The retinal layer thickness was increased at 1 day after I/R and decreased at 7 days after I/R (P < 0.05). This effect was associated with increase in MDA and ROS levels (P < 0.05). The Tunel-positive cells increased and the number of ganglion cell layer (GCL) neurons decreased significantly after I/R injury. The expression of p-p65 and ICAM-1 increased significantly in I/R retinas (P < 0.05). Each effect was markedly attenuated by application of OCT. These data indicate that OCT protects the retina against retinal I/R damage, which could be through inhibition of oxidative stress and downregulation of NF-κB and ICAM-1 expression.Somatostatin (SST), an endogenous peptide, may exert anti-inflammatory and neuroprotective effects on retinal injury induced by ischemia. Retinal ischemic reperfusion (I/R) injury always produces many reactive oxygen species (ROS), which can aggravate the tissue damage. The effects of octreotide (OCT), a SST analogue, on retinal I/R injury and ROS formation, are not very clear. In this study, we observed the effects of OCT on morphological changes, oxidative stress, and cell death, induced by retinal I/R injury. The activation of nuclear factor  Neuronal death induced by retinal I/R injury is the major cause of visual impairment and blindness. There are several biological processes that happened during retinal I/R injury, including oxidative stress, calcium overload, inflammation, cellular necrosis, and apoptosis . Studiesischemia , and stuischemia . Most reischemia . But it C57BL6 mouse, weighted 19–28 g, were purchased from the Model Animal Research Center of Nanjing University and housed in IVC cages, under a 12 h-12 h of light to dark cycle condition. The humidity and temperature were kept at 60 ± 5% and 22 ± 3°C, respectively. Water and food were accessed freely. Protocols about the animal surgery and performance were approved by the Committee of Medical Ethics and Welfare for Experimental Animals, Henan University, School of Medicine . All performance for the surgery must be under anesthesia condition and all efforts should be made to reduce the suffering and number of animals used. Seventy-two mice were used in this study. Animals were divided randomly into three groups: control group (20), ischemia/reperfusion (I/R) group (26), and I/R + OCT group (26). Usually, only the right eyes were chosen to perform I/R procedures. In the control group, sometimes both of the eyes were selected for the analysis.Mice were anesthetized with pentobarbital . One drop of 1% tropicamide was used to dilate the pupils. Proparacaine hydrochloride was dropped onto the cornea for topical anesthesia. Animals were lied down on their left side. A 30-gauge sterile needle connected with a sterile plastic infusion tube was put horizontally into the anterior chamber of right eye. A normal saline bag interlinked with the other end of the infusion tube was raised to a height of 150 cm, which made the intraocular pressure (IOP) increase to about 110 mmHg. Ischemia can be estimated by observation of the anterior globe, which was whitened when the vessels were blocked. Ischemic condition was kept for 60 min, and then the needle was removed gradually. The vessels reperfusion could be confirmed by observation of the color restored in anterior segment of globe. In our preliminary experiments, sham surgery was made in the left eyes. For these animals only the needle was put into the anterior chamber, but the IOP was not increased. The results indicated that there were no significant differences in retinal morphology and protein expression between the control group (intact) and sham group (sham surgery). So the sham group alone was not arranged especially and integrated with intact control animals into control group. Animals were killed at 24 hours, 7 days, and other time points after I/R, retinas, or eyeballs were collected for the following analysis.μg/kg , was injected twice/day subcutaneously after completion of the ischemic condition.For the control group, ischemia-reperfusion was not performed, and no drug was applied. In I/R group, ischemia-reperfusion was induced and then sterile saline was injected subcutaneously as vehicle control. For I/R + OCT group, OCT , 30 To quantify the extent of histopathological damage in I/R retina, the overall retina, inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL) were determined. For each eye, three measurements from center, nasal, and temporal of the hemisphere were averaged. Each experiment group was arranged with 3–5 animals.μm interval focused on the NeuN-positive neurons in GCL were taken. Then, the images were merged to generate a new picture as a representative sample. Each group used 4–6 animals and the cells were counted by using ImageJ software .Retinal neurons in the GCL were evaluated using the confocal microscopy imaging in whole retinal preparations which were labeled with NeuN antibody, the neuron marker. Eyeballs were fixed in 4% paraformaldehyde (PFA) at 4°C for at least one night. Cornea was cut from the eyeball, and then the retina was isolated from the eyecup. After washing with PBS, retina was incubated in 10% normal goat serum in PBS with 1% triton for 30 minutes. Anti-NeuN  was applied to retina overnight at 4°C. Retina was washed with PBS for 5 min, 3 times. Anti-rabbit IgG conjugated with Cy3 was applied to the retina at room temperature. After one-hour incubation and washing with PBS, retina was flat-mounted. Immunofluorescence images acquisition were taken by a confocal microscope . The GCL was confirmed by the trace of inner retinal vessels. Three serial images with 1.5 Apoptotic positive cells were detected by using an In situ Cell Apoptosis Detection Kit  on paraffin-embedded retina sections. Five different areas in each retinal section were chosen to count the Tunel-positive cells. The positive cell numbers per millimeter of retinal surface in the 5 areas were reported. Three or four samples per group were used for this assay.μM, 40 min, at room temperature). Images were obtained with a fluorescent microscope. The relative fluorescent intensity was quantified by using ImageJ  software.Retina was dissected completely from each eyeball and then homogenized by using RIPA buffer (Beyotime). Homogenate was centrifugated (1600 g) at 4°C for 10 min, and the supernatant was selected. BCA Protein Assay Kit  was used to determine the protein concentration. The MDA levels were measured using assay kits  according to the manufacturers' instructions. Reaction oxygen species (ROS) in the retinal tissue sections were assayed by dihydroethidium (DHE). DHE, as a ROS fluorescent probe, can penetrate through the cellular membrane and be oxidized into ethidium bromide by intracellular ROS. Then the ethidium bromide can be bound to DNA in the nucleus and fluoresce red. Cryosections from frozen retinas were incubated with DHE  was applied to the sections overnight at 4°C. On the next day, sections were incubated with secondary antibody for 1 hour at room temperature. After washing  with PBS, section images were obtained by a microscope, and 3–5 samples per group were used for ICAM-1 staining.μg protein per sample was separated on 10% SDS-PAGE and then transferred onto nitrocellulose membranes (Millipore). The membranes were blocked in 5% milk or 3% BSA in Tris-buffered saline with 0.5% Tween 20 (TBST) for 1 hour at room temperature on shaking table. The membrane was incubated overnight at 4°C with primary antibodies, including p65 , p-p65 , ICAM-1 , and β-actin . On the next day, after washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1 : 10000) on the shaking table for 1 hour at room temperature. After washing the membranes with TBST, protein bands were detected by using ECL reagent and obtained images after exposure on X-OMAT film (Kodak). The intensity of the protein band was semiquantitatively measured with image analysis software , and β-actin was used for calibration.After isolation of the whole retina from eyeball, RIPA buffer  was applied to retinas for the homogenates . Protein concentrations were measured using a BSA protein assay kit (Beyotime Biotech). 30 n = 3–6) and differences among groups were statistically analyzed by one-way ANOVA. P < 0.05 was considered as statistically significant. Statistical analyses were carried out using Sigma stat32.All data were expressed as mean ± SD (The overall retina thickness and the component layers in INL, OPL, and ONL were measured. At 24 hours after retinal I/R injury, as shown in Figures The improved retinal morphology by OCT suggested that OCT played an important role during retina I/R injury. Tunel assay was performed to determine the apoptosis cells at 24 hours after I/R injury in different groups. In The mean values of retinal MDA in the control, I/R, and I/R + OCT group were 2.7 ± 0.45, 4.3 ± 0.51, and 3.1 ± 0.44 nmol/mg protein, respectively. The value in I/R group was significantly greater than that in the control group. There was no significant difference between I/R + OCT group and control group. Application of OCT inhibited the increase of MDA level induced by retina I/R injury .DHE staining was used to test whether ROS were suppressed by application of OCT. DHE, as a ROS fluorescent probe, can be oxidized by intracellular ROS and fluoresce red. At 24 hours after retina I/R injury, DHE fluorescence was significantly upregulated in retinal cryosections. However, application of OCT decreased the levels of ROS. κB is an inflammatory transcription factor, and the activation of its subunits p65 has been demonstrated to play an important role in a lot of tissue damage caused by I/R, which were found in both brain and retina [NF-d retina –16. Westd retina –4. Cell In this study we examined the neuroprotective effects of OCT in a mouse model with retinal I/R injury. The results showed that retinal I/R injury caused the activation of ICAM-1, p-p65, which was correlated with leucocytes infiltration, tissue edema, ROS formation, cell apoptosis, and cell loss. These effects were inhibited by application of OCT, demonstrating the antioxidant and neuroprotective acts of OCT.Ischemia is a final common pathomechanism in many retinal diseases. It is also the most important reason, leading to visual impairment and blindness. Deprivation of oxygen and metabolic substrates, disturbance of waste products removal, and inflammation response are the main pathological processes involved in cerebral and retinal ischemia damage. These processes would result in the generation of numerous oxygen radicals, which have been considered as a major mediator in retinal injury induced by ischemia , 18. The2 agonists attenuate the cell apoptosis and cell death induced by chemical ischemia in rat retinal plant [1 knockout retina, where SSTR2 was overexpressed and overfunctional [2 is an important target for the