drinks per week (drinks/week). Patients with an intensity of ≥14 drinks/week or a duration of ≥20 years were divided as heavy drinkers, and the rest as mild/non drinkers.As smoking is strongly related to drinking, the status of smoking was also considered in this study. The information on smoking habits of patients, similar to that on alcohol intake habits, was complete and non-smokers were identified as those who never smoked and the rest as smokers6Details of the RT techniques used at SYSUCC were described previously6After completion of treatment, patients were followed up every 3 months for the first 3 years and the intervals gradually increased to 6–12 months after 3 years. The follow-up duration was calculated from the first day of treatment to the day of death or the last examination.The primary end point was overall survival (OS), and the secondary end point was locoregional recurrence-free survival (LRFS) and distant metastasis free-survival (DMFS). OS was calculated as time from start of treatment to death from any causes. LRFS and DMFS were calculated as time from start of treatment to the first occurrence of locoregional or distant failure, respectively.All analyses were performed using SPSS software, version 19.0. We compared the categorical variables in different groups using the Chi-square test. The rates of OS, LRFS and DMFS were estimated by means of the Kaplan-Meier method and differences were compared using the log-rank test. Multivariate analysis using a Cox proportional hazards model was used to test the independent significance of different variables by enter method of insignificant explanatory variables. The covariates entering into the multivariable analysis included host factors (age), tumor factors (T and N classification), treatment factors (chemo-RT or RT), smoking status, and smoking index.The baseline characteristics of the whole patients analyzed in this study are shown in The median duration of follow-up was 80.5 months . Up to the last day of follow-up, 579 (30.1%) of the 1923 patients developed locoregional failure, 368 (14.0%) developed distant metastasis and 593 (30.8%) died. For the entire cohort, the 5-year OS, LRFS and DMFS rates were 75.0%, 75.7% and 85.6%, respectively.P < 0.001) and smokers  (P = 0.023), but when dividing T classification into early (T1-2) and advanced (T3-4) ones, no significant correlation was found (P = 0.535). Among the 364 drinkers, the types of alcoholic beverages were 6 (1.6%) for beer, 9 (2.5%) for wine, 195 (53.6%) for liquor and 154 (42.3%) for random type. Detail information on alcohol intake in the 364 male NPC drinkers are shown in In total, 364 patients (18.8%) were drinkers, and drinking status had a strong and significant correlation with age and smoking status. Drinkers, as compared with non-drinkers, had a significantly higher percentage of older (≥47 years) patients  . T classP < 0.001) (P = 0.577). As compared with non-drinkers, the risks of death and locoregional recurrence were significantly higher for current drinkers (All P < 0.001) (P = 0.043) and LRFS   were former drinkers while 300 (82.4%) were current drinkers. Between non-drinker, former drinker and current drinker, significant differences were found in 5-year OS rate  and 5-year LRFS rate  (All < 0.001) , while n< 0.001) , and the< 0.001) . In the = 0.013) . No signP < 0.001) and LRFS rate  for heavy drinkers (≥14 drinks/week) were significantly lower than the corresponding rates for mild/none drinkers (<14 drinks/week) (P = 0.316). In the multivariate analysis, drinking ≥14 drinks/week was found to be an independent unfavorable prognostic factors for both OS  and LRFS   drank ≥14 drinks/week and 1792 (93.2%) drank <14 drinks/week. The 5-year OS rate  . No sign= 0.023) . No sign= 0.023) .P < 0.001) and LRFS rate  for heavy drinkers (≥20 years) were significantly lower than the corresponding rates for mild/none drinkers (<20 years) (P = 0.991). In the multivariate analysis, drinking ≥20 years was found to be an independent unfavorable prognostic factors for both OS  and LRFS   drank ≥20 years and 1740 (90.5%) drank <20 years. The 5-year OS rate  . No sign= 0.013) . No sign= 0.013) .As drinking was significantly correlated with age (≥47 vs.<47 years), and smoking status (smoker vs. non-smoker), we analyzed the prognostic value of alcohol intake in male NPC patients stratified by age and smoking status, respectively.P = 0.018, P = 0.008), and patients <47 years . Patients drinking ≥20 years also had significantly unfavorable OS and LRFS in patients ≥47 years , and patients <47 years , while patients drinking ≥14 drinks/week only had significantly unfavorable OS and LRFS in patients ≥47 years . In the univariate analysis, current drinkers had significantly unfavorable OS and LRFS when compared with non-drinker, in both patients ≥47 years , while had unfavorable OS only in smokers (P = 0.011). Patients drinking ≥14 drinks/week had significantly unfavorable OS and LRFS only in smokers , and patients drinking ≥20 years had significantly unfavorable OS and LRFS in both smokers  and non-smokers . For patients stratified by smoking status, univariate analysis indicated that when compared with non-drinkers, current drinkers had unfavorable LRFS in both smokers and non-smokers . Stratified analyses further revealed that the negative impacts of alcohol were manifested mainly among older patients and among smokers.et al.16P < 0.001 in univariate analysis and P = 0.06 in multivariate analysiset al. also noted the associations between the frequency (>1 drink/day) and duration (≥20 years) of alcohol drinking and poor survival of NPC patients in univariate analysis , but both lost significance after adjustment for clinical variables and demographic characteristicsMany reports have discussed the prognostic significance of alcohol drinking for HNC. In an article by Broglie P = 0.091, P = 0.122)  . The inset al. showed that ethanol consumption can invoke a strong depletion of body fat, facilitate wasting and shorten survival time of tumor-bearing mice25et al.Why alcohol drinkers with NPC had unfavorable prognosis ? Besides the increased possibility of smoking in this group of people22In this study, we reviewed a large cohort of male NPC patients (n = 2008) from a single institute, and enrolled 1923 of them for analysis. To the best of our knowledge, this is the first report about the significant impact of status, intensity and duration of alcohol drinking on the prognosis of NPC. The limitations of our study are related to its retrospective nature. We found it difficult to obtain the exact amount of alcohol consumption, since most people drink more than one kind of alcoholic beverage with different alcohol proofs. Different kinds of alcoholic beverage (such as wine and liquor) may have opposite effects on the prognosis of malignanciesNevertheless, this study adds to evidences supporting the unfavorable impact of heavy drinking on the prognosis of NPC patients. Further studies are required to assure the prognostic effect of alcohol drinking, and establish optimal therapeutic regimens for these patients.How to cite this article: Chen, Y.-P. et al. Alcohol drinking as an unfavorable prognostic factor for male patients with nasopharyngeal carcinoma. Sci. Rep.6, 19290; doi: 10.1038/srep19290 (2016).


