We investigated the association between ubiquitin-conjugating enzyme E2Z (UBE2Z) gene SNP rs46522 and the risk of CAD in a Chinese Han population with type 2 diabetes and explored a possible interactive effect with environmental risk factors of CAD.  665 patients with T2D were enrolled; 390 were CAD patients and 275 were non-CAD patients. Genotype analysis of rs46522 (T>C) was performed using PCR-RFLP. p′ = 0.020) and codominant model  after adjustment for confounders of CAD. A synergistic effect of rs46522 and BMI was discovered . In subgroup analysis, minor allele T was significantly associated with CAD in overweight and obesity subgroup (p = 0.034), and the association was also proved in recessive model . Smokers with genotype TT had threefold risk of CAD in comparison to nonsmokers with genotype TC or CC (p < 0.001).  The SNP rs47522 was associated with the risk of CAD supposing recessive inheritance model (TT versus CC+CT, OR′ = 1.277, 95%CI′ 1.039–1.570,  The SNP rs46522 in UBE2Z gene is associated with the risk of CAD in the individuals of Chinese Han descent with type 2 diabetes and is of synergistic effect with BMI. Diabetes mellitus is one of the most harmful metabolic diseases worldwide, and a major challenge to public health, especially in China . CoronarThe study was designed in accordance with the Helsinki Declaration. The research protocol and informed consent forms were approved by the research ethics committee of Peking University First Hospital. Before recruited, all subjects gave informed written consent form.Diagnosis of type 2 diabetes was based on the 1999 WHO diagnostic criteria, which is defined as fasting blood glucose (FBG) of 7.0 mmol/L (126 mg/dL) or over and/or 2 hours postprandial blood glucose (PBG) of 11.1 mmol/L (200 mg/dL)) or over. Subjects were selected from those patients who underwent coronary angiography or coronary artery CT scan during their hospitalization in Peking University First Hospital. 665 unrelated Chinese Han individuals with T2D were enrolled, 390 of whom were CAD-positive and 275 were CAD-negative. Subjects with CAD were defined as a stenosis size of 50% or above in at least one of major coronary arteries or their main branches on cardioangiography at Peking University First Hospital. Non-CAD subjects were confirmed with cardioangiography or coronary artery CT scan with the stenosis less than 50%. Once recruited, complete medical records written by residents of these patients were collected including the following data: age, gender, ethnicity, family, social and medication history, vital signs, body weight, body height, and smoking status .Peripheral blood was withdrawn and stored in EDTA-containing tubes. Genomic DNA was isolated with the salting-out method from peripheral blood leukocytes . The extμL PCR system consisted of 1 μL DNA template of each subject, 1 μL primers of both forward and reverse ones (5 pmol/μL), 12.5 μL 2 × PCR Mix of Taq polymerase and dNTP, and 10.5 μL double distilled water. Annealing temperature was set at 61°C for 35 cycles. The reaction system of RFLP was constituted of 7.5 μL PCR product, 0.5 μL restriction endonuclease , 2 μL 10× buffer (NEB corporation), and 10 μL double distilled water. After 2 hours of water bath heating (65°C), RFLP product was analyzed using agarose gel electrophoresis. Furthermore, direct sequencing method was performed in 5% of randomized selected samples in order to minimize bias for the quality control.The SNP rs46522 (T>C) at an intron of UBE2Z gene is located on chromosome 17q21.32. It was genotyped using polymerase chain reaction following restriction fragment length polymorphism (PCR-RFLP). Primers were designed as 5′-CATAGTGAGGTGGAGCAGCA and 5′-ACGCAACCAGTAAGGACAGG using Primer 3.0 software. A 25 p value of <0.05, and odds ratios (ORs) were calculated with 95% confidence intervals (CIs). Continuous variables were expressed as mean ± SD (standard deviation) and were analyzed with Student's t-test or one-way ANOVA. Categorical variables were presented as percentage of cases and were analyzed with the Pearson chi-square test. Genotype distribution of the SNP rs46522 was tested for Hardy-Weinberg equilibrium. The associations between genotypes and CAD were analyzed using multiple logistic regression analysis and were further adjusted for potential confounders including age, gender, history of hypertension and diabetes, lipid levels, fasting blood glucose, BMI, and smoking status.The statistical analyses were carried out using the Statistical Package for the Social Sciences for Windows (SPSS), version 16.0 . Statistical significance was defined as A total of 665 patients with T2D were recruited in this study. The demographic data and clinical characteristics were not significantly different between the CAD and non-CAD groups but in gender, age, and HbA1c, lipid profile, and smoking status . HoweverThe observed genotype frequency of rs46522 in non-CAD group was consistent with the Hardy-Weinberg equilibrium. The rate of genotyping success was greater than 98% for the SNP rs46522. The genotypes were consistent with the results from direct sequencing for the randomly chosen samples, and the consistent rate was 95%. There were no significant differences between CAD and non-CAD groups in the distribution of allele frequencies and genotype frequencies.p = 0.044), and the significance remained after adjustment for the confounders including age, gender, history of hypertension and diabetes, smoking status, BMI, fasting glucose, and lipid levels  on the CAD risk.Both genetic variations and environmental factors contributed to the pathogenesis of CAD. While taken as a successive covariate, BMI exhibited a synergistic effect with rs46522 . Also, the carriers of genotype TT had a higher risk of CAD than carriers of genotypes TC and CC in overweight and obesity subgroup, assumed in recessive model , which is transferred onto the target protein with an isopeptide bond formation . As of dThe potential mechanism by which the genetic variations in UBE2Z gene contribute to CAD risk was not yet clearly explicated. As the SNP rs46522 is located in an intronic region of UBE2Z gene, one possible explanation is that rs46522 is in strong linkage disequilibrium with the causal SNPs of gastric inhibitory polypeptide (GIP) gene, while the encoding protein GIP could modulate glucose and lipid metabolism. GIP gene is located on chromosome 17q21.32 and contains 6 exons. It was proved that functional variant SNPs in GIP gene were associated with cardiovascular disease in a small-scale case-control study . InteresMeanwhile, efforts were made to explore if there was a possible association between GIP gene variants and the risk of type 2 diabetes. In a case-control analysis in a South Indian population, no significant association was found between common variants in GIP (rs2291725 and rs2291726) and diabetes . If the As a coronary artery disease-related variant, rs46522 at the UBE2Z locus yielded significant association with type 2 diabetes in a genome-wide association study . Exploraβ = 0.012, p for interreaction = 0.028). In other words, every 1 kg/m2 increase in BMI resulted in additional 1.2% higher CAD risk in the given genetic group. To date, accumulating evidence indicated that BMI and waist circumference were significantly related to insulin resistance and chronic inflammatory state [The potential interactive effect of rs46522 was investigated with traditional environmental confounders in CAD risk. BMI, as one of the known CAD risk factors, displayed a possible effect of interaction with the SNP rs46522 in the study (ry state , which mAs a main risk factor of CAD, smoking was also taken into consideration in the study. The study showed that the smokers with genotype TT at rs46522 had significant twofold increase in CAD risk compared to nonsmokers with genotype TC/CC, although the difference did not exist anymore after adjustment.In general, the study can be interpreted that rs46522 is associated with the CAD risk in patients with type 2 diabetes, and increased BMI and smoking could increase CAD risk, especially in the individuals with higher genetic susceptibility. Clinically, the study provides strong evidence for diabetes education that body weight management and smoking cessation can significantly benefit the patients from CAD.However, the following limitations should be acknowledged in the study. The number of enrolled individuals was relatively small, especially the sample size of non-CAD group. Also, clinical values of CAD and non-CAD groups were not perfectly matched, which might bias the results even though adjustments were performed. Functional study and perspective study should further be conducted on the genetic variants in UBE2Z and GIP gene to discover the underlying pathway and confirm the predicting value for CAD in Chinese population, respectively.In conclusion, the study showed that rs46522 in UBE2Z gene contributed to the development of CAD in individuals of Chinese Han descent with type 2 diabetes and had an interactive effect with BMI on CAD risk. Genotype TT at rs46522 is a risk factor for CAD, especially in patients with smoking habits.