protection of ischemic retina. In particular, it has been established that OCT could reduce the edema and MDA in ischemic rat brain and guinea pig retina [Based on the suggestion of previous studies, SST system may play anti-inflammatory and neuroprotective roles in many situations and may have been investigated intensively. There is evidence that SSTRal plant . A new inctional . Accumulg retina , 9. But Oxygen free radical generated from tissue was the major cause in I/R injury. In the cell membranes, polyunsaturated fatty acid content was abundant, so the cell membranes were subjected to oxidative attraction and lipid peroxidation. The accumulated peroxidation products may increase the vascular permeability, inducing tissue edema and irritating the inflammatory reaction. These changes could lead to the cell death , 19. TheκB, an important redox-sensitive transcription factor, regulates the genes of many inflammatory mediators and plays an important role in ischemic tissue damage both in brain and in retina [κB p65 increased from 6 h and last even to 7 d after I/R injury. Studies have demonstrated that inactivation of NF-κB specifically in astrocytes had neuroprotective effects against I/R induced retinal cell death and other CNS injuries [κB correlated the expression of Cybb, Ncf1, and Ncf2 and facilitated the oxidative stress conditions [PHOX, one of the NADPH oxidase units, played a key role in retinal I/R injury and associated with the phosphorylation of NF-κB p65 [PHOX did not seem to be influenced by retinal I/R injury [κB p65 caused by ischemia was inhibited by application of OCT, which indicated that downregulation of p-p65 by OCT had protective role against the retinal ischemic injury. This is consistent with previous report that OCT may protect the rat brain from acute ischemic injury by inhibiting the NF-κB activation [NF-n retina , 21–23. injuries , 24–27. injuries . Evidencnditions . AnotherR injury . In the tivation .κB was correlated to an increase expression of inflammatory factor, including ICAM-1, after retinal I/R injury [ Icam1 knockout mice led to the alleviation of cerebral ischemia injury [ICAM-1, a transmembrane glycoprotein belonging to the immune globulin superfamily, is necessary for the adhesion of leucocytes to the capillary endothelium and has been implicated in the development of leukostasis . ImmunohR injury –33. ICAMR injury . As a reR injury . Previoua injury , 37. OurκB p-p65 and ICAM-1, oxidative stress was obviously inhibited and the neuronal cells obtained protection. During these processes, peripheral leucocytes, vascular endothelia cells, glial cells, and neurons were linked and interacted with each other. Generally, OCT may play an inhibitory role after combining with SST receptors widespread in circulatory and nervous system and exert anti-inflammatory and antioxidative actions. These actions inspire us to further explore the potential therapeutic points about endogenous SST system in the neuroinflammation.In conclusion, the present study provided in vivo evidence that application of OCT protected the retinas in a mouse I/R injury model. Therefore, OCT could be useful as a potentially therapeutic drug for the treatment of ischemic retinopathy. Through downregulating the expression of NF-


Three soils with different tillage activities were collected and compared for their organic matter sources and degradation. Two soils (TD and TP) with human activities showed more diverse of chemicals in both free lipids and CuO oxidation products than the one (NS) without human activities. Branched alkanoic acids only accounted for less than 5% of lipids, indicating limited microbial inputs in all three investigated soils. The degradation of lignin in NS and TD was relatively higher than TP, probably because of the chemical degradation, most likely UV light-involved photodegradation. Lignin parameters obtained from CuO oxidation products confirmed that woody gymnosperm tissue (such as pine trees) may be the main source for NS, while angiosperm tissues from vascular plant may be the predominant source for the lignins in TD and TP. Analysis of BPCAs illustrated that BC in NS may be mainly originated from soot or other fossil carbon sources, whereas BC in TD and TP may be produced during corn stalk and straw burning. BC was involved in mineral interactions for TD and TP. The dynamics of organic matter needs to be extensively examined for their nonideal interactions with contaminants. Recent studies suggested that different SOM fractions interact differently with pollutants, depending on their hydrophobicity, functional groups and maturity108http://www.stats.gov.cn/tjsj/ndsj/2013/indexch.htm, (2013) (Date of access: 07/03/2015)]. Therefore, SOM in tillaged soils play a significant role in global carbon cycling.Previous studies have used biomarker method to examine SOM turnover and selective preservation in forest, grassland soils and sediments in response to climate changeYuanyang terrace area is located in Honghe, Yunnan, Southwestern China. This area is famous for the indigenous people with rich ecological knowledge, and the conservation of the unique terraced agricultural landscapes. This rice terrace landscape has been used for over 1000 years with a rich biodiversity in the nearby forest. The temperate and rainy climate promoted active SOM behavior in this area. In this study, soils samples were taken in areas with different agricultural activities. Solvent extraction was used to obtain solvent-extractable lipids and CuO oxidation was applied to quantify lignin-derived phenol organics. Molecular biomarkers for black carbon were also determined and compared. Understanding SOM behavior in this area will definitely provide important information for soil management and to evaluate carbon cycling in human-affected ecological systems.Both human activity-impacted soils (TD and TP) showed higher organic contents in comparison to NS, as suggested by their 5 times higher C contents than NS . After cThe chemicals detected in the solvent extractable free lipids contained n-alkanols, n-alkanoic acids, iso- alkanoic acids, n-alkanes, and steroids as presented in 1812-C32 showed a significant even over odd dominance . Again, NS showed less abundance of chemicals in CuO oxidation products (Table S2). Five lignin parameters, namely S (syringyls), V (vanillyls), C (cinnamyls), S/V, and C/V, contain important information regarding plant-sources of SOM23According to the plot of S/V vs. C/V , lignin-v, (Ad/Al)s, and 3,5-DHBA/V are generally used to describe the extent of plant organic matter degradationv ratios were in the range of 0.87 ± 0.09–2.04 ± 0.39 with the trend of TP < TD < NS s, 3,5-DHBA/V)2 fluxes3334Lignin parameters of VSC, (Ad/Al) TD < NS . 3,5-DihBenzene-polycarboxylic acids (BPCAs) were used as biomarkers to describe BC in agricultural soils3639BPCAs were also compared for soil sample before and after mineral removal. The sum of BPCAs in TP and TD increased greatly after mineral removal, suggesting that BC was involved in mineral interactions for these two soils. Previous studies have suggested the BC may be physically included in the soil aggregates4243Soil without human activity showed less abundance of biomarker chemicals in comparison to the soils with human activity. Human activities may have introduced or accelerated SOM turnover in soils. The higher degradation extent of SOM in NS and TD may be related to chemical degradation, and SOM was better preserved in the reduction environment of TP. Organic fractions showed some woody angiosperm tissues in tillage soils, which may be preserved in the organo-mineral complexes from the original soil before tillage. This mineral protection was also involved in BCs. Unlike common BC analysis methods, biomarkers (BPCAs) identified different BC fractions and origins. Aromatic cluster with small sizes in BCs could form organo-mineral complexes more easily than the aromatic condensed ones. BCs in NS was mostly from soot or other fossil carbon sources, and hardly complexed with mineral particles. Human activities significantly altered SOM compositions and properties, and thus extended work is demanded to relate the dynamics of carbon cycling to their nonideal interactions with contaminants.Three soil samples, which are natural soils , a terrace paddy soil (TP) and a terrace dry farming land soil (TD), were collected in Yuanyang , Yunnan province. TP and TD have been tilled for around 50 years according to the field investigation with the local managers. All the samples were run in duplicate and the organic elemental contents are summarized in 2 purge. The extract yields were determined by weighing the dry residue.Free lipids were extracted according to the protocols described in previous studies4)2(SO4)2.6H2O] and 15 mL of 2 M NaOH in teflon-lined bombs at 170 °C for 4.5 h. The extracts were acidified to pH 1 with 6 M HCl, and kept for 1 h at room temperature in the dark to minimize cinnamic acid reactions. After centrifuged at 2500 rpm for 30 min, the supernatants were liquid–liquid extracted with diethyl ether. The ether extracts were concentrated by rotary evaporation, transferred to 2 mL glass vials and dried under the purge of pure N2.The residues of the above-mentioned lipid extraction were air-dried and then subjected to CuO oxidation to release lignin-derived phenols3 were added in the bombs and reacted at 170 °C for 8 h. After cooling, the mixture was filtered by an ash-less cellulose filter. The digestion solution was diluted 5 times and then treated using cation exchange resin . The treated aqueous samples were freeze-dried for further processing.In order to investigate the interactions of BC with mineral particles, soil particles before and after HF treatment were extracted for BPCA analysis. In brief, 0.5 g of the original soil sample was digested using 10 mL of 4 M trifluoroacetic acid (TFA) at 105 °C for 4 h. After cooling, the residue was rinsed several times with deionized water by filtration through a glass fiber filter (Whatman GF/A 1.6 μm), and dried at 40 °C for 3 h. The residue was then transferred to Teflon-lined bombs. Two mL of 65% HNOThe extracts of free lipid extraction, CuO oxidation, and BC biomarkers were separately dissolved and then trimethylsilyl (TMS) derivatized. All the derivatized solutions were subjected to GC–MS analyses . TMS derivatives of n-heptadecanoic acid and ergosterol were used as external quantification standards for free lipid fractions and the TMS derivatives of vanillic acid were used as the standard for CuO oxidation products.More detailed experimental conditions are presented in How to cite this article: Li, F. et al. Organic matter source and degradation as revealed by molecular biomarkers in agricultural soils of Yuanyang terrace. Sci. Rep.5, 11074; doi: 10.1038/srep11074 (2015).