Diabetes mellitus (DM), a kind of metabolic disease, is increasing over the last four decades in the world. The purpose of this study was to investigate the effect of Jiang Tang Xiao Ke (JTXK) granule, a naturally occurring ingredient from Chinese herbal medicines, on serum glucose, lipids, and oxidative stress in DM rats induced by high-fat diet and streptozotocin. JTXK granule 9 g/kg  and pioglitazone 1.5 mg/kg (as a positive control for comparison) were orally administrated to DM rats for 4 weeks. Results showed that administration of JTXK granule reduced serum glucose, total cholesterol, triglyceride, and low density lipoprotein levels  but increased high-density lipoprotein level by 69%, compared with the drug-untreated DM rats. Serum malondialdehyde and nitric oxide levels were lowered  associated with the elevation in serum superoxide dismutase levels (by 60%) after JTXK granule treatment. In addition, JTXK granule suppressed serum alanine aminotransferase activity (up to 50%) and alleviated pathological changes of pancreas and liver tissues in DM rats. The beneficial changes of pioglitazone on biomarkers were also found in DM rats. These findings suggested that JTXK granule may be an alternative medicine for the management of DM. Ginseng and Rhizoma coptidis are famous not only for their wide application in treatment of DM but also for the profound research of their antidiabetic mechanisms [Diabetes mellitus (DM) is a multifactorial metabolic disorder characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion and/or insulin action. In 2013, according to International Diabetes Federation, 381 million people suffered from diabetes, which was estimated to almost double by 2030 . Althougchanisms .β-cell dysfunction, which can lead ultimately to diabetes [Up to now, various studies have been carried out to identify the underlying mechanism of DM , 11. Incdiabetes . Jiang Tdiabetes . Current Radix rehmanniae (Di Huang), Fructus corni (Shan yu rou), Ginseng (Ren Shen), Radix salviae miltiorrhizae (Dan Shen), and Rhizoma coptidis (Huang Lian) with a proportion of 3 : 1 : 1 : 3 : 1. The raw herbs were purchased from Beijing Tong Ren Tang medicinal materials Co., Ltd.  and authenticated by Professor Chun-Sheng Liu in the Beijing University of Chinese Medicine. For the preparation of the aqueous extract of JTXK granule, the herbs  were boiled in twelve volumes of distilled water for 1 h. The procedure was repeated three times. The pooled aqueous extract was filtered through gauze cloth and the filtrate was evaporated by heating until the relative density reached 1.15. For the preparation of the ethanolic extract of JTXK granule, the herbs  were extracted three times with twelve volumes of 60%  ethanol under reflux. A final yield of 20% (w/w)  was obtained. JTXK granule was made from the pooled aqueous and ethanolic extracts and then stored at 4°C until use.JTXK granule mainly consists ofPioglitazone pills, the positive control drug used in this study, were purchased from Beijing Taiyang pharmacy Co. Ltd. . Streptozotocin  was bought from Sigma-Aldrich Chemical Co., Ltd. . STZ was dissolved into 0.1 mol/L sodium citrate-hydrochloric acid buffer when used. Insulin ELISA assay kits were purchased from Beijing north biotechnology research institute . Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO) kits were purchased from Nan Jing Jian Cheng biological research institute .Male Sprague Dawley rats, weighing 180−200 g, were purchased from Beijing Wei Tong Li Hua experimental animal center (certification number SCXK (Jing) 2012-0001). The animals were housed under the clean level conditions (certification number SCXK (Jing) 2011-0024) with the temperature of 22 ± 1°C, humidity of 55 ± 5%, and 12 : 12 h light/dark cycle in Beijing University of Chinese medicine. All rats were allowed free access to tap water and food. The high-fat diet (HFD) containing 20% sucrose, 2.5% cholesterol, 10% lard, 0.3% sodium cholic acid, and 66.5% (w/w) in standard feed was provided by Ke'ao xieli feed Co., Ltd. .The rats subjected to the experiments were allowed to adapt to the environment for a week. Ten animals were chosen and fed with standard diet as the normal control group. The other 35 rats were fed with HFD for four weeks, and then a single intraperitoneal injection of a prepared solution of STZ (30 mg/kg suspended in 0.1 mol/L citrate buffer at pH 4.5) was applied to induce diabetic models. If the volume of fasting blood glucose (FBG) was not less than 16.7 mmol/L after 72 hours of STZ injection, the diabetic models were successful. One week late, DM rats were randomly divided into 3 groups of 10 animals in each: (1) drug-untreated DM rats and (2) and (3) DM rats treated with pioglitazone 1.5 mg/kg and JTXK granule 9 g/kg, respectively. Both drugs were dissolved in distilled water and given to DM rats via gastro gavage once a day. The normal and drug-untreated DM rats were administrated with the same volume of vehicle. The study protocol was approved by the animal ethics committee of Beijing University of Chinese medicine, .Before and after the drug administration, the fasting blood glucose (FBG) and random blood glucose (RBG) levels in the tail vein were monitored using a glucometer (Johnson & Johnson). At the end of the experimental period, rats were anesthetized with ether after 12 h of fasting. Serum samples were prepared by centrifuging the clotted blood collected from the abdominal aorta and then centrifuged at 3,000 rpm/min for 15 min. The serum fasting insulin (FINS) levels were determined according to the manufacturer's instruction, and insulin sensitivity index (ISI) was calculated according to the formula as follow:Serum TC, TG, HDL, and LDL levels, as well as ALT activity, were determined with automatic biochemistry analyzer . Serum SOD activity and the volume of MDA and NO were measured using commercially available kits.Rats were deprived of food overnight and a baseline (0 min) blood