Severe fever with thrombocytopenia syndrome (SFTS) is an emerging epidemic infectious disease with high mortality in East Aisa, especially in China. To predict the prognosis of SFTS precisely is important in clinical practice.From May 2013 to November 2015, 233 suspected SFTS patients were tested for SFTS virus using RT-PCR. Cox regression model was utilized to comfirm independent risk factors for mortality. A risk score model for mortality was constructed based on regression coefficient of risk factors. Log-rank test was used to evaluate the significance of this model.2 = 88.35, p < 0.001).One hundred seventy-four patients were confirmed with SFTS, of which 40 patients died (23%). Baseline age, serum aspartate aminotransferase (AST) and serum creatinine (sCr) level were independent risk factors of mortality. The area under ROC curve (AUCs) of these parameters for predicting death were 0.771, 0.797 and 0.764, respectively. And hazard ratio (HR) were 1.128, 1.002 and 1.013, respectively. The cutoff value of the risk model was 10. AUC of the model for predicting mortality was 0.892, with sensitivity and specificity of 82.5 and 86.6%, respectively. Log-rank test indicated strong statistical significance (×This risk score model may be helpful to predicting the prognosis of SFTS patients. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging epidemic infectious disease in China, Korea and Japan caused by a novel bunyavirus, SFTS virus (SFTSV). It has become an important public health threat in Asia, especially in China. The national surveillance data from 2010 to 2013 in China showed that the incidence of SFTS and its epidemic areas are growing, but the case fatality rate (CFR) has steadily declined . Other fWe and others  have fouThough the viral load is a predictor of morbidity , in contPatients with clinical manifestations of suspected SFTS, including fever (body temperature >38.0 °C prior to admission or at admission), thrombocytopenia, leukocytopenia and gastrointestinal symptoms were tested for SFTSV via reverse transcription–polymerase chain reaction (RT–PCR) using total RNA extracted from a peripheral blood sample  at localParameters which entered in Cox univariate regression analysis included: age, gender, body temperature, time intervals between symptom onset to admission, hematological parameters, biochemical parameters, coagulation indicators and neurological symptom.This study was conducted according to the Helsinki II Declaration and approved by the ethics committee at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention. Written informed consent was obtained from the patients.U test was used for variables with an abnormal distribution between the survival and death groups. Cox univariate and multivariate regression analysis was applied to determine the risk factors among parameters at admission that were associated with mortality of SFTS patients. Factors with p < 0. 1 in univariate analysis were included in Cox multivariate regression analysis. Risk score model was established based on the acquired regression coefficients of the determined risk factors to predict mortality. Criterion for factors that include in the regression model was that the factors present in the last step of forward conditional with p < 0.05 and Hazard ratios (HR) >1.0. Receiver-operating characteristic (ROC) curves and area under the curve (AUC) were constructed to assess the predictive power of the model. Patients were divided into high-risk and low-risk groups according to cutoff values obtained from ROC analysis. The Kaplan-Meier survival analysis was used to compare the cumulative risk for death in the two groups, and the significance of difference was tested with the log-rank test. ×2 test was utilized to compare mortality rate between patients with above and less than cutoff value of risk model. p < 0.05 was considered to be statistically significant.Statistical analyses were performed using the SPSS, version 16.0, software . Mann–Whitney p < 0.001).Among the 233 suspected cases with fever, thrombocytopenia, fatigue and diarrhea admitted to our hospital from May 2013 to November 2015, 174 were finally diagnosed as SFTS by detecting SFTSV using RT-PCR. Forty patients (23%) subsequently died. The survival group included 63 women (47.0%) and 71 men (53.0%), while the non-survival group comprised of 19 women (47.5%) and 21 men (52.5%). Gender composition was identical in the two groups. The median age was 64 (range 28–84) years, and was significantly higher among those who died , fatigue , loss of appetite  diarrhea , superficial lymphadenopathy , neutropenia , thrombocytopenia  and neurological symptoms . Among those who survived, 38 cases manifested neurological symptoms. Nine cases had severe neurological symptoms included abnormal level of consciousness. Others exhibited mainly limb shaking. Among those patients who have died, 25 cases had neurological symptoms (60%) at admission and other developed central neurological symptoms primarily discordance of consciousness and coma within 3–7 days. Ages of those with severe neurological symptoms who survived are younger than those who died .The major baseline clinical symptoms and findings included fever (p >0.05) were comparable. The time intervals between symptom onset to admission were also similar between the two groups . Hospitalization duration was longer in patients who recovered than those who died .Body temperature at admission in surviving and fatal cases  and phosphorus (P3+) in the fatal cases were significantly elevated, whereas sodium (Na+), calcium (Ca2+) and hydrocarbonate (HCO3-) were notably reduced.For the biochemical parameters, levels of alanine aminotransferase (ALT), AST, total bilirubin (TB), high-sensitivity C-reactive protein (hs-CRP), blood urea nitrogen (BUN), serum creatinine (sCr), troponin (Ctnl), CK, hydroxybutyrate dehydrogenase (HBDH), LDH, prothrombin time (PT) and international normalized ratio (INR) were dramatically increased in the fatal cases. While the prothrombin time activity percentage (PTA) was markedly decreased. Concentrations of serum potassium  count, neutrophil (NEU) count, neutrophil/lymphocyte (N/L) ratio, mean corpuscular volume (MCV) and red blood volume distributing width (RDW) in the fatal cases were remarkably increased. Red blood cell (RBC) count, lymphocyte (LYM) count, PLT count and thrombocytocrit (PCT) were prominently decreased. These results are shown in Tables Cox regression model analysis showed that baseline parameters, including age, serum AST level and sCr level were independent risk factors of mortality. HR (95% CI), and mean survival times of these factors are shown in Table The cutoff values and area under ROC curve (AUCs) of these parameters for predicting death are included in Table 2 = 88.35, p < 0.001). Data are shown in Tables n = 123), 7 cases died (5.7%). And of those with M > 10 (n = 51), 33 cases died (64.7%). Mortality rates between two groups had strong significant difference .The cutoff value of the score model was 10. Patients with >10 were at high-risk of mortality. AUC of the model for predicting mortality was 0.892, with sensitivity and specificity of 82.5 and 86.6%, respectively. Mean survival time of patients with M > 10 is shorter than those with M ≤ 10. Log-rank test indicated strong statistical significance (×n = 67) enrolled in our hospital this year. Of whom, 51 cases survived (76.1%) and 16 died (23.9%). Of those