P = 0.5). Treatment with rosiglitazone for 8 weeks, but not placebo, was more likely to lead to a reduction in cfDNA levels (P = 0.046); however, the absolute changes in cfDNA concentrations during treatment were not statistically significant (P > 0.05). cfDNA levels correlated with markers of endothelial dysfunction (hsCRP P = 0.0497) and vWF (P = 0.0005). In conclusion, cell-free DNA levels are not influenced by renal impairment but do reflect endothelial dysfunction in patients with CKD.Patients with chronic kidney disease (CKD) are at increased risk of cardiovascular disease. Circulating free nucleic acids, known as cell-free DNA (cfDNA), have been proposed as a novel biomarker of cardiovascular risk. The impact of renal impairment on cfDNA levels and whether cfDNA is associated with endothelial dysfunction and inflammation in CKD has not been systematically studied. We analysed cfDNA concentrations from patients with varying degrees of CKD. In addition, to determine whether there is a relationship between cfDNA, inflammation, and endothelial dysfunction in CKD, levels of proinflammatory cytokines and von Willebrand Factor (vWF) were measured in patients treated with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone or placebo for 8 weeks. cfDNA levels were not increased with renal impairment or associated with the degree of renal dysfunction ( Patients with renal impairment have an elevated risk of cardiovascular disease, which increases as renal function declines , 2. Receγ) agonist rosiglitazone for 8 weeks [As chronic kidney disease (CKD) is considered a proinflammatory state , 13, we  8 weeks .n = 31), gemfibrozil, 600 mg twice daily (n = 27), and placebo (n = 32) for 6 weeks in patients with stages 3–5 CKD.  Plasma samples were available for 19 patients in the atorvastatin group, 17 from the gemfibrozil group, and 21 who received placebo at baseline and week 6 . 26% of the SAFIRE subjects were on haemodialysis and 14% on peritoneal dialysis. This study was approved by the Ethics Committees of Royal Perth and Sir Charles Gairdner Hospitals. Written informed consent was obtained from all participants and research was conducted in accordance with the Declaration of Helsinki.Clinical samples were obtained from participants in two trials referred to internally as REVERT  and SAFIhttp://dx.doi.org/10.1155/2014/643189). Briefly, plasma samples were thawed at room temperature (RT), inverted to mix, and centrifuged at maximum speed for 10 minutes at 4°C in a microcentrifuge. 200 μL aliquots of plasma were removed for cfDNA isolation and incubated with RNase A and Proteinase K before binding of cfDNA to the supplied columns. Two wash steps were performed; then cfDNA was eluted with 50 μL of nuclease-free water (incubated for 5 minutes at room temperature before centrifugation), and the eluate passed through the membrane a second time to maximise recovery. cfDNA was stored at 4°C for up to a month or at −20°C for extended storage.Cell-free DNA was isolated using a QIAamp DNA Blood Mini Kit (Qiagen) with modifications as described previously . Plasma μL) were added to Qubit Working Solution (190 μL), vortexed for 5 seconds, and incubated at RT for 2 minutes. A Qubit 2.0 Fluorometer  was used to calculate the dsDNA concentration.cfDNA was quantified using the Qubit dsDNA HS Assay Kit  according to the manufacturer's protocol. cfDNA samples  according to the manufacturer's instructions. All sample wells contained two internal markers of known size (50 bp and 17 kb) and DNA ladder.To determine the amount of apoptotic cfDNA present, 1 β, IL-6, IL-8, IL-10, and TNFα were quantified using the Milliplex MAP High Sensitivity Human Cytokine magnetic bead panel  with plates read on a Luminex 200 system using xPonent software, version 3.1 . 50 μL of plasma was analysed per well, and all samples were assayed in duplicate as per the manufacturer's recommendation. Normalisation of data from different assay plates was performed by Merck Millipore . Data for IL-10 was subsequently excluded from analyses due to excessive variance from the reported sensitivity and performance of the IL-10 assay. To confirm the accuracy of the Luminex system, IL-6 results were compared with those obtained using a quantitative enzyme immunoassay  and found to be highly correlated (P < 0.0001). von Willebrand factor (vWF) activity was measured with the ATA-Liatest vWF kit  and Diagnostica Stago STAR-automated coagulation analyser. High sensitivity C-reactive protein (hsCRP) levels were determined with an immunonephelometric method .The cytokines IL-1P < 0.05.Statistical analyses were performed using Prism 6 . Normality was assessed using the D'Agostino and Pearson omnibus test. Corrections for multiple comparisons were performed using Dunn's test. Statistical significance was defined as P = 0.5). As separating patients by CKD stage could reduce the ability to detect a more subtle relationship between cfDNA and renal function, the association between cfDNA levels and individual patient's estimated glomerular filtration rates (eGFR) was explored. No correlation between cfDNA and eGFR levels was evident (P = 0.87) (Baseline cfDNA levels from REVERT and SAFIRE were grouped by CKD status (stages 2–5) and the mean, median, and interquartile ranges calculated. One hundred and twenty-seven samples had baseline cfDNA results available; one sample (from a patient with CKD stage 5) was excluded from further analyses, as cfDNA levels were more than 10-fold higher than other values. Median cfDNA levels for each CKD stage were 2 (8.4 ng/mL), 3 (7.9 ng/mL), 4 (6.8 ng/mL), and 5 (7.7 ng/mL) . There w = 0.87) .cfDNA can be derived from necrotic or apoptotic cells, which may reflect the underlying processes leading to its production and influence its biological properties . To deteP = 0.6)  .γ agonist rosiglitazone has anti-inflammatory properties, which might influence the generation of cfDNA providing a link between cfDNA, inflammation, and vascular dysfunction in CKD. Therefore, we next compared cfDNA levels at baseline and following 8-week treatment with either placebo or rosiglitazone. No difference in cfDNA levels from baseline was detected in either group .The PPARβ, IL-6, IL-8, TNFα, hsCRP, and vWF) were compared with levels of cfDNA in all patients from the REVERT cohort. For IL-1β, 56 of the 140 samples (40%) had at least one data point of ≤0.14 pg/mL, which was the limit of detection for this assay. For IL-6, 7 of the 140 samples (5%) had at least one data point of ≤0.14 pg/mL, which was the limit of detection for this assay. All data points for IL-8, TNFα, hsCRP, and vWF had detectable levels of the respective cytokines. Results stratified by CKD stage are shown in β, IL-6, IL-8, or TNFα was identified   and vWF (P = 0.0005), which have previously been shown to reflect endothelial dysfunction and associate with elevated cardiovascular risk  and vWF (P = 0.0005) but not with proinflammatory cytokines in patients with CKD.We report the largest study to date analysing the effect of renal impairment on circulating cfDNA levels and demonstrate that, even in the presence of advanced kidney disease, levels of cfDNA are equivalent to those reported in healthy controls and that apoptotic cfDNA fragments are not present. Eight-week treatment with the peroxisome proliferator-activated receptor (PPAR) agonist rosiglitazone, an agent which has been demonstrated to reduce inflammation and endothelial dysfunction in patients with CKD , was assPrevious studies in patients with end stage kidney disease have shown that cfDNA levels are increased within a few minutes of commencing haemodialysis but that baseline levels are generally equivalent to those found in healthy controls , 24. Howγ agonists may reduce endothelial dysfunction in patients with CKD. In keeping with this hypothesis, we demonstrated that patients who received rosiglitazone were more likely to show a decrease in cfDNA levels after 8 weeks than those who received placebo  and that cfDNA levels correlated to levels of vWF and cfDNA, established markers of endothelial dysfunction [Endothelial dysfunction is an early finding in atherosclerosis and is strongly associated with inflammation and insulin resistance , 33. PPAfunction , 41.There are several limitations to our study. Despite the use of a high sensitivity cytokine detection kit and an optimized protocol for the detection of cfDNA, levels of cfDNA and several cytokines were at the lower limits of detection, which may have reduced the ability to detect small differences compared to background. Eight-week therapy with rosiglitazone may have been insufficient for meaningful differences in cfDNA levels to develop or the anti-inflammatory benefits of this agent may only become evident in the presence of more overt inflammation where levels of cytokines and cfDNA are orders of magnitude higher , 42. Furγ agonists may have a role in reducing cfDNA levels. cfDNA levels do not reflect the elevated cardiovascular risk in patients with CKD but may therefore retain their utility as a biomarker for other inflammatory conditions in patients with renal impairment.Chronic kidney disease is an inflammatory state associated with increased cardiovascular morbidity and mortality. cfDNA levels are elevated in the presence of inflammation, have been proposed as a biomarker for cardiovascular risk and a tool to monitor inflammatory conditions, and have intrinsic proinflammatory properties. The impact of renal impairment on cell-free DNA levels has not previously been systematically studied. The stage of CKD does not influence levels of cfDNA; however, there is a correlation with levels of hsCRP and vWF, but not other inflammatory cytokines associated with endothelial dysfunction. PPARSupplementary Figure 1: Plasma cell cfDNA levels are equivalent in EDTA or citrate-containing tubes. cfDNA levels were calculated in triplicate samples processed independently from two healthy controls. No differences were detected between samples collected in EDTA or citrate-containing tubes.Supplementary Figure 2: Apoptotic cfDNA is not present in patients with CKD. 12 cfDNA samples from patients in the REVERT and SAFIRE studies were run on Agilent DNA 12,000 chip to determine the proportion of apoptotic cfDNA present. Panel A) shows a microfluidic DNA gel with a representative electropherogram from one of these samples in Panel B). Panels C) and D) show equivalent traces from patients with sepsis, arrows indicate apoptotic DNA peaks.