glucose level was measured. Then a single dose of glucose (2 g/kg) was dissolved in 1 mL of water and administered by gavages. Over the following 30 min, 60 min, and 120 min, the blood samples were taken from the tail vein and used to detect the glucose levels, respectively .After 4 weeks of drug treatment, the pancreas and liver were removed and fixed in 10% neutral buffered formalin. The organs were routinely processed and sectioned at 4-5 mm thickness. Sections of pancreas and liver were stained with hematoxylin and eosin (HE) and examined by light microscopy in order to demonstrate the histopathological changes of DM rats. The photomicrographs of each tissue section were taken on laboratory microscopy .P < 0.05 was considered to be statistically significant.SPSS version 17.0 software  was used for statistical analysis. All data were presented as Mean ± SE. Statistical significance among groups was determined by one-way analysis of variance (ANOVA) followed by Duncan's analysis to compare various groups with each other. To evaluate the effect of JTXK granule on glucose homeostasis, the FBG and RBG were measured as routine protocols. FBG and RBG levels in DM rats were significantly higher (4 folds higher) than the normal rats, which indicated that the rat model of DM was successfully established. JTXK granule and pioglitazone treatment for 4 weeks reduced both FBG  and RBG , compared with before medications. JTXK granule decreased FBG and RBG  compared with untreated DM rats, while pioglitazone decreased FBG and RBG  .OGTT is a more physiological method of assessing the glucose induced insulin secretion and glycemic control. After treatment with JTXK granule and pioglitazone for 4 weeks, blood glucose levels significantly decreased  at 120 min of glucose load, compared with the drug-untreated DM rats .Although there were no differences in serum FINS levels between normal and DM rats, ISI was markedly reduced in DM rats (by 23%), compared with the normal rats. Pioglitazone and JTXK granule treatment did not alter the serum FINS levels in DM rats. However administration of pioglitazone and JTXK granule increased the ISI by 7% and 5% in DM rats, respectively .As shown in As shown in In light microscopy, the normal rats showed typical histological structure with normal islet A. The siCompared with the normal rats, DM rats lost their body weight (up to 30%) at the end of the experiment. Administration of JTXK granule and pioglitazone for four weeks improved the body weight loss in DM rats .β-cells, which associates strictly with the induction of oxidative stress, both systemically and locally.Among all patients with DM, type-2 diabetes mellitus (T2DM) makes up about 90% of the cases. Immense amounts of research on mechanisms and control of T2DM have been launched considering its increased levels of incidence and associated mortality. Several DM models were constructed and explored in these researches . Among tAccording to traditional Chinese medicine (TCM) theory, it is considered that the main pathogenesis of T2DM is due to the malfunction of liver, spleen, and kidneys organ systems. The treating principle of JTXK granule is to restore the function of the organs in TCM point of view. Accumulating evidence suggests that the main ingredients of JTXK granule, such as berberine, tanshinone, and catalpol, can not only reduce the FBS and lipid levels but also regulate the oxidative stress in the body according to the pharmacological study of modern medicine. Therefore, JTXK granule has potency to control glucose and lipid metabolism and relieve symptoms of diabetes. However, mechanism of the formulated JTXK granule for preventing and controlling the development of DM remains to be investigated.γ. It can increase glucose uptake and utilization in the peripheral organs and decrease gluconeogenesis in the liver through increasing glucose transporters 1 and 4, lowering free fatty acids and remodeling of adipose tissue [ Ginseng, were efficacious for treating hyperglycaemia [In the present study, DM rats induced by HFD and STZ showed impaired glucose tolerance and stable fasting and random hyperglycemia compared with the normal rats. Four weeks administration of pioglitazone and JTXK granule improved oral glucose tolerance and reduced FBG and RBG levels in DM rats. Some researchers found that the serum fasting insulin level elevated  in diabee tissue . In accolycaemia , 24, whilycaemia . In the α [Hyperlipidemia is an important contributor to insulin resistance, and hence reduction of lipid profiles is helpful in the remission of DM . The hypα , but theα . ALT, wh2− within the physiological concentrations by converting superoxide anion radicals produced in the body to hydrogen peroxide [Increasing evidence implicates the role of oxidative stress in the different stages of the development of DM, starting from the prediabetes state, impaired glucose tolerance, and overt diabetes mellitus to diabetic complications states . As it iperoxide , 32. ItsGenerally, NO at physiological levels produces many benefits to the body. Metabolic disorders of diabetes influenced the content and the activity of NO through NO/cGMP pathway, implicating the elevation and following decrease of NO level in the early and late stage of diabetes . HyperglIn conclusion, JTXK granule, a Chinese medicinal formula, at 9 g/kg  treatment for 4 weeks reduced serum glucose via increasing insulin sensitivity and protection of pancreas islets in DM rats. In addition, the JTXK granule decreased serum TC, TG, and LDL levels but increased HDL levels, compared with the drug-untreated DM rats. At the same time, JTXK granule showed improved antioxidant activity, which was manifested by decreased MDA and NO levels and with elevation in SOD levels in DM rats. Islet morphology showed marked improvement in DM rats treated with JTXK granule. These findings suggested that JTXK granule may be an effective and safe alternative treatment for T2DM.