patients with M ≤ 10 , 36 cases survived (92.4%) and 3 cases died (7.6%). Of those with M > 10 , 13 cases died (46.4%) and 15 cases survived (53.6%). Mortality rates between these two groups had significant difference .The cutoff value of M (=10) was prospectively validated in a cohort of SFTS patients -1, IL-6, IL-10, granulocyte colony stimulating factor (G-CSF), interferon inducible protein-10 (IP-10) and monocyte chemoattractant protein (MCP-1) were profoundly elevated in fatal cases, and their levels correlated with various clinical parameters [Though only 58.6% patients had fever at adimissiom, and body temperature was not associated with prognosis, all patients had fever prior admission or body temperature elevated after admission. Fever and fatigue were the main chief complaint of SFTS patients. Though the fatality rate of SFTS in China has fallen from first reported 30% to present 12% owing to increased reporting, advances in diagnostic procedures and improved clinicians’ skills , 16. TheExcept for new treatment methods tried in a few therapies with few patients in Japan and South Korea –23, suppIn the prospective study, we showed that the model is more suitable for the prediction of survival than for mortality. The model we constructed will be helpful for the prediction of SFTS prognosis. And the results will be further prospectively validated by a more larger cohort of patients.This study had several limitations. Firstly, we had no quantitative results of the virus RNA and only had the positive results. Secondly, patients with subclinical or minor infection signs who sought medical care were perhaps not tested for SFTSV. Thirdly, some patients with mild manifestations may have been excluded due to their low/undetectable viral loads. All these factors influence the accuracy of registered incidence and mortality.In summary, the risk score model that we established is useful for predicting death in patients with SFTS and may be applicable in clinical practice.


Gardnerella vaginalis biofilm. Recent data indicates that G. vaginalis biofilms are more tolerant to antibiotics and are able to incorporate other bacterial vaginosis -associated species, yielding a multi-species biofilm. However, despite its apparent role in bacterial vaginosis, little is known regarding the molecular determinants involved in biofilm formation by G. vaginalis. To gain insight into the role of G. vaginalis in the pathogenesis of bacterial vaginosis, we carried out comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression levels of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. Similar to other bacterial species, expression of genes involved in antimicrobial resistance were elevated in biofilm cells. In addition, our data indicate that G. vaginalis biofilms assume a characteristic response to stress and starvation conditions. The abundance of transcripts encoding proteins involved in glucose and carbon metabolism was reduced in biofilms. Surprisingly, transcript levels of vaginolysin were reduced in biofilms relative to planktonic cultures. Overall, our data revealed that gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity. This phenotype may contribute towards the chronic and recurrent nature of bacterial vaginosis. This suggests that G. vaginalis is capable of drastically adjusting its phenotype through an extensive change of gene expression.Bacterial vaginosis is the most common gynecological disorder affecting women of reproductive age. Bacterial vaginosis is frequently associated with the development of a  Gardnerella vaginalis bacteria. Nuno Cerca and colleagues at the University of Minho in Portugal, with co-workers elsewhere in Portugal and in the USA, studied changes in gene activity in BV biofilms. Their results linked differences in the activity of 815 genes to an ability of the bacteria to enter a protected form of growth when producing biofilms. The differences included increased activity of specific genes associated with antibiotic resistance. This insight into the bacterial ability to undergo drastic changes in gene activity could guide work to understand chronic and recurrent BV. It may also lead to new ways to treat this common and troublesome condition.Altered gene activity may promote chronic and recurrent infections in the most common gynecological disorder in women of reproductive age. Bacterial vaginosis (BV) frequently involves the formation of biofilms by  Before trimming the raw data, we identified the genes, with the reads per kilobase per million (RPKM) above 1.00, expressed in each condition. We only detected three genes uniquely expressed in biofilm cells, whereas 11 genes were found uniquely in planktonic cells. However, the majority of gene transcripts that were only detected in planktonic or biofilm cells, encoded uncharacterized proteins or transfer RNA, as shown in the Supplementary Material Table .14 and used the PSORTb program (v.3.0)15 to predict their subcellular localization. The results are shown in Table Our data indicated that within the 1045 genes that were transcribed in both conditions, 815 (78%) were differentially expressed between planktonic and biofilm cells. For downstream analysis, only genes with fold-changes above two were considered. Transcript levels of 309 (30%) genes were elevated, whereas 36 (3%) were reduced in biofilms. Among the transcripts that were more abundant in biofilms, 78 encoded hypothetical proteins. In an effort to find homology with known proteins, we performed a BLAST analysis, a search in the Pfam database (version 29.0) for Pfam domainsIn order to confirm the results obtained by RNA-seq, transcripts detected in greater or lesser abundance in biofilms were randomly selected and their relative levels quantified by quantitative PCR (qPCR). Both RNA used for cDNA libraries construction  and RNA obtained by performing new experiments  were used for validation. As can be seen in Fig. 16 Significant enrichment was only found -corrected) in KEGG pathways  pathways, and enrichment analysis of the genes with down and upregulated transcription was accomplished using STRING.ays Fig. . As coul17 Cytoscape was used to create a gene interaction network including all differently expressed genes and neighbors, yielding a total of 764 nodes and 7685 edges  in biological processes or KEGG pathways associated with translation and metabolic process  and metabolism (glycolysis and carbon metabolism).Table HMPREF0424_0563,a gene with a molecular function related to hydrolase activity. Furthermore, in biofilm cells we found an overexpression of the HMPREF0424_1220 gene encoding an aminotransferase involved in amino acid biosynthesis. A similar trend was reported for Neisseria meningitides.18 Interestingly, we found HMPREF0424_0420, a gene that encodes the LPXTG-motif cell anchor domain-containing protein, which can be involved in biofilm formation, as described in Gram-positive bacteria.19 Moreover, in BV-associated G. vaginalis biofilm cells, transcript levels of the gene HMPREF0424_0573, which encodes a LysM domain-containing protein possibly associated with autoaggregation of G. vaginalis, were also increased, similar to what was observed for Lactobacillus reuteri biofilms.20 Transcripts encoding the ComEA protein (HMPREF0424_0943), which is involved in DNA repair and NLPA lipoprotein (HMPREF0424_0888 gene), which is involved in ABC transporters were also found in greater abundance in biofilms