P < 0.001), which was validated as an independent prognosis factors in a Cox regression model. Further analysis showed that the LN count was only a prognosis factor with the number from 8 to 16(except for 13).After the number 16, the 5-year survival rate decreased gradually. Collectively, our results confirmed that the number of LNs in yp III stage rectal patients was a prognosis factor only with the numbers from 8 to 16(except for 13). Using the total mesorectal excision technique with an adequate pathologic examination, a large number of LNs retrieved (≥17) might indicate worse tumor response grade and poorer survival.Lymph node (LN) status after surgery for rectal cancer is affected by preoperative radiotherapy. The purpose of this study was to perform a population-based evaluation of the impact of pathologic LN status after neoadjuvant radiotherapy on survival. A total of 1,650 patients receiving neoadjuvant chemotherapy in Surveillance, Epidemiology, and End Results Program (SEER)-registered ypIII stage rectal cancer was analyzed. We identified the optimal cutoff for retrieved LNs as 10 (χ2 = 14.006,  Preoperative radiotherapy (preop-RT) has become part of standard practice offered to improve treatment outcomes in patients with rectal cancer1346The purpose of this study was to assess the impact of the number of LNs examined on survival of rectal cancer patients treated with preop-RT at ypN(+) stage and to determine the optimal number of LNs that should be examined.In our 8-year study period, a total of 1,650 with rectal cancer met our selection criteria, including 1,042 male and 608 female. The median patient age was 57 years (IQR 48-66). There were 1,125 patients with ypN1 stage and 525 with ypN2 stage. Patient demographics and pathological features are summarized in X-tile plots were constructed and the maximum χ2 log-rank value of 14.006 (P < 0.001) was produced, applying 10 as the optimal cutoff value to divide the cohort into high and low risk subsets in terms of rectal cancer cause-specific survival (RCSS) . To asse(P < 0.001), black patients (P = 0.043), poor and undifferentiated tumor grades (P < 0.001), mucinous and signet-ring cancer (P = 0.001), advanced TN stages (P < 0.001), higher negative to positive LNs ratio (P < 0.001), and number of LNs (P < 0.001), were significant risk factors for poor survival according to univariate analysis (< 0.001)  .P < 0.001) and ypIIIC subgroups , but not in the ypIIIA subgroup  (P = 0.003) and ypIIIC subgroups  . We then further analyzed these three subgroups. Patients with ypTis n = 5) and ypTx (n = 23) were excluded from this analysis. The results showed that the retrieved LNs (with a cutoff of 10) were significantly associated with 5-year RCSS, according to the univariate analysis in the ypIIIB   and 5, . and ypTx= 0.013)  and 5, et al. showed that for colon cancer patients with N1 stage, there was an absolute 23% improvement in the 5-year overall survival if >40 LNs were analyzed compared with ≤10 LNs (P < 0.001); and in patients with N2 stage, the 5-year overall survival rates following analysis of >35 and <35 LNs were 71% and 51%, respectively (P = 0.002)et al. reported that the mean numbers of LNs examined in stage III patients who died or were alive within 5 years was 13.1 vs 14.8, respectively, and this difference was statistically significant (P < 0.001)et al. showed that the median survival times for colon cancer patients with 1–7, 8–14 and ≥15 LN retrieval were 46, 52 and 67 months, respectively (P < 0.001)131419The presence of LN metastases in colorectal cancer is well recognized as one of the most important prognostic factors for long-term outcome9111213et al. have shown that retrieving fewer than 12 LNs in the proctectomy specimen of rectal cancer patients treated with preoperative chemoradiation does not affect their overall survival and may be a marker of greater tumor response and, consequently, decreased LR rateet al. were also unable to identify any relationship between a reduced number of LNs examined and decreased patient survivalet al. have demonstrated good disease-free survival rates in patients with a complete absence of LNs in their specimensVS 10), and a poorer tumor regression score results in shorter RCSS rates2526The intended purpose of preop-RT is tumor down-staging by decreasing the primary tumor bulk and associated LN metastases. Some authors have demonstrated that neoadjuvant therapy may result in radiation-induced lymphocyte destruction and stromal fibrosis resulting in alterations of the morphology of the LNs, making it difficult to detect them23This large population-based study has several potential limitations. First, the SEER database does not include information on the administration of chemotherapy. It is possible that additional or concurrent chemotherapy decreases the number of LNs more than preop-RT alone. Second, individual surgeons, pathologists, and other factors may affect LN harvest, but we cannot adjust for these factors. Finally, SEER database also does not report data on preoperative clinical stage, or tumor regression score, so we cannot analyze the relationship between tumor regression score and LNs retrieval in this study.In conclusion, our analysis of the SEER database revealed that the number of LNs retrieved was a prognostic factor only for numbers ranging from 8 to 16(except for 13) with a maximum χ2 log-rank value of 14.006 at a cutoff of 10. The good tumor response associated with a reduced number of examined LNs may offset the influence of potential under staging with a cutoff of less than 7. Increased LN retrieval (≥17) could indicate worse preop-RT response and poorer RCSS.Data from the Surveillance Epidemiology and End Results (SEER) Program of the United States National Cancer Institute, release 2015, were utilized for this study. SEER, a population-based cancer registry, collects cancer incidence and survival data from 18 regional population-based registries covering approximately 26% of the US population. The SEER data contain no identifiers and are publicly available for studies of cancer-based epidemiology and TNM staging of colorectal22SEER registry patients eligible for this cohort included those with adenocarcinoma, NOS (8150/3), adenocarcinoma in adenomatous polyps (8210/3), villous adenoma (8261/3) or tubulovillous adenoma (8263/3), mucinous adenocarcinoma (8480/3), signet ring cell carcinoma (8490/3) of the rectum diagnosed from 1998 through 2005 and treated with surgical resection. The inclusive study dates were chosen to allow a known treatment sequence of “radiation prior to surgery”. The patient age was limited to 18 to 80 years old. Patients diagnosed after 2006 were excluded to ensure adequate follow-up times. Additional exclusion criteria were as follows: no LNs examined pathologically, ypN0 patients, with synchronous distance metastases, and patients who died within 30 postoperative days.The following information was retrieved from the SEER database: the patient age, sex, race, extension of primary tumor invasion, radiation sequence, total number of LNs examined, number of involved LNs, grade, survival time, and SEER cause-specific death classification. All cases were restaged based on the AJCC-7 staging. The primary endpoint of the study is RCSS which was calculated from the date of diagnosis to the date of cancer specific death. Deaths attributed to the cancer of interest are treated as events and deaths from other causes are treated as censored observation.http://www.tissuearray.org/rimmlab/), which identified the cutoff with the minimum P values from log-rank χ2 statistics for the categorical LNs in terms of survivalU test was used for continuous variables, and the chi-square test was used for categorical variables. The 5-year RCSS rate was estimated from Kaplan-Meier curves. Deaths attributed to the rectal cancer of interest are treated as events and deaths from other causes are treated as censored observation. Statistical analyses were performed with the statistical software package SPSS  for Windows, version 17 . Two-sided p values of less than 0.05 were considered to be statistically significant.The LNs cutoff points were analyzed using the X-tile program .


Evidence linking caries in primary dentition and children’s anthropometric measures is contradictory. We aimed to evaluate the prevalence of primary dental caries and its relationship with body mass index (BMI) among 7-9 years old school children in urban Guangzhou, China.This cross-sectional study enrolled 32,461 pupils  aged 7-9 years from 65 elementary schools in Guangzhou. Dental caries was detected according to criteria recommended by the World Health Organization (WHO). The total mean decayed, missing or filled teeth (dmft) of primary dentition were assessed. Weight and height were measured and BMI was calculated. Children were classified into underweight, normal weight, overweight and obesity groups by BMI based on Chinese criteria. Z-score of BMI-for-age (BAZ) was calculated by WHO standardized procedure. Multivariable odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated using logistic regression. Restricted cubic spline regression was applied to evaluate the shape of the relationship between BAZ and primary dental caries.P < 0.0001) and 34 %  lower odds for the presence of primary dental caries after adjustment for age and gender, respectively. Although in general, increased BAZ was associated with decreased risk of dental caries, full-range BAZ was associated with dental caries in an A-shaped manner with a zenith at around -1.4.The prevalence of primary dental caries was 30.7 % in total sample. Regarding dmft values, the mean ± standard deviation (SD) in the combined sample were 1.03 ± 2.05 in boys and 0.93 ± 1.92 in girls. Both indices decreased by age. Compared with normal BMI group, children in overweight and obesity groups have 27 %  contains supplementary material, which is available to authorized users. Dental caries remains the most commonly encountered chronic disease of childhood, even though it is largely preventable . Accordir = 0.40) between increasing obesity prevalence and increasing caries prevalence [Oral health is strongly influenced by the daily intake of food such as energy-dense foods and drinks, which also play a vital role in the development of obesity. The prevalence of childhood obesity has also increased dramatically in many countries around the world, with China being no exception. In China, children and adolescents were diagnosed as overweight/obesity increased from 1.8 % and 0.4 % respectively in 1981–1985 to 13.1 % and 7.5 % respectively in 2006–2010 , while oevalence , but in evalence . NeverthIn this study, we aimed to investigate the oral health status and its association with BMI in Guangzhou elementary school children aged 7-9 years old with a large sample size.n = 335, like hepatitis, tuberculosis, etc.) or physical/mental deformities  and missing anthropometric data  were excluded. The parents of the excluded children also provided written informed consent. Finally, 32,461students were included in this cross-sectional study.The data were derived from the surveys on students' constitution and health carried out by the government in 2011, in Guangzhou, Guangdong Province, China. Through a multistage cluster sampling, we first randomly selected 65 schools from the urban area, and in a second stage, students aged 7–9 years from those schools were invited to participate in this survey. Among 36,555 students, 33959 (92.9 %) pupils were recruited and written informed consent was obtained from the parents of all children who participated in the survey. Approval for the study was obtained from the Ethics Committee of the School of Public Health, Sun Yat-Sen University (NO.201414). All subjects suffering from obvious diseases /height (in meters) squared. The subjects were categorized as underweight, normal weight, overweight, and obesity according to the sex- and age-stratified cutoff values of BMI recommended by Group of China Obesity Task Force in 2003 and the reference norms established from the 2010 national physical fitness and health surveillance data . UnderwePrevious studies have documented that partitioning the continuous BMI score into four groups might result in less information being carried by the data, a reduction or spurious increase in statistical power, with resultant Type I or Type II errors , 33. AddFull mouth dental caries detection was performed by eight trained doctors, of whom two were senior dentists and the others were physicians, with reference to the WHO criteria . It tookP-value for the model χ2 was ≤0.01. Tests for linearity used the Wald test. Statistical analysis was carried out using SPSS Statistics 19.0 Software  and Stata Software . A P value < 0.05 was considered