Alphaproteobacteria decreased with soil depth. Among the soil properties measured, soil pH was the most significant factor correlating with bacterial community in both upper and lower-layer soils. Bacterial community similarity based on jackknifed unweighted unifrac distance showed greater similarity across horizontal layers than through the vertical depth. This study showed that soil depth and pH were the most important soil properties determining bacterial community structure of the subarctic tundra soil in Council, Alaska.The subarctic region is highly responsive and vulnerable to climate change. Understanding the structure of subarctic soil microbial communities is essential for predicting the response of the subarctic soil environment to climate change. To determine the composition of the bacterial community and its relationship with soil properties, we investigated the bacterial community structure and properties of surface soil from the moist acidic tussock tundra in Council, Alaska. We collected 70 soil samples with 25-m intervals between sampling points from 0–10 cm to 10–20 cm depths. The bacterial community was analyzed by pyrosequencing of 16S rRNA genes, and the following soil properties were analyzed: soil moisture content (MC), pH, total carbon (TC), total nitrogen (TN), and inorganic nitrogen ( Evidencet al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Understanding the soil microbial community structure is essential to elucidate microbial processes. The bacterial communities have been characterized in various arctic soil environments including subarctic regions using culture-independent techniques, such as DGGE, T-RFLP, clone libraries, and next-generation sequencing  of the University of Alaska Fairbanks (UAF).c. 30 m above sea level, and the annual mean air temperature and precipitation are −3.1 ± 1.4 °C and 258 mm, respectively . At the time of sampling (mid-August 2011), the depth of the active layer was c. 50–70 cm. The sampling site is composed of moist acidic tussock tundra, and the dominant vegetation was cotton grass (Eriophorum vaginatum) or tussock, blueberry (Vaccinium uliginosum), and lichen and moss (Sphagnum spp.) beds.The study site is located in Council, on the Seward Peninsular in Northwest Alaska , which is a subarctic region. The site is c. 300 × 50 m. The points were spaced at 25-m intervals, resulting in a latticework of 12 points × 3 points. Before acquiring soil samples, we removed the aboveground vegetation and litter layer, and cleaned the shovel with 70% ethanol to prevent contamination between samples. At each site, soil samples were collected from a depth of 0–10 cm (upper-layer soil) and a depth of 10–20 cm (lower-layer soil). The collected soil samples were wrapped in autoclaved foil, placed in ziplock bags, and transported to the laboratory in a frozen state. The soils in sites 4 and 35 were saturated with water, and only the upper-layer soil was collected. Overall, 70 soil samples were collected for this study. The collected soil samples were stored at −20 °C until DNA extraction and soil analysis.Thirty-six sampling points were selected over an area of c. 7 g of fresh soil was immediately set aside after sampling and kept frozen until extraction. Inorganic N (Gravimetric MC was determined by measuring the difference in weight between the field-moist soil samples and the same soil samples dried at 105 °C for 48 h. For inorganic nitrogen (N) analyses,  Thomas, . Soil waTo extract genomic DNA from the soil samples, the soils were subsampled from the bulk soil, freeze-dried, and homogenized. Genomic DNA was extracted from 0.5 g of the homogenized soil samples using a FastDNA® SPIN kit for soil  and a QuickPrep adapter , according to the manufacturer's recommended protocol. The total DNA was quantified by Hoechst dye 33258 staining using a spectrophotometer with excitation and emission at 350 and 460 nm, respectively . The extracted DNA was stored at −20 °C until further analysis.−1), and 22.6 μL of deionized distilled water (DDW). The PCR program was as follows: an initial denaturation step at 95 °C for 3 min followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 90 s, with a final extension at 72 °C for 7 min. All samples were amplified in triplicate, pooled in equal amounts, and purified using the QIAquick PCR Purification Kit (Qiagen). The PCR products were quantified with a NanoDrop. DNA sequencing was performed by DNALink  using a GS-FLX 454 pyrosequencer (Roche).Genomic DNA extracted from the soil was amplified by PCR using the adapter-multiplex identifier–primer combinations targeting the V1–V3 regions (27F–518R) of bacterial 16S rRNA gene  length of 378 ± 45 bp were clustered based on operational taxonomic units (OTUs) at 97% similarity using uclust  Sequence Read Archive database .r  and primer-e v6  with 999 permutations and nonmetric multidimensional scaling (nmds) were conducted to compare bacterial community structure. Classification and regression tree (cart) analyses were conducted using rpart in the r software package  to determine which environmental variables explained the deviance of the dominant bacterial groups. A Mantel test was used to determine which physical and chemical properties of soil were significantly correlated with the bacterial community. To assess how the bacterial community changed with sampling distance (c. 22–427 m), a distance-decay relationship analysis, which assumes that community similarity will decrease with increasing geographical distance, was performed, as described in Martiny et al. , Shannon (P < 0.001), and Simpson (P < 0.05) indices  from all the soil samples. On average, we obtained 1311 sequences  per sample. When we compared the soil bacterial communities using the same number of reads (700 sequences per sample), bacterial abundance and bacterial diversity were significantly higher in the upper-layer soils, as indicated by the Chao1 , Gemmatimonadetes, Cyanobacteria, Spirochetes, Fibrobacteres, Caldiserica (former candidate division OP5), Lentisphaerae, and Epsilonproteobacteria were also identified at relatively low abundances, as well as members of 27 candidate phyla and several unclassified bacteria  plots indicated that the bacterial communities showed greater similarity across horizontal layers than through vertical depth  between the two depths. Bacterial communities were similar between sampling sites .In general, the bacterial community structures in the upper and lower-layer soils were different. er soils . At the yer soil . The 16Sr = 0.392, P < 0.001) with bacterial community composition in all soil samples , MC , TC , and TN  showed significant correlation with the overall soil bacterial community composition  and r = 0.167, P < 0.05) were significantly associated with the community composition, whereas C/N ratio  and MC  were significantly associated with the community composition in the lower-layer soils  than across the horizontal layers (> 25 m). Soil depth is one of the major parameters influencing microbial