cells.Among the transcripts elevated in biofilm cells, we found 21 It was, therefore, of interest to determine the expression levels of previously annotated potential virulence genes.22 As can be seen in Fig. HMPREF0424_0125 transcript, which encodes TadE-like protein. This might play an important role in adhesion to vaginal epithelial cells, similarly to what has been described in Actinobacillus actinomycetemcomitans.23 Furthermore, our data supported the previous hypothesis that G. vaginalis biofilm development is likely associated with type II glycosyltransferase.22 Of note, glycosyltransferases are likely to be important for the biosynthesis of exopolysaccharide which in turn is important for biofilm formation. The ability to grow as a biofilm would likely confer an increase in antibiotic tolerance and resistance to mucosal immune defenses.24 Herein, levels of transcripts encoding antimicrobial-specific resistance proteins belonging to efflux pump families were increased. In addition, the HMPREF0424_1196 transcript, which encodes a Rib-protein, was elevated in biofilm cells. Rib proteins belong to the α-like protein (Alp)-family of highly repetitive surface antigens and are commonly found in Gram-positive pathogens.25 These proteins elicit protective immunity through their inter-strain size variability.22Biofilm formation by pathogenic bacteria is often associated with altered virulence. Bacterial biofilms may suppress certain virulence factors while others are activated in order to evade immune defenses, and survive challenging conditions.G. vaginalis produces athiol-activated cholesterol-dependent cytolysin, vaginolysin (vly), which might induce vaginal cells lysis. Strikingly, in our experiments, the expression levels of vly (HMPREF0424_0103) were significantly lower in biofilm cells  and translation , as also reported for other microorganisms such as S. epidermidis,11S. aureus,10 and S. mutans.12 In G. vaginalis biofilms, cell density is substantially higher than in planktonic culture.4 As a consequence, most biofilm cells are likely to encounter restricted availability of nutrients.29 Similar to what was found for S. mutans30 and S. pneumonia,13 we also observed that the transcripts encoding ABC transporter proteins were elevated in biofilm cells. In addition, our study revealed that transcripts of genes involved in the synthesis of peptidoglycan and cell wall were also greater in biofilms. This has also been shown for P. aeruginosa31 and S. aureus.10 It has been hypothesized that the cell envelope is a highly dynamic and active component of biofilm cells, contributing to its persistence.13 However, the reasons for the overexpression of genes involved in cell wall biogenesis require further investigation.Here, we showed that BV-associated 22 were also more abundant in biofilm cells, with the exception of vly. Several studies have highlighted the role of vly gene in G. vaginalis virulence.33 The vly gene belongs to the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins, which cause cytotoxicity on vaginal epithelium.33 Our previous findings showed that planktonic cultures of BV isolates of G. vaginalis expressed twofold more vly than planktonic cultures of non-BV isolates.26 Herein, we found that vly transcript levels were higher in planktonic than in biofilms cells. The low levels of expression of vly in biofilms might reflect the more chronic nature of vaginal colonization by BV-associated G. vaginalis and serve as a means towards preventing a host immune response. Similarly, Resch and colleagues10 showed that the production of various S. aureus toxins were significantly upregulated in planktonic rather than in biofilm cells, suggesting that toxins may not be conducive to biofilm persistence in the host.Notably, transcripts of other potential virulence genes, previously annotated by Yeoman and colleagues,G. vaginalis biofilm phenotype might induce aquiescent mode of growth that is less sensitive to antibiotics, as the efficacy of many antibiotics relies on active cell metabolism and the cell-wall construction process.11 Here, we observed that efflux pumps and ABC transporters, reported as mechanisms responsible for antimicrobial resistance,34 were upregulated in biofilms cells. Comparable evidence for the role of efflux pumps in biofilm resistance has been found in several microorganisms such as Pseudomonas aeruginosa,35Escherichia coli,36 and Candida albicans.37Similar to what was observed in other microorganisms, BV-associated G. vaginalis changes its transcriptomic profile when growing as a biofilm. These changes are likely important for biofilm persistence and, consequently, for the virulence of this bacterium. Furthermore, the fact that vly is downregulated in biofilms represents an important finding, that might contribute towards a better understanding of the pathogenesis of BV. However, this study is limited by the fact that the growth medium did not contain all of the factors found in vivo, and some in vivo cues may turn on the expression of biofilm-related genes. Nevertheless, as animal models for BV are lacking, in vitro models can be very informative, and are key to furthering our understanding of virulence potential of G. vaginalis. In conclusion, our findings showed that the gene expression profile of BV-associated G. vaginalis biofilms characterizes a distinct physiologic status that may promote the chronic and recurrent nature of BV.Taken together, these data indicated that BV-associated G. vaginalis strain AMD, isolated from a woman diagnosed with BV based on Amsel criteria at VCU Women’s Health Clinic,38 was used for RNA-seq analysis. G. vaginalis strains UM121, UM137, and UM241, also isolated from women with BV based on Amsel and Nugent criteria,26 were used for subsequent analysis.2 .26 At this time, planktonic cells were still in the exponential growth phase. Thereafter, 18 mL of planktonic cells were harvested by centrifugation  and suspended in 1 mL of RNA protect .Planktonic cells were grown in sBHI  for 24 h at 37 °C with 10% CO6 CFU/mL. After homogenization, 200 μL of G. vaginalis suspensions were dispensed into each well of three 96-well flat-bottom tissue culture plates . The tissue culture plates were then incubated at 37 °C in 10% CO2. After 24 h, the culture medium covering the biofilms was removed, replaced by fresh sBHI and allowed to grow, under the same conditions, for an additional 24 h. This time was required for this strain to develop a notable biofilm. Forty-eight hour biofilms were then washed once with 1 × PBS, scraped from the bottom of 96-well plates in sBHI and pooled together. Finally, biofilm cells were harvested by centrifugation  and suspended in 1 mL of RNA protect (as described above).For biofilm formation, the cell concentration of 24 h old cultures was assessed by optical density at 600 nm  and was further diluted in order to obtain a final concentration of approximately 10et al.49). To remove genomic DNA, TURBO DNA-free™ kit  was used as indicated by the manufacturer followed by acid-phenol:chloroform:isoamyl alcohol (125:24:1) treatment. RNA integrity was determined using an ExperionTM automated electrophoresis system , and samples with RNA Quality Indicator (RQI) above eight were selected for cDNA library preparation.Total RNA was extracted using a combination of mechanical lysis  and the columns of the RNeasy Mini kit (QIAGEN), as optimized before , which already includes the kit