statistically significant.Data input was performed using Epidata 3.1 software. Variables were expressed as mean ± standard deviation (SD) or percentage. Chi square test was applied to compare the prevalence of dental caries across different groups stratified by age, gender or weight. For evaluation of the differences of dmft between two groups, Mann-Whitney U test was performed, and for three or more groups, Kruskal-Wallis test was used. Logistic regression models were performed to analyze the association between BMI and the risk for the prevalence of primary dental caries after adjustment for age in sex stratified analysis and for age and sex in the combined sample. Odds ratio (OR) and their 95 % confidence intervals (95 % CI) were calculated. We further fitted a logistic regression model with restricted cubic splines using BAZ as a continuous variable to explore potential nonlinear association . The refA total of 32,461 pupils  aged 7 to 9 years were enrolled in the present data. The gender and age-specific BMIs are presented in Table P < 0.001). The mean dmft was also significantly higher in boys than in girls .Table As listed in Table The association of the dental caries prevalence and BMI are demonstrated in Fig. P < 0.0001). The P-value for the linearity was P < 0.0001. Children with BAZ > 1 were associated with reduced risk of dental caries, which remained significant after adjusting for sex and age.To illustrate the non-linear associations between BAZ and the odd of dental caries, we graphed the ORs and 95 % CI from models using restricted cubic splines for the total sample Fig. . BAZ wasBased on this large population-based cross-sectional study in Chinese children aged 7-9 years, we found the following main results: 1) the total prevalence of primary dental caries was 30.7 %; 2) the prevalence of primary dental caries decreased with age; 3) there was an inverse association between dental caries and BMI, in comparison with normal weight group, overweight and obese children were more likely to be primary dental caries free.P > 0.05) among youths aged 6–11 years [Bagramian et al. have documented that temporal trends showed a global decline in dental caries since the 1970s, yet recent data indicate an increase of the disease in both the primary and permanent dentition . Based o11 years . Accordi11 years . SimilarCaries is usually regarded as a consequence of frequent ingestion of fermentable sugars, which can also lead to obesity. As a result, the link between caries and obesity has long been suspected. However, research results into the obesity-caries relationship in children have been mixed and inconclusive. In the data of NHANES III, Kopycka-Kedzierawski et al. suggested that overweight may be associated with decreased rates of caries in children aged 2-18 years . LikewisThe strengths of this study include a relatively large sample size, wide age range, and inclusion of both sexes and the use of a standardized diagnostic interview administered by trained interviewers. Also, we used restricted cubic spline regression to evaluate the shape of the relationship between BAZ and primary dental caries. Some potential limitations also warrant discussion. First, no causal association can be made as with any cross-sectional study. Second, dental caries detection was carried out visually and no X-rays were taken, thus the prevalence of dental caries and dmft value might be underestimated. Thirdly, other factors including energy-dense foods and drinks, family/parental socioeconomic status, vitamin D have also been observed to exert significant influences on the presence of dental caries and obesity as well , 43. HowIn conclusion, our results suggested that children at risk of being overweight and obesity generally had lower caries experience than their normal weight peers in urban Guangzhou, China. Further longitudinal studies are required to confirm the results of the present study, and to determine the mechanisms of the association between BMI and dental caries.BMI, Body Mass Index; WHO, World Health Organization; DMFT, Decayed Missing or Filled Teeth; BAZ, BMI-for-Age Z-score; OR, Odds Ratios; CI, Confidence Interval; SD, Standard Deviation; CDC, Center for Disease Control and Prevention; IOTF, World Obesity/Policy & Prevention; NHANES, National Health and Nutrition Examination Survey


P = 0.039), and women who initiated CGMS earlier gained less weight (P = 0.017). The mode of blood glucose monitoring  and pre-pregnancy BMI  were independent factors for weight gain. In conclusion, early CGMS for GDM mothers reduces gestational weight gain. A follow-up study with a large cohort is needed.In this study, we sought to investigate the effects of a continuous glucose monitoring system (CGMS) on maternal and neonatal outcomes. A total of 106 women with gestational diabetes mellitus (GDM) in gestational weeks 24–28 were randomly allocated to the antenatal care plus CGMS group or the self-monitoring blood glucose (SMBG) group. The CGMS group was subdivided into early and late subgroups. There were no significant differences in prenatal or obstetric outcomes, e.g., caesarean delivery rate, Apgar score at 5 min, macrosomia or neonatal hypoglycaemia, between the CGMS and SMBG groups. The CGMS group had lower glycated haemoglobin (HbA1C) levels than the SMBG group; however, the difference was not statistically significant. The proportion of GDM women with excessive gestational weight gain was lower in the CGMS group than in the SMBG group (33.3% vs. 56.4%,  Gestational diabetes mellitus (GDM) is characterized by carbohydrate intolerance with an onset or first recognition during pregnancy511Glucose monitoring during pregnancy is indispensable for improving glycaemic control and reducing the risk of related adverse perinatal outcomes1419202122Prospective randomized clinical trials are warranted to confirm the effects of a CGMS, especially when initiated early, on glycaemic control as well as maternal and neonatal outcomes, including prenatal and obstetrical outcomes, among GDM mothers; this represents the purpose of this study, and we sought to determine whether CGMS reduces GWG. To the best of our knowledge, no study has examined the effects of behavioural adjustments, such as attentive and early glycaemic monitoring, on the GWG of GDM mothers.The protocol and informed consent documents were approved by the research ethics committees of ZhongDa Hospital, which is affiliated with Southeast University. This study was performed in accordance with approved guidelines. All patients provided written informed consent prior to participation in the study protocol.This study was a prospective, observational, open-label randomized controlled trial undertaken in the Department of Endocrinology, Zhongda Hospital, the Affiliated Hospital of Southeast University, China, from September 2011 to December 2012.The mothers enrolled in this study were diagnosed after 24 weeks of gestation. The model of care included lifestyle advice, clinical follow-up, and glucose monitoring combined with CGMS or SMBG. The CGMS group was subdivided into early and late subgroups. The patients in the two subgroups were asked to wear a CGMS during their second and third trimesters of pregnancy.All the enrolled women underwent a 75 g oral glucose tolerance test (OGTT) at 24 to 28 weeks of gestation according to the criteria of the American Diabetes Association (ADA)All the women were interviewed in the hospital and underwent dietary counselling for a eucaloric diet with a low glycaemic index and low saturated fat levels. A basic explanation of the nutrient composition of commonly consumed food and food products was provided. The dietary advice dictated that 50% to 60% of the energy per day should be derived from carbohydrates, 25% to 30% should be from fat, and 15% to 20% should be from protein; furthermore, energy intake should be distributed as equally as possible throughout the day, based on the recommendations of the China Diabetic Association. Total calories were approximately distributed as 10% for breakfast, 30% each for lunch and dinner, and 30% for snacks. The diet was divided into three meals and three snacks. During pregnancy, moderate intensity physical exercise was encouraged. The heart rate goals for moderate intensity exercise were evaluated by the formula (220-age) × 0.65–0.75. The physical conditioning programme was a 35- to 45-min session performed thrice weekly. The physiotherapist motivated the women individually to continue exercising during pregnancy or to start exercising. The physiotherapist also provided written instructions for exercise and self-care. Walking, swimming, and cycling are recommended types of exercise29The treatment programme was determined based on self-monitored plasma glucose values within a week after the initiation of monitoring. Insulin treatment was administered under conditions of two fasting blood glucose values >105 mg/dL (5.8 mmol/L), two 1 h postprandial blood glucose levels >155 mg/dL (8.6 mmol/L), 2 h postprandial blood glucose >130 mg/dL (7.2 mmol/L), or fasting blood glucose >90 mg/dL (5.5 mmol/L) with at least two postprandial values >141 mg/dL (7.8 mmol/L), according to the ADA. Women received treatment with NPH as an intermediate-acting insulin with an initial dose of 0.2 units/kg. If fasting blood glucose levels were high, then treatment was given before bedtime. If postprandial blood glucose levels were high, then regular insulin or short-acting insulin was administered before meals (1 unit for every 101 umg/dL (0.561 ummol/L) over the target value)Follow-up meetings were set every 2 to 4 weeks until 28 gestational weeks, fortnightly until 32 gestational weeks and weekly thereafter. The side effects of pregnancy were documented at the prenatal visits. All the patients underwent an ultrasound evaluation of foetal weight in the third trimester. The patients with dietary counselling had a routine check at 36 gestational weeks.The patients in the SMBG and CGMS groups were taught to perform self-monitoring of blood glucose by using Accu-Chek Advantage metres . These patients were instructed to check their glucose level four times a day  from the first visit at which they received the GDM diagnosis until labour and delivery, except during the period mentioned below for the CGMS group. The CGMS group was monitored using a CGMS . The CGMS sensor was inserted into the upper outer buttock of the subjects for 48 to 721 uhours on weekdaysth percentile, extreme LGA was defined as birth weight at or above the 97.7th percentile, and small-for-gestational age (SGA) was defined as birth weight at or below the 10th percentile based on standard growth and development tables for the Chinese population.The obstetrical and neonatal outcomes included caesarean section, birth weight, standard deviation of weight for gestational weeks, and Apgar score at 5 min. Neonatal hypoglycaemia was defined as a blood glucose concentration ≤45 mg/dL (2.5 mmol/L)HbA1C levels were examined once every 4 weeks. Glycaemic control was also assessed via participant-monitored blood glucose levels, which were recorded four times a day. The measures were evaluated by comparing the CGMS data, including hypoglycaemia, which was defined as <59 mg/dL (3.3 mmol/L) during pregnancy; standard deviation (SD) of the mean glucose value; mean of daily continuous 24 h blood glucose (MBG); and mean amplitude of glucose excursions (MAGE) computed as the arithmetic mean of the differences between consecutive peaks and nadirs. Only increases of more than one SDBG of the mean glycaemic values were considered. The mean postprandial glucose excursion (PPGE) was measured. The absolute mean of the daily differences (MODD) was the mean absolute value of the differences between glucose values during two successive 24 h periods2), overweight (25 ≤ BMI < 30 kg/m2), or obese (BMI ≥ 30 kg/m2). GWG was calculated as the simple difference between the weight at the end of pregnancy and the pre-pregnancy weight. We measured the weight on the delivery day using the same medical scales  while the women were wearing light indoor clothes and no shoes. The 2009 Institute of Medicine (IOM) guidelines were used to classify the total gestational weight gain based on the pre-pregnancy BMI as follows: normal, 11.5 kg to 16 kg; overweight, 7 kg to 11.5 kg; and obese, 5 kg to 9 kgEach woman reported her pre-pregnancy weight, and BMI was calculated as weight in kilograms divided by the square of height in metres. Self-reported pre-pregnancy weight is widely used in studies35χ2 test, and Student’s t-test were used when appropriate. Continuous variables were analysed using a two tailed t-test, and a P value less than <0.05 was considered statistically significant. For the prospective study, binary logistic regression