community. Waldrop & Harden  showed t0 cm thanAlphaproteobacteria decreased with depth, although those of Acidobacteria and Actinobacteria were similar in both soil layers  from the statistical analyses, the minor groups also showed significant correlations with soil pH (P < 0.001) in both upper- and lower-layer soils , and Legionellales (Gammaproteobacteria) increased with increasing soil pH, whereas Ktedonobacteria (Chloroflexi) decreased with increasing soil pH  and OTU_2 (98.5% sequence similarity with Pseudolabrys sp.) were predominant in this study  which are known to fix nitrogen as plant root symbionts. The genus Steroidobacter of Gammaproteobacteria can reduce nitrate to dinitrogen monoxide and further to dinitrogen   were detected in this study as well (The higher resolution of pyrosequencing data allowed us to look into the information on the potential ecological roles of bacteria in the subarctic tundra soil. At the species level, OTU_1 (99.5% sequence similarity with  as well . These ret al. (et al. (et al., et al., et al., The distance-decay relationships showed that bacterial community similarity decreased with increasing sampling distance in lower-layer soils, whereas no significant relationship was detected in the upper-layer soils Fig . Decreaset al.  reported (et al.  also obsIn summary, soil depth and pH were the most influential soil properties to determine the bacterial community structure in subarctic tundra soil in Council, Alaska. Although various plants covered the top soil, the bacterial communities were relatively similar across the horizontal layers compared with the communities through the vertical depth. Bacterial communities were more significantly correlated with soil pH than the other measured soil properties. These results indicated that the bacterial communities in this study differed at the two different soil depths and were relatively stable against various soil properties except soil pH. Moreover, we found that certain phylogenetic groups at lower taxonomic levels showed a different response to pH from that at the phylum level. This indicated the necessity of analyzing bacterial communities at lower taxonomic levels such as species, which actually perform various functions in the environment. More metagenomic and transcriptomic studies are needed to understand the community structure of other soil microorganisms such as archaea and fungi, and the ecological functions of microorganisms in tundra soil.


These results suggest that compared to an oatmeal-based breakfast, eggs do not have any detrimental effects on lipoprotein or glucose metabolism in T2DM. In contrast, eggs reduce AST and TNF-α in this population characterized by chronic low-grade inflammation.There is concern that egg intake may increase blood glucose in patients with type 2 diabetes mellitus (T2DM). However, we have previously shown that eggs reduce inflammation in patients at risk for T2DM, including obese subjects and those with metabolic syndrome. Thus, we hypothesized that egg intake would not alter plasma glucose in T2DM patients when compared to oatmeal intake. Our primary endpoints for this clinical intervention were plasma glucose and the inflammatory markers tumor necrosis factor (TNF)-α and interleukin 6 (IL-6). As secondary endpoints, we evaluated additional parameters of glucose metabolism, dyslipidemias, oxidative stress and inflammation. Twenty-nine subjects, 35–65 years with glycosylated hemoglobin (HbA1c) values <9% were recruited and randomly allocated to consume isocaloric breakfasts containing either one egg/day or 40 g of oatmeal with 472 mL of lactose-free milk/day for five weeks. Following a three-week washout period, subjects were assigned to the alternate breakfast. At the end of each period, we measured all primary and secondary endpoints. Subjects completed four-day dietary recalls and one exercise questionnaire for each breakfast period. There were no significant differences in plasma glucose, our primary endpoint, plasma lipids, lipoprotein size or subfraction concentrations, insulin, HbA1c, apolipoprotein B, oxidized LDL or C-reactive protein. However, after adjusting for gender, age and body mass index, aspartate amino-transferase (AST) ( Epidemiological studies report controversies on the effects of dietary cholesterol and egg intake on the risk for heart disease in patients with diabetes ,2. ThereOatmeal is recognized as a heart-healthy breakfast due to the effects of β-glucan on reducing plasma LDL cholesterol (LDL-C) . OatmealIt has been documented that the Latino population has a genetic predisposition for developing T2DM . In Mexiversus 1/2 cup (40 g) of oatmeal per day on plasma glucose and inflammatory markers, our primary endpoints. Our secondary endpoints included plasma lipids, markers of oxidative stress, and parameters of glucose metabolism, such as glycosylated hemoglobin (HbA1c). We hypothesized that eating one egg per day would not adversely affect primary or secondary endpoints when compared to an oatmeal breakfast. We further hypothesized that eggs would reduce inflammatory markers in this population, likely due to the presence of highly bioavailable carotenoids [We aimed to compare two breakfasts with perceived differences in effects on heart disease risk, eggs and oatmeal, by conducting a randomized, crossover clinical trial in subjects with T2DM. We evaluated the effects of consuming one egg per day for a relatively extended period (five weeks) otenoids . Due to otenoids , we chosWe recruited 33 subjects (aged 35–65 years) diagnosed with T2DM to participate in this randomized crossover design study in the city of Hermosillo, Mexico. Based on the standard deviation from our previous studies where we observed changes in inflammatory markers and using a Z value of 1.96 , we estimated that 25 subjects would be sufficient to observe differences between groups ,21. We an = 26) and insulin (n = 6). In addition, 18 subjects were taking blood pressure medication and 9 were on reductase inhibitors. The intervention protocol was approved by the University of Connecticut Institutional Review Board, the Ethical Committee from Centro de Investigacion en Alimentacion y Desarrollo (CIAD), and the Review Board from Hospital Chávez. All subjects gave written informed consent prior to initiating the study. This study is registered at Clinicaltrials.gov Twenty-nine subjects finished both dietary interventions. Three subjects dropped out of the study due to personal reasons and 1 subject was removed due to non-compliance with the dietary treatments. In order to maintain control of T2DM, all subjects were taking glucose-lowering medications, as prescribed by their physician, including metmorfin  of lactose-free milk. We provided lactose-free milk to control for potential lactose intolerance, which is very common in adults from Hispanic origin. The average amount of calories consumed for breakfast was 313 kcal/day for the egg period and 335 kcal/day for the oatmeal period. An exercise diary was also