for ribosomal (rRNA) depletion: Ribo-Zero™ Kit (Bacteria)—Low Input . The construction of the libraries was rigorously validated by qPCR and Hi-Sensitivity D1K TapeStation (Agilent 2200 TapeStation). Libraries were then multiplexed and sequencing data generated from paired-end reads (2 × 150 bp) using a MiSeq system (Illumina).G. vaginalis strain 409-05 (GenBank accession number NC_013721).Gene expression was normalized using RPKM, that account for both library size and gene length, as described by Mortazavi and colleagues.39 To detect significant gene expression alterations, Kal’s test40 with FDR41 correction was applied. A p-value ≤ 0.05 was considered statistically significant. Transcripts uniquely expressed in each condition were identified using BioinfoGP.42 Data were deposited at Gene Expression Omnibus database .After sequencing, adapters were trimmed by MiSeq® internal software during the base calling. Quality, ambiguity and length trimming, as well as mapping to the reference genome, and normalization of gene expression were performed using CLC Genomics Workbench version 8 . Quality, ambiguity and length trimming were performed using the CLC genomics workbench default settings. RNA-seq reads were aligned to the reference genome of 43 and KEGG pathway44 assignations and enrichment analysis were performed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (version 10).16 In addition, UniProt repository45 was used to determine the function of proteins that were not identified by STRING. Classes with p-value ≤ 0.05, FDR-adjusted, were considered statistically significant for enrichment. Further analysis was carried out using Cytoscape (version 3.2.1),46 in which a gene interaction network including all differentially expressed genes and neighbors created by STRING16 was imported. Gene clusters (regions of high connectivity) were obtained in Cytoscape with the MCODE plugin.47 Default parameters  were used as the cutoff criteria for network module screening. Thereafter, an enrichment analysis of clusters was performed using STRING with a threshold of p < 0.05, FDR-adjusted.16In order to determine the function of differentially expressed genes, gene ontology (GO). Oligonucleotide primers were designed using Primer348 having G. vaginalis 409-05 genome as template  as priming strategy. The qPCR reaction was prepared by mixing together 5 µL of iQ SYBR green supermix (Bio-Rad), 2 µL of 1:800 diluted cDNA, 0.5 µL of 5 µM forward and reverse primes and water up to 10 µL. The run was performed in a CFX96TM thermal cycler (Bio-Rad) with the following cycling parameters: 3 min at 95 °C, followed by 45 cycles of 10 s at 95 °C, 10 s at 60 °C, and 15 s at 72 °C. Reaction efficiency was determined by the dilution method.50 At 60 °C all set of primers used had the highest and more similar efficiencies. Normalized gene expression was determined by using the delta Ct method (ECtΔ), a variation of the Livak method, where ΔCt = Ct (reference gene)—Ct (target gene) and E stands for the reaction efficiency experimentally determined. A non-reverse transcriptase control was included in each reaction. Three biologic replicates of each condition were analyzed.In order to validate RNA-seq data, qPCR was performed to quantify the transcription of eight randomly selected genes, by using the same total RNA utilized for libraries construction  and new total RNA obtained from independent experiments performed under the same biological conditions . Furthermore, the gene expression profile of known virulence genes was also addressedte Table . The samSupplementary Figure 1Supplementary Figure 2Supplementary Tables


Herba Cistanche (AEHC) on STC treatment and to determine the possible mechanisms by a loperamide-induced slow transit constipation (STC) model. HPLC was performed for identification and confirmation of the bioactive components in the AEHC. It was found that AEHC attenuated STC responses based on increased fecal quantity, moisture content, and intestinal transit rate, as well as serum levels of GAS, MTL, SS, and CGRP. The protein and mRNA levels of c-kit, a labeling of interstitial cells of Cajal (ICC), also increased. Meanwhile, only the protein level of SCF, a ligand of c-kit, increased. The analysis of our data suggested that AEHC could obviously improve the function of ICC via a signaling pathway involving PI3K, SCF, and c-kit and enhance colonic motility indices such as GAS, MTL, SS, and CGRP. It is interesting to note that AEHC appeared to be effective on constipation, so further experiments are necessary to clarify the exact mechanisms involved.Traditional Chinese medicine was reported to have good effects in treating functional constipation. This work attempted to prove the effects of aqueous extracts of Constipation is a common functional gastrointestinal disease and public health problem, characterized by continuously difficult, infrequent, or incomplete defecation . The preA number of current chemical drugs such as osmotic or secretory laxatives and bulking agents are universally used to treat constipation , althoug4 receptor agonist with high selectivity and specificity, which can enhance the gastric, small intestine and colon motility and works faster in dynamics [−2 channel to promote gastrointestinal secretion and increase the effects of intestinal transmission [Tegaserod is a selective 5-hydroxytryptamine receptor antagonist that can stimulate intestinal peristalsis and secretion , 12. Prudynamics –15. As asmission .Motility in constipation is a widespread problem, but its etiology is not yet clear. Nevertheless, convincing evidence has demonstrated the contribution of interstitial cells of Cajal (ICC) to pathogenesis of constipation . ICC is  Herba Cistanche is a traditional herbal medicine used in the treatment of chronic renal disease, female infertility, profuse menorrhagia, and impotence [ Herba Cistanche prevents brain neuron apoptosis by expressing apoptosis-related factors and neurotrophic factors in MES23.5 cells [ Herba Cistanche has also demonstrated pharmaceutical potential in the treatment of Yang-Qi Kidney-Yang Deficiency Syndrome. A recent study has demonstrated that Herba Cistanche heightens the mitochondrial respiration and glutathione antioxidant status in H9c2 cardiomyocytes [Various herbal medicines and traditional Chinese formula have recently drawn attention as new-fangled therapeutics for the treatment of chronic constipation as a result of improved stool production , 21. Hermpotence , 23. Her.5 cells . Herba Cmyocytes .TCM believes that constipation has a close relationship with kidney. Opening at external genitalia and anus, kidney governs urination and defecation. Transportation of the waste relies on the promotion of kidney qi and nourishment of kidney yin. If kidney yang is deficient or the fire of life gate dies out, cold congestion of large intestine will result in stagnation of waste. Constipation is also caused by dry bowel movement, since deficiency of kidney essence fails to generate enough fluid .Herba Cistanche is a common tonifying herb, which is sweet and salty in flavor and warm in nature. Attributive to large intestine and kidney meridians, it can tonify kidney yang and moisten the intestine to relieve constipation. This single herb and its Chinese pharmaceutical preparations have been widely used to treat constipation with remarkable efficacy [efficacy . Herba Cistanche roots on constipation have been recorded [ Cistanche species” [ Herba Cistanche roots on constipation has not existed so far, the clinical effective dose is 20 g per day in Chinese Pharmacopoeia, and the clinical effect dose not increase further with the dosage.In China, the effects ofrecorded , 29 and species” ; however Herba Cistanche is so scarce [ Herba Cistanche's function of intestinal promotion and its action on ICC in the present study.Sinceo scarce , standar Herba Cistanche were purchased from Nanjing Haichang Chinese Medicine Group Co., Ltd. , which were harvested, collected, and processed following standard ethnobotanical practices from the plantations in the Sinkiang area of China. The identity of the plant was ascertained morphologically by Professor Tu Lin Lu of Nanjing University of Chinese Medicine, Nanjing, China. A voucher specimen (NUCMCHS-2015628) was deposited at the College of Health Sciences, Nanjing University of Chinese Medicine, Nanjing, China.All Herba Cistanche, weighing 500 g, were pulverized using an electric blender and extracted in 5000 ml distilled water, after which the aqueous extract was purified at 100°C for 2 h using a circulating extraction equipment . The extracts were filtered through a 0.22 μm membrane pore , and the residue obtained from the extracts was dissolved in 4000 ml water. After reflux extraction, the extracts were collected and evaporated to obtain the final sample product. Aqueous extracts were concentrated to dry pellets (1 g/ml) using a rotary evaporator  and stored at −20°C for subsequent usage. Solid samples were reconstituted in distilled water to give the required doses of 10, 20, and 40 mg/kg body weight for the experiment.Slices ofThe experimental protocol used in this study was reviewed and approved based on ethical procedures and scientific care by the Suzhou Hospital of Traditional Chinese Medicine-Institutional Animal Care and Use Committee, Suzhou, China . All male rats (Sprague Dawley strain) with a mean weight of 160 ± 20 g were purchased from Shanghai Laboratory Animal Research Center in accordance with the guidelines of the National Institutes of Health. The animals had free access to a standard diet (AIN-93 M) and tap water ad libitum and were housed individually in stainless-steel wire-mesh cages, well ventilated at 23 ± 1°C, 45 ± 5% relative humidity, and exposed to 12 h natural light and 12 h dark daily.n = 12 per group): A: normal group (NG), B: model group (Lop + vehicle-treated group), C: group of constipation rats treated with low aqueous extract of Herba Cistanche (Lop + LAEHC-treated group), D: group of constipation rats treated with medium aqueous extract of Herba Cistanche (Lop + MAEHC-treated group), and E: group of constipation rats treated with high aqueous extract of Herba Cistanche (Lop + HAEHC-treated group). Group A was treated with normal saline (10 ml/kg); Group B was treated with loperamide (4 mg/kg); Group C was treated with loperamide (4 mg/kg) and low aqueous extract of Herba Cistanche (1 g/kg); Group D was treated with loperamide (4 mg/kg) and medium aqueous extract of Herba Cistanche (2 g/kg); and Group E was treated with loperamide (4 mg/kg) and high aqueous extract of Herba Cistanche (4 g/kg). All rats from normal group were injected with 0.9% sodium chloride, while the others were injected with loperamide in 0.9% sodium chloride twice a day for 3 consecutive days to induce constipation. The aqueous extract of Herba Cistanche was suspended in water and was orally administered once daily from day 4 to day 18 in Group C, Group D, and Group E, whereas Groups A and B received a consistent volume of water via gavage.The loperamide was used to induce constipation in rats in accordance with previous studies , 33. A t18 column .HPLC profiling was performed in the Shimadzu Liquid Chromatographic System , consisting of an LC-20AT pump and a double UV-VIS spectrophotometric detector. The eluents were monitored at 330 nm at 30°C. The isocratic mobile phase consisted of methanol −0.1% formic acid  and ran at a flow rate of 1.0 ml/min. Chromatographic separation was performed on a Diamonsil CAlterations in exercise, food intake, water intake, body weight, and hair were observed and recorded daily throughout the experimental period. We observed “alterations in exercise” following the specific steps: the self-made box  is used for open field test; the white ground was divided into diamonds (12 cm × 12 cm) by black line in dark and quiet room; a 100 W bulb hung 1 m above the center of the box; the rats were placed to the center of the box on the 4th, 6th, 12th, and 18th days of the modeling. The number of squares that rats passed through within 5 minutes was used as the horizontal movement score and the upright time of the rats within 5 minutes was used as the vertical movement score. Horizontal and movement scores plus vertical movement score indicate the amount of exercise of rats. Each rat was measured once , 35.On the final day of the oral administration, fresh stool pellets from each SD rat were collected into round-bottomed stoppered tubes during 6 h, and the total weights from each group were recorded. To determine the fecal moisture content, we used an electric balance to dry the stool pellets until constant weight is attained and measured the dry weight. The moisture content was calculated by the following equation:Intestinal transit ratio was conducted according to previously reported protocols ; brieflyAt the end of the experimental period, rats were subjected to 12 h fasting but were allowed free access to water. All rats were then anesthetized 30 min later by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and placed on a temperature-regulated table. Blood samples were collected and centrifuged at 3500 rpm for 15 min to obtain serum. The colon was split immediately and flushed with normal saline at 4°C and then divided to two pieces. One fragment was fixed in 10% formalin and processed in split paraffin for subsequent immunohistochemical (IHC) analysis, while the other was stored at −80°C until they were assayed.The concentrations of gastrin (GAS), motilin (MTL), somatostatin (SS), and calcitonin gene related peptide (CGRP) in the serum were estimated by ELISA using commercially available kits.μm thick slices that were stained with hematoxylin and eosin . Morphological features of these sections were observed under light microscopy, after which the villus length, crypt thickness, and muscle thickness were measured using Leica Application Suite .Transverse colons collected from SD rats were fixed with 10% formalin for 12 h, embedded in paraffin wax, and sectioned into 5 μm sections were cut from paraffin blocks for IHC analysis. The sections were stained with rabbit anti-c-kit (1 : 100) at 4°C overnight. The secondary antibody and avidin-biotin peroxidase complex method were used according to the manufacturer's instructions. An immunoglobulin-negative control was used to eliminate nonspecific binding. Expression levels of c-kit were determined by the semiquantitative method. The tissue sections were then examined using the Carl Zeiss imaging system, and staining intensity was quantified using Image-Pro Plus 6.0 software .We followed the methods of Zhu et al. (2016) ; unstainμl) was extracted according to the manufacturer's instructions using the TRIzol Reagent . A reverse transcription-PCR kit  was used to synthesize the first strand of cDNA in accordance with the manufacturer's protocol. All the targeted parameters are shown in Gene expression levels of c-kit, SCF, and PI3K mRNAs in the colon were determined using a total RNA extraction kit  according to the manufacturer's protocols. Real-time PCR was performed with ABI StepOnePlus Real-Time PCR System (ABI). SYBR Green I Real-Time PCR Master Mix  was used to detect PCR products. Reactions were performed in triplicate runs. Subsequently, fresh rat colon tissue weighing 0.5 g was homogenized in liquid nitrogen. Total RNA  antibodies, respectively. Binding of the primary antibody was detected using the corresponding HRP-conjugated secondary antibody . An enhanced chemiluminescence kit  and the Odyssey Infrared Imaging System  were used for chemiluminescence detection. Housekeeping protein β-actin was used as a loading control. The amount of protein expression is presented relative to the levels of β-actin.Protein contents of c-kit and SCF in colon samples homogenates were evaluated by Western blot. In brief, the total proteins of homogenates were determined using a bicinchoninic acid assay . Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used to separate proteins in BioRad electrophoresis system . Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. After 2 h treatment with blocking buffer in TBS containing 5% nonfat milk at room temperature, PVDF membranes were incubated overnight with 1 : 1000 diluted anti-c-kit , 1 : 500 diluted anti-SCF , and 1 : 8000 diluted anti-P < 0.05.Measurement data were reported as mean ± standard error of mean (SEM) and analyzed by SPSS 16.0 software . One-way analysis of variance (ANOVA) followed by Duncan multiple range test was used to determine significant differences in all the parameters. Values were considered statistically significant at  Herba Cistanche contain high concentrations of several bioactive components related to laxative effects. The concentrations of total echinacosides and total verbacosides were 0.64 mg/g and 0.16 mg/g, respectively. Two peaks indicating high echinacoside and verbascoside levels were detected in the LP at an appropriate retention time by high-performance liquid chromatography (HPLC).As shown in P < 0.01), while differences among Groups B, C, D, and E  but increased when treated by AEHC (P < 0.05) . Although the intestinal transit rate in Groups C, D, and E was also significantly higher than that in Group B (P < 0.05), Groups C, D, and E had significantly lower ratios than Group A.As seen from P < 0.01, resp.). On the other hand, Groups C, D, and E  have effectively higher levels of GAS and MTL from the administration of Herba Cistanche extracts compared with Group B. The SS values in Group B rats were higher compared with Group A (P < 0.01); Groups C, D, and E have attenuated levels of SS (P < 0.05) (P < 0.01) and in Herba Cistanche-administered Groups C, D, and E were constant (P > 0.05).The serum levels of GAS and MTL obtained from Group B rats significantly decreased compared with those from Group A ( < 0.05) . CompareP < 0.01). However, the average length of the villus in Group C greatly increased by more than 30% when compared with Group B . Meanwhile, the average length of the villus greatly increases in Groups D and E when compared with Group B . Furthermore, alteration of muscle thickness was very similar to villus length. In Group B, the muscle thickness was dramatically thinner when compared with Group A. However, the muscle thickness levels in Groups C, D, and E increased by 30–35% relative to that of Group B . The expression level of c-kit in Group C was higher than that in Group B (P < 0.05), while more c-kit was present in Groups D and E than in Group B (P < 0.05). After treatment with different concentrations of aqueous extract of Herba Cistanche for 2 weeks, the protein expression levels of c-kit and SCF were detected by Western blot analysis. Western blot analysis showed that c-kit and SCF expression levels were remarkably lower after loperamide treatment in colons of rats (μg/ml AEHC (P < 0.05). In addition, treatment with 100 and 200 μg/ml AEHC elevated c-kit protein expression levels. SCF protein expression significantly increased by 20.1, 24.7, and 8.4% at treatment with 100, 200, and 500 μg/ml AEHC, respectively , while the mRNA levels in Groups C, D, and E were higher than in Group B (P < 0.05) (P < 0.05).To investigate whether AEHC treatment can affect the regulation of mRNA related to muscle contraction, alterations in c-kit, SCF, and PI3K expression levels were observed in colons of constipation rats using specific primers. The results showed that the mRNA levels of c-kit in Group B was significantly lower compared with Group A ( < 0.05) . We also < 0.05) . In addiConstipation is a highly common disorder characterized by poor bowel movements. It greatly diminishes quality of life and brings about huge economic burdens on both patients and national health insurance . NaturalLoperamide, atropine-diphenoxylate, and morphine are widely used to induce constipation of laboratory animals. Among the three drugs, loperamide induces prolonged duration of stool evacuation and a delay of intestinal luminal transit, since it inhibits both water secretion and smooth movement in the intestinal wall . Herba Cistanche can recover the propellant function of colon to different degrees.Analysis of body weight in this study did not find any difference within each group. After the experiment, no difference was found either. The low-dosage group of AEHC still saw the lowest body weight, yet the model group and the normal group witnessed the highest body weight. Constipation is generally a functional disease. Slow transit constipation induced in this study did not affect nutrient uptake of the rats and their body weight during the experiment. Difference in amounts of feces of the rats and their moisture content did not exist between groups before modeling; however, they both decreased remarkably after modeling and the moisture content of feces increased after using AEHC at different dosages. After studying the propellant rate of carbon powder of colon of rats, the model group saw obvious decrease of the rate, while it remained normal in low-dosage, medium-dosage, and high-dosage groups of AEHC. The results showed that loperamide-induced chronic transit constipation treated with different dosage ofGastrointestinal hormones are high-performance bioactive substances secreted by endocrine cells of gastrointestinal mucosa and pancreas, possessing effects of both excitement and inhibition to regulate gastrointestinal motor function , 41, of c-Kit is a transmembrane protein. The stem cell factor (SCF) of its ligand is produced by neuron cells and smooth muscle cells. It promotes the development and differentiation of ICC and maintains its normal physiological function. c-Kit labeling indirectly reflects the quantity and density of ICC , 45. In AEHC promotes intestinal motility by improving ICC function and regulating neurotransmitters in this study, which proved that AEHC has the potential to treat and prevent constipation. The extracts improved the slow-wave production of colon and regulated the rhythm of the contractive activity of smooth muscle by increasing ICC quantity through the PI3K/SCF/c-kit signal pathway.However, further more detailed experimental studies might improve understanding of the other molecular pathways of AEHC in curing STC and help guide prospective clinical studies evaluating its effects and safety.