analysis was performed to detect independent predictors of weight gain. All the statistical analyses were performed using statistical software .Quantitative and nominal data are presented as the mean ± SD and percentages, respectively. For the statistical analyses, Fisher’s exact test, Pearson’s Of the women who were preliminarily eligible to participate in the study, 55 in the CGMS group and 62 in the SMBG group provided informed consent to participate. However, 4 of the participants in the CGMS group were lost to follow-up and discontinued the study, and 7 of the participants in the SMBG were found to be ineligible. The final analyses included 51 participants in the CGMS group  and 55 in the SMBG group. Of the randomized patients, 106 were included in the analysis . The cliP = 0.37). No births occurred before the 35th gestational week. No perinatal deaths were observed in either group. Gestational weeks at delivery, Apgar score at 5 min, macrosomia, neonatal hypoglycaemia, and extreme LGA (≥97.7th percentile) and SGA (≤10th percentile) were not significantly different between the two groups. Fewer LGA (≥90th percentile) infants were born to mothers in the CGMS group than to those in the SMBG group, but the difference was not statistically significant .The caesarean delivery rate was greater in the SMBG group than in the CGMS group, but the difference was not statistically significant . Compared to those in the SMBG group, HbA1C levels were lower in the CGMS group but were not significantly different throughout the last two trimesters. Similar reductions in HbA1C levels were observed in the CGMS and SMBG groups  in later pregnancy (32 to 36 weeks gestation)  .P = 0.046) , lunch PPGE (LPPGE), dinner PPGE (DPPGE), and MODD were observed between the subgroups. As expected, MAGE was significantly higher among mothers with the CGMS in the third trimester than among those wearing the CGMS in the second trimester  .P = 0.02). Among the 16 patients in the CGMS group who used insulin, 11 used regular insulin alone as a short-acting insulin 1–3 times per day. Five patients used intermediate-acting insulin; 1 of these patients was treated with intermediate-acting insulin alone, and 4 were treated with intermediate-acting insulin and regular insulin. Among the 7 patients in the SMBG group who used insulin, one patient each used regular insulin alone and NPH alone; the remaining 5 patients were treated with NPH and regular insulin.Insulin was more commonly used in the CGMS group than in the SMBG group (31.3% (16 patients) vs. 12.7% (7 patients), P = 0.027), and NPH was more commonly used in the SMBG group .In a comparison of the two groups, regular insulin was more commonly used in the CGMS group .By the last visit, there was no significant difference in the required insulin dose between the CGMS and SMBG groups . Compared with the SMBG group, the CGMS group had a lower proportion of patients who experienced excessive gestational weight gain (33.3% vs. 56.4%) and a higher proportion of patients with appropriate weight gain (62.8% vs. 38.2%). Fewer patients wearing the CGMS gained an inadequate amount of gestational weight . Furthermore, the GDM women who wore the CGMS in the early stage gained less weight than those who wore the CGMS in the later stage  (No significant difference in pre-pregnancy BMI was observed between the CGMS and SMBG groups . The GDM= 0.003) .P = 0.042) and pre-pregnancy BMI  were independent factors for weight gain.Binary logistic stepwise regression analysis was performed to assess the independent effects of clinical factors on weight gain for GDM mothers. The following factors were considered: age, family history of diabetes, education, pre-pregnancy BMI, OGTT 0 h, OGTT 1 h, OGTT 2 h, fasting C-peptide, postprandial C-peptide, SBP, DBP, TG, LDL-C, HDL-C, HbA1C, insulin medication, and mode of blood glucose monitoring. Pre-pregnancy BMI and mode of blood glucose monitoring entered the model, whereas the other independent variables failed to enter. The mode of blood glucose monitoring  were unavailable. Follow-up data at 6 weeks postpartum were also deficient, and the incidence of diabetes among the mothers was not examined.Despite these limitations, this study proved that the CGMS, especially when initiated early, provides benefits in conjunction with a professional healthcare system to reduce maternal weight gain and glycaemic variability. Extensive clinical studies are warranted to test the effectiveness of CGMS management of maternal weight gain in reducing perinatal problems, especially foetal macrosomia, in GDM women.How to cite this article: WEI, Q. et al. Effect of a CGMS and SMBG on Maternal and Neonatal Outcomes in Gestational Diabetes Mellitus: a Randomized Controlled Trial. Sci. Rep.6, 19920; doi: 10.1038/srep19920 (2016).


Objective. Nondiabetic obese individuals have subclinical involvement of peripheral nerves. We report the factors predicting peripheral nerve function in overweight and obese nondiabetic Nepalese individuals. Methodology. In this cross-sectional study, we included 50 adult overweight and obese nondiabetic volunteers without features of peripheral neuropathy and 50 healthy volunteers to determine the normative nerve conduction data. In cases of abnormal function, the study population was classified on the basis of the number of nerves involved, namely, “<2” or “≥2.” Multivariable logistic regression analysis was carried out to predict outcomes. Results. Fasting blood glucose (FBG) was the significant predictor of motor nerve dysfunction  = 1.003–1.127). Homeostatic model assessment of insulin resistance (HOMA-IR) was the significant predictor  of sensory nerve dysfunction. Body mass index (BMI) was the significant predictor  in case of ≥2 mixed nerves' involvement. Conclusion. FBG, HOMA-IR, and BMI were significant predictors of peripheral nerve dysfunction in overweight and obese Nepalese individuals. Peripheral neuropathy is a very common condition, which is often distressing and disabling . The popA cross-sectional study was conducted on 50 adult overweight or obese nondiabetic volunteers recruited from the outpatient department of College of Medical Sciences Teaching Hospital, Bharatpur, Nepal, after obtaining an informed consent. The ethical approval for this study was obtained from the institutional review board. Detail history and clinical examination findings of all the participants were recorded.The data on height, weight, abdominal girth, and waist-hip ratio were obtained and the participants were classified into two groups using WHO  criteria  of four NCS was performed with Neuroperfect, 4-channel EMG/NCV/EP machine 2000 by Medicaid using surface electrode for motor nerve conduction and ring electrodes for sensory NCS in the air-conditioned neurophysiology lab with control of temperature to ideal and the details of the obtained values were systematically recorded.Motor nerve conduction studies (MNCS) were done in median nerve , ulnar nerves , common peroneal nerve , and posterior tibial nerve . Sensory nerve conduction studies (SNCS) were done in median nerve , ulnar nerve , and sural nerve . F-wave latency was recorded from all the motor nerves in the same setting.The 100 normal values for each nerve variable , SNAP , motor CV (conduction velocity), sensory CV, and F-wave latency) were obtained by recording the neurophysiological values of 50 healthy individuals of similar age and height. This data was used to calculate the cut-off values to determine the peripheral nerve function  of 50 nondiabetic overweight and obese individuals. Volunteers were also classified on the basis of a number of nerves involved, namely, “<2” or “≥2.” Those volunteers whose nerve function was found to be normal (no nerves involved) and those with abnormal nerve function with only one nerve involved were categorized as “<2 nerves involved,” while the rest of the volunteers  were categorized as “≥2 nerves involved.”The study did not include individuals with clear signs of polyneuropathy in the NCS and individuals with any diseases or drugs known to affect PNS  function.t-test if variables passed normality test; otherwise, Mann-Whitney U test was done. The descriptive values were expressed as mean ± SD  when the variable passed the test of normality; otherwise, median (IQR)  was used. Similarly, χ2 test was performed for categorical data. Multivariable logistic regression analysis was carried out in an explorative manner to control the confounders as well as predict outcomes. The variables (predictors) from bivariate analysis were selected at P ≤ 0.25 and these variables along with clinically relevant variables , fasting insulin, insulin at 2 h, HOMA-IR, and quantitative insulin sensitivity check index (QUICKI)) were subjected to binary logistic regression  using backward stepwise method [P < 0.05 for all the analyses.The descriptive statistics for the baseline data from the study population were presented according to the main outcomes (dichotomous): nerve function  and number of nerves involved . All the continuous variables were tested for normality (Shapiro-Wilk test) and their significance of association with the outcome measurements was tested using Independent e method . The staOut of 50 volunteers, the majority had abnormal nerve function of all kinds of nerves: motor (54%), sensory (72%), and mixed (80%). Most of them (70%) had ≥2 mixed nerves' involvement. Sensory nerves were more affected than motor nerves (54% versus 28%) .P = 0.048), FBG (P = 0.018), and 2hPP (P = 0.034) in volunteers with normal and abnormal motor nerve function. Higher BMI (33.57 kg/m2 versus 31.33 kg/m2), higher FBG (100.17 mg/dL versus 92.37 mg/dL), and higher 2hPP (128.10 mg/dL versus 114.40 mg/dL) were seen in volunteers with abnormal motor nerve function as compared to normal function   .P = 0.039 and P = 0.049, respectively. A higher value of HOMA-IR (2.98 versus 2.19) and a lower value of QUICKI (0.32 versus 0.34) were observed in volunteers with ≥2 nerves involved than those with lesser number of nerves involved. Similarly, the association between BMI and the number of mixed nerves involved was statistically significant at P = 0.047 and higher BMI was seen in volunteers with ≥2 nerves involved as compared to those with <2 nerves involved  .The number of motor nerves involved was significantly associated with HOMA-IR and QUICKI at 2 kg/m2) . BMI and2 kg/m2)  and the 2 kg/m2)  were sigP = 0.039, 95% CI = 1.003–1.127) predictor of abnormal motor nerve function. The report showed that an increase in 1 mg/dL FBG increased the odds of having an abnormal motor nerve function by 1.063 times. BMI showed a similar trend, but it was not significant. In case of sensory nerve function . It revealed that the value of HOMA-IR ≤2 increased the odds of abnormal sensory nerve function by 8.368 times compared to >2 HOMA-IR values. Though an increase in age and serum insulin level at 2 h also increased the odds of abnormal sensory nerve function by 1.084 and 1.014 times, respectively, the prediction was not statistically significant. Although weight gain, an increase in BMI, and decrease in HOMA-IR increased the odds of abnormal mixed nerve function, there were no significant predictors of mixed nerve function  and serum insulin level at 2 h by one unit  significantly increased the odds of ≥2 motor nerves' involvement by 1.120 and 1.011 times, respectively . Although one-unit decrease in HOMA-IR increased the odds of ≥2 mixed nerves involved and males have higher odds of mixed nerve involvement than females, both HOMA-IR and gender were not statistically significant.Increase in age by one year  predictor of abnormal motor nerve function. An increase in 1 mg/dL FBG increased the odds of having an abnormal motor nerve function by 1.063 times. Also, for obese volunteers to have ≥2 motor nerves' involvement, we found that age and serum insulin level at 2 h significantly increased the odds by 1.120 and 1.011 times, respectively. Singleton et al. in their study in patients with impaired glucose tolerance found 21% of them to have motor nerve involvement [Interestingly, in logistic regression, we found that increased FBG is the significant  involvement in our study. Peripheral nerve dysfunction is established in obesity , 6. HoweWe did not measure skin temperature, which is ideal during NCS, which is one of our study's major limitations. However, in routine practice, this may not be feasible.Our study found evidence of deranged peripheral nerve function in overweight and obese population in various combinations. BMI, HOMA-IR, and FBG were found to be the significant predictors. Appropriate management strategies to control BMI, FBG, and insulin resistance could prevent adult overweight and obese individuals from a future neuropathic process.