provided to subjects to ensure that there were no changes in their activity level during the interventions. Subjects provided 3 exercise dairies at the end of each breakfast period. All participants were very closely monitored by their physician (HV) throughout the whole study to ensure that they did not change their medications. They were also monitored by the researchers to ensure compliance with egg and oatmeal intake, and to ascertain that they did not change the rest of their dietary habits or their level of physical activity during the 13-week intervention.2. Body fat was measured by electric bioimpedance using an Impedimed IMP5™ . Blood pressure was measured with an automated blood pressure monitor  after a 5 min rest. The average of 3 separate recordings is reported.Weight was measured to the nearest 0.1 kg, height was measured to the nearest centimeter, and body mass index (BMI) was calculated as kg/mg to measure plasma total cholesterol (TC), LDL (LDL-C), HDL (HDL-C) and triglycerides by using the Cobas c-111 Clinical Analyzer . Oxidized-LDL was measured by using an ELISA kit from Mercodia  and Apolipoprotein B by utilizing an ELISA kit from Abcam .Subjects fasted 12 h prior to blood draws. Plasma was separated from red blood cells by centrifugation at 2400× Fasting glucose concentrations, one of our primary endpoints, was analyzed by an enzymatic colorimetric method . Plasma insulin was determined with an ELISA method . The homeostasis model assessment (HOMA) was used to calculate insulin resistance . HbA1c wLipoprotein subclass profiles and diameters were measured by proton NMR spectroscopy as previously reported . This meAlanine aminotransferase (ALT), aspartate aminotransferase (AST), and C-reactive protein (CRP) were measured using the Cobas c-111 Clinical Analyzer. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the other two primary endpoints, were measured utilizing ELISA kits . Adiponectin was measured by using a Quantikine ELISA by RD Systems Inc. .t-test was used to evaluate differences in all measured parameters between the oatmeal and the egg periods. Values are reported as mean ± SD. A p-value < 0.05 was considered to be significant. Those parameters that were significantly different by paired t-test were further analyzed by using gender, age and initial BMI as covariates.SPSS version 13.2 was used for Statistics. A paired n = 16) or oatmeal (n = 17). Before the crossover, three patients decided not to continue the study and one was removed by the investigators due to non-compliance. Twenty-nine patients finished the whole intervention. All measured variables presented are for these 33 patients.As indicated in n = 27) had plasma TC < 2.5 mmol/L, LDL-C < 1.2 mmol/L, and blood pressure < 130/85 mmHg. Plasma triglycerides were high with a mean 2.2 ± 1.2 mmo/L, typical of a diabetic population.The baseline characteristics of subjects are presented in p = 0.051). (Results for the primary endpoints are presented in  0.051). B.p < 0.01), while percent of energy from total fat, and saturated, monounsaturated and polyunsaturated fatty acids (g) were higher during the egg period (p < 0.001). Total and soluble fiber intake was higher during the oatmeal period (p < 0.01), while dietary cholesterol was higher during the egg period (p < 0.01)  . Interes < 0.01) .versus oatmeal periods (p > 0.05) (data not shown). There were no significant differences in body weight, body fat, BMI or blood pressure between the egg and oatmeal periods . Similar periods . Values There were no differences in total lipoprotein number or concentrations of lipoprotein subclasses between the egg and the oatmeal periods including the atherogenic lipoproteins large VLDL, IDL and small LDL .vs. 6.87 ± 8.98 mg/L), adiponectin (6.2 ± 3.2 vs. 5.6 ± 2.2 μg/mL), and ALT 24.3 ± 11.7 vs. 26.3 ± 13.0 IU/L) were not different when compared at the end of egg and oatmeal periods, respectively. However, AST was significantly reduced following the egg period (p < 0.05). Data for liver enzymes is presented in CRP  can be easily incorporated into the diets of patients with T2DM, with no apparent concerns for causing dysregulation of glucose metabolism or formation of atherogenic particles. The beneficial effects on inflammatory markers confirm what we have observed in past studies regarding the effects of eggs in decreasing inflammation in other populations at high risk for heart disease and diabetes, including obesity  and metaOatmeal has been shown to decrease LDL-C in healthy and hypercholesterolemic individuals  in cliniet al. [Contrary to the findings of Pearce et al. , who weret al. . Althouget al. , and theet al. , we failThese results indicating a lack of effect of egg and oatmeal consumption on plasma lipids and lipoproteins may be attributable to several factors. In our previous studies, we challenged our subjects with two to three eggs per day ,26,32, wIn addition to maintaining fasting plasma glucose levels, our main finding in this study is that TNF-α and AST were reduced following the 5-week, one-egg-per-day breakfast. Chronic low-grade inflammation is a hallmark of diabetes and diabetic patients are characterized by having elevated concentrations of inflammatory markers ; thus, tThe main strength of this study is that it was a controlled clinical intervention. We performed thorough dietary recalls and ensured that subjects did not change their activity level during the intervention period. We also provided the intervention foods and monitored consumption. In addition, the recruited subjects had good diabetic control as monitored by their HbA1c plasma concentrations, as well as the absence of extreme diabetes complications, which was a key element for subject compliance. Further, we ensured that subjects did not change their medication dose/type during the 13-week intervention. The weaknesses of this study include the short duration of the trial, which might not have been sufficient to determine changes in some of the measured parameters, and the fact that the data cannot be extrapolated for those diabetic patients with uncontrolled diabetes or additional complications associated with T2DM. Another perceived weakness is the lack of blinding of the subjects and that no intention to treat analysis was performed.In this study, we have shown that eggs should not be a concern for those individuals with T2DM when HbA1c levels are well controlled. When compared to an oatmeal breakfast, one egg per day did not result in changes in plasma glucose, our primary end point, LDL-C, triglycerides or in the concentration of atherogenic particles including large VLDL, IDL or small LDL. Further, there were no changes in plasma insulin, HOMA or HbA1c, indicating that eggs can be consumed without any detrimental changes in lipoprotein or glucose metabolism in this population. The most interesting finding, however, was that eggs—possibly due to their content of highly bioavailable lutein and zeaxanthin—reduced inflammation in diabetic subjects when compared to oatmeal intake.