Background: Cardiovascular disease is the leading cause of death in children with end-stage renal disease (ESRD). Isolated aortic dilation (AD) is rare in children. We aimed to determine the prevalence and the risk factors for AD in children with ESRD. Methods and study design: We reviewed records of all ESRD patients followed at our institution from January 2007 to October 2012. AD was defined as Z-score > 2 in the dimension of at least one of the following echocardiographic aortic parameters: annulus, root at the sinus, sino-tubular junction, ,or ascending aorta. Results: The records of 78 patients on dialysis and 19 kidney transplant recipients were available. 30 patients (30.9%) had AD. Multivariate analysis revealed independent associations of AD with body mass index (BMI) Z-score : 0.35 – 0.78) and ESRD secondary to glomerular disease . We developed a classification and regression tree (CART) model to identify patients at low vs. high AD risk. Our model classified 62 patients of the cohort (64%) to be high- or low-risk, with a positive predictive value of 89% and a negative predictive value of 100%. Conclusion: Our data suggest that AD, as a possible marker of aortopathy and early aneurysm formation, is a novel and prevalent cardiovascular complication in ESRD children. Glomerular disease and low BMI Z-score appear to be potent predictors. CART modeling helps identify high-risk children, potentially guiding decisions regarding targeted echocardiographic evaluations.  AD = aortic dilation; BMI = body mass index; BP = blood pressure; CART = classification and regression tree; CCHMC = Cincinnati Children’s Hospital Medical Center; CKD = chronic kidney disease; CVD = cardiovascular disease; DBP = diastolic blood pressure; EDW = estimated dry weight; ESRD = end-stage renal disease; FSGS = focal segmental glomerulosclerosis; IDWG = interdialytic weight gain; iPTH = intact parathyroid hormone; SBP = systolic blood pressure; PD = peritoneal dialysis; UF = ultrafiltration Aortopathy, defined as pathological dilation of the aortic root and/or the ascending aorta is extremely rare in healthy individuals but can have devastating outcomes, such as aortic dissection and aneurysm formation with subsequent rupture . While cWith approval by Cincinnati Children’s Hospital Medical Center (CCHMC)’s institutional review board, we retrospectively and cross-sectionally studied all patients who received ESRD care at our institution between January 2007 and October 2012. ESRD was defined as undergoing chronic hemodialysis (HD) or peritoneal dialysis (PD) or having received a kidney transplant. Additional specific inclusion criteria were age below 23 years, absence of congenital or structural heart disease, including bicuspid aortic valve, and absence of disorders known to be associated with a high incidence of aortic diseases, such as Marfan, Turner, Loeys-Dietz, or Ehlers-Danlos syndromes . 2) = weight (kg)/height (m) [th percentiles for both systolic (SBP) and diastolic BP (DBP) to determine the respective BP indices for each subject. SBP or DBP indices > 1.0 were defined as uncontrolled hypertension [Demographic and clinical data were extracted from each patient’s medical records. The clinical parameters of interest were collected closest to the time of echocardiography and are shown in ight (m) . BP was rtension . Dyslipirtension . The dialysis parameters of interdialytic weight gain (IDWG), ultrafiltration (UF) volume, and Kt/V were determined from the dialysis records for the month prior to echocardiography. For children on HD, the collected data included pre- and posttreatment weights, estimated dry weight (EDW), and average UF for each of 12 consecutive HD sessions. Average IDWG was calculated from the difference between mean pretreatment weights and mean posttreatment weights from the preceding HD sessions. Average UF was calculated from the difference between mean pretreatment weights and mean posttreatment weights. Average excess weight was calculated from the difference between mean posttreatment weight and mean EDW. Normalized values for IDWG, UF, and excess weight were also calculated for each subject by dividing their mean values by the subject’s EDW and then multiplying by 100 . ChildreOnly one echocardiogram study was analyzed for every enrolled patient. When multiple echocardiograms were available, we chose: 1) the most recent study for patients on dialysis who had not received a kidney transplant, 2) the latest pretransplant study on dialysis for patients who subsequently underwent kidney transplantation, and 3) the first posttransplant study for patients with no available pretransplantation study. With this strategy, we aimed to capture the biggest possible effect of ESRD on aortic pathology and to simultaneously avoid as much as possible any potential improvements in aortic dilation after transplantation. Echocardiograms were retrospectively identified and had been obtained while at rest using a Vivid 7 GE ultrasound imaging system  or a Philips iE33 x MATRIX ultrasound system . Testing was performed by pediatric registered sonographers. Images were obtained in standard views according to the American Society of Echocardiography’s guidelines , 10. Ech® [® is a secure web-based application designed to support data capture for research studies. Study data were collected and managed using REDCap® . REDCap®Categorical data are presented as counts and percentages and were analyzed with Fisher’s exact test. Continuous data are presented as means and standard deviations (SDs) and analyzed with Student’s t-test. p-values less than 0.05 were considered statistically significant. Two multivariate logistic regression models were created to identify predictors of AD. Model 1 includes the variables of p-values ≤ 0.15 in the univariate analysis. Model 2 includes the clinical variables that are known to be associated with CVD in addition to the variables included in model 1.We developed risk prediction models using recursive partitioning , for the presence or absence of AD. CART creates nonparametric discriminating trees by dividing the cohort into subgroups representing high or low risk of AD based on available clinical variables. The statistical analyses were conducted using version 2.13.0 of the R statistical package, SAS version 9.3 . Of the 133 patients with ESRD during the study period, 130 met the inclusion criteria, although 33 of them only had incomplete echocardiographic records, leaving data from 97 children to be analyzed. The mean age of the population was 11.5 (6.5) (range 0.2 – 22.7) years. The majority of subjects were male (56.7%), Caucasian (77.3%) and had nonglomerular disease (55.7%). 19 patients had no pretransplant echocardiograms available and were thus studied posttransplant. From the remaining 78 patients, echocardiograms obtained on chronic dialysis were available and thus included. 34 of these patients had been on HD only, 41 on PD only, and 3 had switched modalities. Even though 80 subjects (82.4%) were prescribed antihypertensive medications, hypertension (defined as SBP or DBP indices > 1), was still present in 66 (82.5%). AD was found in 30 of 97 (30.9%) patients. The mean age of the AD group was 12.3 (6.1) years. The mean values of aortic dimensions in the patients with and without AD are shown in 2 (p = 0.001). This difference was still significant when we compared the BMI Z-scores (–0.7 (1.7) vs. 0.9 (1.3), p < 0.0001). Interestingly, 7 (23.3%) children with AD were malnourished, defined as BMI Z-score < –2, compared to none in the group without AD. While more than half  of our patients had nonglomerular ESRD, AD was found predominantly in those with glomerular ESRD: 46.5% of children with glomerular disease (20/43) compared to 18.5% (10/54) of children with nonglomerular disease had AD (p = 0.006). Specifically, focal segmental glomerulosclerosis (FSGS) tended to be more frequent in the AD group compared to the group without AD . Both groups were comparable with regards to age and sex, but children with AD had a lower BMI compared to children without AD: 18.4 (3.7) vs. 23.3 (7.15) kg/mUncontrolled hypertension was more common in the AD group on univariate analysis. While children with AD had similar SBP and DBP when compared to those without AD, both SBP and DBP indices were significantly higher in the AD group. There were no significant differences between the AD and non-AD groups with regards to dialysis modality or other dialysis parameters . Model 1 of our logistic regression analysis shows that both BMI Z-score and the presence of glomerular disease are associated with AD. An increase of BMI Z-score by 1 is associated with a decrease in AD risk . The presence of glomerular disease is a significant independent predictor of AD . In contrast to the univariate analysis, the association between AD and SBP and DBP indices is insignificant in model 1. Logistic regression analysis using our model 2 shows similar results as shown in The results of the CART are shown in Survival in children with ESRD has increased over the last 20 years, but their standardized morality rate remains very high. CVD is the leading cause of death in adolescents and young adult patients with ESRD, and annual CV mortality rates are elevated several hundred fold in young adults with long-standing CKD , 15. Left ventricular hypertrophy accelerated ischemic heart disease, premature dilated cardiomyopathy, aortic valve calcification, increased arterial stiffness, and arterial intima and media thickening are the most frequently observed CV alterations in young adult survivors of childhood-onset ESRD . VasculaThe literature describing the prevalence of and risk factors for thoracic AD in a young ESRD population is sparse . Based oAD is uncommon in healthy children and adolescents , 20, 21,We found a 30.9% incidence of AD in our center’s ESRD population compared to reference incidences of 2.3% in healthy children and 2.8% in hypertensive children . We alsoOur patients with AD were more likely to have glomerular disease and hypertension. At first glance, this association could be explained by the tendency of patients with glomerular disease to retain more fluid, leading to pre- and postcardiac overload. However, fluid-related variables, such as IDWG and normalized UF percentage, were not significantly higher in patients with AD compared to those without. This might suggest that both glomerulonephritis and AD share a common pathological pathway. Along these lines, Adedoyin et al.  also repFurthermore, both the findings of Adedoyin et al.  and our Our data suggest that patients with AD are more likely to have uncontrolled or elevated BP than patients without AD in univariate analysis but not in multivariate analysis. Elevated BP has been a well-studied risk factor for AD in hypertensive adults , 33, 34 Aortopathy, beginning with dilatation of the aorta and leading to aneurysm formation, is a subclinical condition that can be diagnosed only by using comprehensive aortic interrogation by noninvasive imaging strategies such as echocardiography. Such surveillance is likely not performed routinely in most centers. Our nonparametric CART is a highly predictive model based on clinical risk factors that are very likely available to physicians caring for ESRD patients and might be helpful in the identification of a high-risk population that will benefit from targeted close observation for developing AD. Despite of the increased awareness of the need to establish multidisciplinary care of adults and children with CKD , recent Our study has some significant limitations. First, this is an observational retrospective single center report. Second, our study describes a clinical observation, i.e., AD, that is not necessarily associated with a pathophysiologic process, i.e., aortopathy. Third, given the cross-sectional design of the study, longitudinal outcomes, and complications of AD cannot be evaluated. Lastly, gold-standard diagnostic measures, such as cardiac MRI and ambulatory blood pressure monitoring, were not available to further validate our data. Despite these limitations, we believe that our observations are significant enough to warrant further research into the occurrence of aortopathy in children with ESRD. Such further research could include prospective noninvasive surveillance with detailed assessment of the aortic root  and concomitant measurements of inflammatory markers. These efforts could supplement and expand the findings presented here, especially because, to our knowledge, no prior research has evaluated the prevalence of AD among children with ESRD. Describing this prevalence represents the first significant step towards an improved understanding of this novel manifestation of CVD in young individuals with ESRD and thus at very high risk for CV morbidity and mortality. The first authors wish to acknowledge the International Society of Nephrology (ISN) as this work has been made possible through an ISN-funded fellowship for S.U. We also acknowledge grant support from the Center for Clinical and Translational Science and Training (UL1-RR026314). The authors declare no conflicting interest. No honorarium, grant, or other form of payment was given to any author to produce the manuscript.