Inorganic arsenic exposure has been related to the risk of increased blood pressure based largely on cross-sectional studies conducted in highly exposed populations. Pregnancy is a period of particular vulnerability to environmental insults. However, little is known about the cardiovascular impacts of arsenic exposure during pregnancy.We evaluated the association between prenatal arsenic exposure and maternal blood pressure over the course of pregnancy in a U.S. population.The New Hampshire Birth Cohort Study is an ongoing prospective cohort study in which > 10% of participant household wells exceed the arsenic maximum contaminant level of 10 μg/L established by the U.S. EPA. Total urinary arsenic measured at 24–28 weeks gestation was measured and used as a biomarker of exposure during pregnancy in 514 pregnant women, 18–45 years of age, who used a private well in their household. Outcomes were repeated blood pressure measurements  recorded during pregnancy.p = 0.022) increase in systolic blood pressure per month and a 0.14-mmHg  increase in pulse pressure per month over the course of pregnancy.Using linear mixed effects models, we estimated that, on average, each 5-μg/L increase in urinary arsenic was associated with a 0.15-mmHg (95% CI: 0.02, 0.29; In our U.S. cohort of pregnant women, arsenic exposure was associated with greater increases in blood pressure over the course of pregnancy. These findings may have important implications because even modest increases in blood pressure impact cardiovascular disease risk.http://dx.doi.org/10.1289/ehp.1408472Farzan SF, Chen Y, Wu F, Jiang J, Liu M, Baker E, Korrick SA, Karagas MR. 2015. Blood pressure changes in relation to arsenic exposure in a U.S. pregnancy cohort. Environ Health Perspect 123:999–1006;  Millions of individuals are chronically exposed to inorganic arsenic via contaminated water sources and through diet . In the Cardiovascular disease is the leading cause of morbidity and mortality worldwide , and assPregnancy profoundly alters both maternal anatomy and physiology to support fetal development . PregnanIn New Hampshire, about 40% of households rely on unregulated private water systems, of which 10–15% contain arsenic levels exceeding the maximum contaminant level . As partThe New Hampshire Birth Cohort. In January 2009, we began recruiting 18- to 45-year-old pregnant women receiving prenatal care at study clinics, as previously described  and diastolic (DBP) BP was measured in the study clinics, using either automated or mercury sphygmomanometers, throughout pregnancy and was generally recorded at each prenatal visit.Arsenic exposure assessment. Women provided a spot urine sample upon enrollment, which was collected and stored as previously described (III), arsenate (iAsV), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine by high-performance liquid chromatography (HPLC) inductively coupled plasma mass spectrometry (ICP-MS) at the University of Arizona Hazard Identification Core (III + iAsV) and organic  metabolites  during pregnancy, which were analyzed as continuous variables with repeated measurements. For each measurement, we calculated the trimester and gestational week, based on the participant’s last menstrual period. We restricted our analysis to measurements taken after 13 weeks gestation due to the low number of measurements recorded before this time. Measurements outside of a reasonable range  (n = 9). All other values recorded for these women were within a physiologically reasonable range. There were few cases of diagnosed pregnancy-induced hypertension (n = 15) or preeclampsia (n = 9) in our study population; thus, it was not possible to analyze these outcomes separately.80 mmHg)  were likWe fitted mixed-effect models  of the rij = [β0 + β1(TIME)ij + β2As0j + β12As0j(TIME)ij + αTZ0j] + [μ0j + μ1j(TIME)ij] + ijr, [1]BPij represents BP at time i for subject j, As0j is urinary arsenic  at baseline  for subject j; TIME is gestational month of BP measurement; β1 is the coefficient for the association between TIME and BP when arsenic is held constant; β2 is the difference in BP for every unit increase in arsenic at baseline; β12 is the difference in monthly BP change over pregnancy per unit increase in arsenic ; αT is a row vector of regression coefficients for covariates at baseline (T denotes vector transpose); Z0j is a vector of covariates at baseline. The random intercept μ0j and slope μ1j estimated the within-subject correlation among repeated measurements and between-subject heterogeneity, and ijr is the error that cannot be accounted for by other covariates and random effects. The terms in the first and second brackets are the fixed and random parts of the model, respectively. We assessed nonlinear trends in the data using the same modeling strategy described above, including model terms to examine the interaction between TIME and categories of arsenic exposure variables , as well as linearity of the time effect by including an additional interaction term between As0j and TIME2. Neither test provided evidence of a nonlinear association (p > 0.05). For ease of interpretation, 5 μg/L (~ 1 SD) was used as the unit to report effect estimates for total urinary arsenic and metabolite levels.where BPa priori considerations, including age at enrollment, prepregnancy body mass index (BMI), smoking during pregnancy, marital status, educational attainment, gestational diabetes, parity, and number of BP measurements. As described above, we included the month of gestation during which each BP measurement was obtained in our models. We considered pregnancy BP measurements after 13 weeks gestation (our baseline) in our models because few subjects received BP measurements prior to that time point. Urinary arsenic concentrations were used as a measure of gestational arsenic exposure because urine samples earlier in pregnancy were not available and prior studies suggest that total arsenic concentrations remain relatively stable : 0.02, 0.29] with creatinine adjustment}, as well as with or without arsenobetaine adjustment  (data not shown). For individuals with missing covariate data (r < 0.20) (data not shown) and thus were not adjusted for in our analysis.Our models were adjusted for available covariates that could potentially influence BP based on y stable . Becauseate data , we usedate data . We examate data . We alsoate data , all had2).We conducted analyses stratified by PMI or SMI, using the median values  as cut points, to assess whether the association between urinary arsenic and BP changes over time differed by these arsenic methylation indices. We also performed analyses stratified by age (below or at/above a median of 30.9 years), history of prior pregnancy (nulliparous or parous), prepregnancy BMI .Because BP increases over the latter part of pregnancy , we furta priori selection requirement, we required women to have at least two BP measurements taken during pregnancy; however, all 527 women in this sample had a minimum of four measurements. An additional 13 women with a history of hypertension were excluded, resulting in a final sample size of 514. This subset was similar to the overall cohort (n = 620) with respect to demographic and lifestyle variables (data not shown). Women in this sample had urinary arsenic concentrations ranging from 0.35 to 288.5 μg/L, which was very similar to the range observed in the overall cohort (0.08–288.5 μg/L). Of the subset of participants included in these analyses, 472 had provided water samples, 463 of which had been analyzed at the time of this study and had a mean arsenic level of 4.3 μg/L . Nearly 1 in every 8 households  tested in this subsample had water arsenic levels > 10 μg/L. In our study group, well water arsenic levels were significantly correlated with urinary arsenic measurements . No differences in the descriptive variables were observed across urinary arsenic tertiles by chi-square or one-way analysis of variance (ANOVA) tests, except for water arsenic levels, in which the third tertile was higher than the first tertile . On average, 13  BP measurements were recorded per participant during pregnancy, with more than half occurring in the last trimester. Both SBP and DBP increased during pregnancy (p = 0.022) and a 0.14-mmHg greater monthly increase in PP  over the course of pregnancy ; DMA β12, 0.15 ; iAs β12, 1.18 ] ] over the course of pregnancy.We observed no association between urinary arsenic and differences in DBP change over pregnancy. Arsenic exposure was related to greater monthly increases in SBP and PP change over the course of pregnancy . Based oregnancy . In sens, 2.38)] . Higher 12, 0.23  vs. β12, 0.06 ; PP β12, 0.18  vs. β12, 0.10 ]  vs. low SMI β12, 0.05 ] over the course of pregnancy, although the test for interaction was not significant. No effect modification by SMI was observed for PP  vs. low SMI β12, 0.12 ]. In analyses stratified by potential effect modifiers, including prepregnancy BMI, age, and parity, we observe no statistically significant associations between total urinary arsenic and longitudinal changes in BP (data not shown). We also examined whether individuals with missing data affected the outcomes and found that, when individuals with missing covariate information (n = 78) were excluded, the estimates were nearly unchanged  (data not shown).Women with higher PMI had greater average increases in both SBP and PP over the course of pregnancy compared with those with lower PMI, although the differences from women with lower PMI were not significant [SBP β= 0.47)] . Similarp = 0.035) higher SBP. Again, total urinary arsenic was unrelated to DBP [β = 0.34 ]. Likewise, as in the longitudinal analysis, PP was positively associated with urinary arsenic, but with wide confidence intervals [β = 0.44 ]. Graphical representation of the linear regression model depicts an increase in the average of the last three SBP measurements in relation to urinary arsenic level .When we conducted simple linear regression models, based on the average of the last three BP measurements, each 5-μg/L increase in urinary arsenic was associated with a 0.78-mmHg  higher SBP. Although we are unaware of any previous studies of arsenic and BP during pregnancy, recent studies have found that exposure to other environmental contaminants may affect BP during pregnancy with similar magnitudes of effects as observed in our study. Several studies have observed associations between particulate air pollution and increased BP in pregnant women  and 2.11  mmHg, respectively (10 (particulate matter ≤ 10 μm in aerodynamic diameter) and ozone exposure in the first trimester were associated with average SBP increases of 1.9 mmHg  and 1.8 mmHg , respectively, an association that was more pronounced in nonsmoking mothers . Moreover, use of urine as an arsenic biomarker allows us to account for exposure from other sources, such as diet. Nearly 1 in 8 individuals (12.5%) in this sample had water arsenic levels that exceeded the U.S. EPA maximum contaminant limit of 10 μg/L, which likely represents the primary source of arsenic exposure among these individuals. Further, work from our study area of New Hampshire has found that a variety of foods, including rice, can also significantly increase an individual’s arsenic exposure  that have the potential to impact BP levels, and due to sample size, we may have been limited in our ability to examine the impact of effect modifiers, such as age or BMI. Our study population of mothers tended to be well-educated and primarily white, which may underrepresent different racial or socioeconomic groups that are at higher risk of gestational hypertension. Nonetheless, internal validity of the study is strengthened by the fact that we have multiple measurements for each woman over the course of pregnancy, detailed medical history, and sociodemographic information from our participants to include in our models. However, some women in our study were missing covariate information. We used multiple imputation methods to impute missing data, and we cannot rule out the possibility that data were not missing completely at random. Further, our choice of mixed models helps to account for random variability. Longitudinal data analysis provides a sensitive tool for characterizing health outcomes that change gradually, such as BP, and repeated measures can be a powerful way to identify small changes that can have a large impact at the population level . BP has It is becoming increasingly evident that pregnant women and developing fetuses are particularly vulnerable to environmental insults. Inorganic arsenic consumed in both drinking water and diet may contribute to overall arsenic burden in U.S. pregnant women. Although the adverse cardiovascular effects of arsenic have been investigated in adults, to our knowledge, our study is among the first to examine these impacts during pregnancy. As cardiovascular morbidity and mortality rise worldwide, the potential risk of later-life cardiovascular diseases in mothers and children who are exposed to arsenic during pregnancy makes this a critical area of investigation.(351 KB) PDFClick here for additional data file.