P = 0.001]. Furthermore, among seven selected SNPs, only G allele of rs2736100 related to RTL in Caucasians was significantly associated with both the decreased RTL (P = 0.002) and the increased susceptibility to SCCHN in Chinese . These findings provide evidence that shortened TL is a risk factor for SCCHN, and genetic variants can contribute to both TL and the susceptibility to SCCHN in southeast Chinese population.Telomere dysfunction participates in malignant transformation and tumorigenesis. Previous studies have explored the associations between telomere length (TL) and cancer susceptibility; however, the findings are inconclusive. The associations between genetic variants and TL have been verified by quite a few genome-wide association studies (GWAS). Yet, to date, there was no published study on the relationship between TL, related genetic variants and susceptibility to squamous cell carcinoma of the head and neck (SCCHN) in Chinese. Hence, we detected relative telomere length (RTL) by using quantitative PCR and genotyped seven selected single nucleotide polymorphisms by TaqMan allelic discrimination assay in 510 SCCHN cases and 913 controls in southeast Chinese. The results showed that RTL was significantly associated with SCCHN risk [(adjusted odds ratio (OR) = 1.19, 95% confidence interval (CI) = 1.08–1.32,  Among 510 cases, 403(79.02%) were with the oral cavity cancer, and 107 (20.98%) with others (P = 3.21 × 10−15). Moreover, the liner regression analysis identified a significant correlation between TL and age  (P = 0.01) (2930P > 0.05)  . Besides = 0.01) , these r > 0.05)  and 2A.P = 0.001). In comparison with subjects with the first quarter (RTL ≥ 1.81), subjects with the third and fourth quarters had significantly increased risks of SCCHN with ORs (95%CIs) of 1.58(1.15–2.17) and 1.56(1.13–2.15), respectively (P < 0.001), but not for other tumor sites , and the P value for heterogeneity test was 0.008 , consistent with the findings in CaucasiansTo evaluate the effect of genetic variants on RTL, we also analyzed the relevance between the seven SNPs reported in previous studies and RTL among 913 controls. Overall, we found that only the G allele of rs2736100 in P = 0.013; additive model: adjusted OR = 1.17, 95%CI = 1.00–1.38, P = 0.049) (P < 0.05). However, heterogeneity test demonstrated no significant difference among different strata  on the susceptibility to SCCHN. Interestingly, we identified that G allele of rs2736100 was significantly associated with an increased risk of SCCHN  . In addit strata .TERT SNP) related to RTL in European was associated with both telomere length and SCCHN risk in this southeast Chinese population. Taking account of these findings, we speculate that genetic variants in TERT may affect the telomere length and consequently induce the altered risk of SCCHN.It is the first case-control study to investigate the relationship between telomere length, genetic variants related to RTL and SCCHN risk in Chinese. We discovered that SCCHN cases, especially oral cancer cases, had shorter telomere length than controls. Meanwhile, our study identified that rs2736100  with other biologically plausible and cancer-causing mutations, a bioinformatics study indicated that rs2736100 may be localized in a regulatory region of the hTERT geneUp to date, three GWAS studies have verified several SNPs related to RTL in European populations, including rs755017 at 20q13.33-TERT and the etiology of cancers has drawn increasing attention. Among those, rs2736100 is one of the most irrefutable identified SNP associated with cancer risk4243TERT contribute to RTL and the carcinogenesis of SCCHN. Taking together, it may be plausible that rs2736100 can affect the hTERT expression, and subsequently generate a higher telomerase activity and an increased risk of SCCHN.Recently, the relationship between genetic variants in et al., observed a significant correlation between blood and buccal cells measurements of RTL Additionally, it needs to be mentioned that RTL was significantly shorter both in the older (age ≥ 60) and in male, consistent with the findings in previous studies. For example, a lot of researches have confirmed that telomere length can act as a candidate biomarker of aging4647485152This study was approved by the institutional review board of Nanjing Medical University and informed consent has obtained from all participants. SCCHN cases were constantly recruited from hospitals including Jiangsu Stomatological Hospital, Nanjing, China and the First Affiliated Hospital of Nanjing Medical University, since May 2009 to October 2013. All of the SCCHN cases were positive histology, and those undergoing radiotherapy or chemotherapy or having any history of tumor were removed from this study. Age (±5 years) and sex matched controls were randomly selected from a cohort including 30,000~ participants in a screening program based on community for non-infective diseases in Jiangsu, China. After knowing-agreeing, the interview was performed with a structured questionnaire to acquire general information and environmental exposure history, such as age, sex, ethnicity, smoking and alcohol drinking. After interview, ~5 ml venous blood sample were collected. Finally, 510 incident SCCHN cases and 913 frequency-matched controls with enough qualified DNA were included in this study. Participants who drank more than twice per week for longer than 1 year were defined as drinkers and those who smoked at least per day for more than 1 year were considered smokers.2) of standard curve must be higher than 0.99. If the threshold cycle (Ct) value was out of the demarcation of the standard curves, the sample was repeated at a different concentration.All methods and experimental protocols were approved by Nanjing Medical University, and carried out in accordance with the approved guidelines. GDNA was extracted from leukocyte of venous blood by phenol chloroform extraction. Each DNA sample was quantified and qualified by spectrophotometer and electrophoresis before genotyping. RTL was detected in genomic DNA using a modified RT-PCR protocol on an 7900HT(ABI PRISM) Sequence Detection System as we performed in previous study® Green (Applied Biosystems) PCR Master Mix. All samples for both the telomere and 36B4 reactions were carried out in duplicate. Laboratory technicians were blinded to the case-control status and equivalent controls and cases were tested in each well of 384-plate. Cawthon’s formula 2−(ΔCt1−ΔCt2) = 2−ΔΔCt was used to calculated RTL.The q-PCR primers were shown in P ≤ 5 × 10−8 (genome-wide association significance level) and MAF ≥ 5% in Chinese population. After linkage disequilibrium (LD) analysis at r2 of 0.3, seven genetic variants related to telomere length were selected for genotyping, including rs755017 at 20q13.33-RTEL1, rs10936599 at 3q26.2-TERC, rs2736100 at 5p15.33-TERT, rs4387287 at 10q24.33-OBFC1, rs7675998 at 4q32.2-NAF1, rs11125529 at 2p16.2-ACYP2, and rs8105767 at 19p12-ZNF208. MAF and LD information were calculated by the 1000 Genomes Project (CHB&JPT subjects Phase I interim release).Up to date, three GWAS studies have discovered several genetic variants related to TL in European populations, such as rs398652, rs1317082 and so on3839The above SNPs were genotyped by means of the TaqMan allelic discrimination assay on the 7900HT (ABI PRISM Sequence Detection System). Data of genotyping and demographic characteristics of subjects between the cases and the controls were calculated by chi-square test. T-test was used to evaluate the distributions of relative telomere length in different status among controls. The goodness-of-fit chi-square test was used to test the HWE of all SNPs among the controls. Linear regression model was used to explore the relationship of single variant and telomere length among controls. Logistic regression model was used to evaluate the association between RTL or genetic variants and cancer risk. The heterogeneity of ORs and 95% CIs derived from relevant subgroups were tested by the meta-analysis. All statistical analyses were calculated with Stata 12.0.Additionally, a mini-meta analysis was conducted by Stata version 12.0 using the “metan” code. Six case-control studies  were included in this meta-analysis. ORs and 95%CI s of risk of RTL were collected to calculate the summary OR.How to cite this article: Gu, Y. et al. Telomere length, genetic variants and risk of squamous cell carcinoma of the head and neck in Southeast Chinese. Sci. Rep.6, 20675; doi: 10.1038/srep20675 (2016).