In all participants combined, the median (95% CI) UIC was 101  µg/L, median  calculated iodine intake was 112  µg/day and median  estimated usual iodine intake was 101  µg/day. According to WHOs criteria for evaluation of median UIC, iodine intake was inadequate in the elderly, pregnant women, vegans and non-pregnant women of childbearing age. Children had the highest (82%) and vegans the lowest (14%) probability of adequate iodine intake according to reported food and supplement intakes. This study confirms the need for monitoring iodine intake and status in nationally representative study samples in Norway. Inadequate iodine intake has been identified in populations considered iodine replete for decades. The objective of the current study is to evaluate urinary iodine concentration (UIC) and the probability of adequate iodine intake in subgroups of the Norwegian population defined by age, life stage and vegetarian dietary practice. In a cross-sectional survey, we assessed the probability of adequate iodine intake by two 24-h food diaries and UIC from two fasting morning spot urine samples in 276 participants. The participants included children ( Iodine is an essential nutrient required for the synthesis of the thyroid hormones triiodothyronine and thyroxine, which are critical to fetal brain development and metabolic processes in the body throughout life . Iodine Milk and seafood are the main dietary iodine sources in Norway, while the contribution from low levels in drinking water and iodine fortified table salt (<1%) is considered to be negligible ,20. SurvIodine from food is readily absorbed and ~92% is excreted in the urine, making urinary iodine concentration (UIC) a good indicator of recent iodine intake at the population level . The WorExisting information on iodine status in subgroups of the Norwegian population is limited, particularly in young children, adolescents and the elderly, as well as in vegetarians and vegans; the latter intentionally eliminate the main iodine-containing foods such as milk and fish from their diet. The aim of this study was therefore to evaluate iodine status by assessing urinary iodine concentration and the probability of adequate iodine intake in subgroups of the Norwegian population defined by age, life stage and vegetarian dietary practice.The study design is cross-sectional and participants were primarily recruited to an international study of dietary exposure to deoxinivalenol, a mycotoxin originating from cereals . The infAfter giving informed, written consent, participants provided information about age, weight, height, dietary restriction, vegetarian dietary practice, smoking and other lifestyle habits in a short interview. Together with a project worker, they choose two days to record all food, drink and dietary supplements consumed in an open diary and to provide first morning void spot urine samples on the two mornings after completing the food records. Most participants (90%) collected data on two consecutive days.The study was conducted according to the guidelines laid down in the Declaration of Helsinki and written informed consent was obtained from all participants, including parental consent for children younger than 16 years of age. The Regional Committee for Medical and Health Research Ethics approved the study (REK 2014/207). Participants received an open food diary and information on how to record all food and drink items for two whole days (24-h food record). They received instruction to report all dietary supplements by name and manufacturer, and to record all food and drink items and all mixed dishes to the nearest gram (or mL). When a kitchen scale was not available, we asked participants to report consumed amounts in household measures and to include a description of the portion size . For mixed dishes, we asked for the name of the dish and a list of single ingredients in addition to the total weight or household measure. Two trained research assistants coded all food and drink items in the food records manually. Food items were assigned a food number used in the Norwegian Food Composition Table . If an iFasting spot urine samples were collected on each of the two mornings following food recording. Participants received two 0.5 L plastic bottles with a wide opening and a double lid for collecting the samples. The urine samples were kept cold until collection by a project worker and distributed into vials stored at −20 °C until analysis. Urinary iodine concentrations were determined at the National Institute for Health and Welfare (THL) in Helsinki (Finland). Iodine measurements were carried out by inductively coupled plasma—mass spectrometry using an Agilent 7800 ICP-MS system . In brief, 100 µL of urine sample was extracted by using ammonium hydroxide solution. Tellurium was used as an internal standard. On the ICP-MS, m/z = 127 was scanned for iodine determination. The limit of quantification (LOQ) was 2 µg/L and the linearity was excellent up to 1500 µg/L (r = 0.9999). Coefficient variation (CV%) was 2–3%. The National Institute of Standards and Technology (NIST) reference standard materials SRM2670a  and SRM3668 Levels 1 and 2 were used to ensure the accuracy of urinary iodine concentrations. The laboratory at THL participates in Ensuring the Quality of Urinary Iodine Procedures Program (EQUIP) organized by the Centers for Disease Control and Prevention (CDC) three times per year.Urinary creatinine concentrations were analyzed at the Department of Drug Analysis, Norwegian Institute of Public Health, according to an accredited method using a colorimetric assay (modified kinetic Jaffe method) on a Beckman Coulter AU680 analyzer . In brief, the method is a reaction with alkaline picrate forming a red-orange complex. The color intensity is directly proportional to the creatinine concentration and measured spectrophotometrically at 505 nm. The LOQ is 0.2 mml/L. We present urinary iodine concentrations as µg iodine per L urine and as µg iodine per g creatinine. According to WHO, a population is defined as iodine sufficient when the median UIC is between 100 μg/L and 299 µg/L in school-age children and non-pregnant individuals. In addition, no more than 20% of the UIC samples should be lower than 50 µg/L [The recommended daily intake (RDI) is the intake level of a nutrient considered to meet the requirement of nearly all healthy individuals over time, while the estimated average requirement (EAR) is the intake level estimated to meet the requirement of half of the healthy individuals in a group. In Norway, the RDI for iodine is based on the Nordic Nutrition Recommendations 2012 (NNR12)  and is 9p-values < 0.05 are considered statistically significant. We present normally distributed data as mean and standard deviation and non-normally distributed data as median, 95% confidence intervals (95% CI), interquartile range (IQR), and 25 and 75 percentiles . We used the Wilcoxon matched pair signed-rank test to examine differences, and Spearman rank correlation and intraclass correlation coefficients (ICC) to assess agreement between paired measurements. ICC was obtained by linear mixed model regression and is the ratio of between-person variation and total variation (between-person + within person variation). Data processing and analyses were performed using IBM SPSS statistics version 23  and STATA 14 . We assessed variable distribution using visual inspection of plots (histograms and box plots). We calculated iodine intakes from food and supplements recorded in the two 24-h food records (µg/day) and used the two 24-h iodine intakes to estimate usual iodine intake and the probability of adequate iodine intake by the Institute of Medicine’s probability approach ,38. The p-values < 0.10 were included in the final model.UIC was not normally distributed and values were log-transformed when used in mean (least square) regression. However, we used median regression analysis (quantile regression) with non-transformed UIC as the dependent variable to evaluate predictors of median UIC. Median regression is less sensitive to outliers than mean regression and estimates changes to the median of the dependent variable (rather than the mean), conditional on the values of the independent variable . We incln = 25), while participants who reported no meat or animal-derived food items were denoted vegans (n = 19). n = 26), 23 used multivitamin-mineral supplements and three used kelp-supplements. Iodine supplements contributed on average 147 µg/day in iodine-supplement users. Five participants reported daily smoking and five reported occasional smoking. The study sample included 156 (57%) female and 120 male participants. Vegetarian dietary practice has multiple definitions . The curThe measured UICs as well as the calculated iodine intakes on day 1 and day 2 did not differ between the two days . We therThe median and 95% CI for UIC was 101  µg/L in all participants combined. Children had the highest and vegans, the elderly and pregnant women had the lowest median UIC . ChildreExcessive iodine intake (defined as UIC at or above 300 µg/L) was evident for 12 individuals, of which two were OL-vegetarians and one was a vegan. These three had the highest observed UICs and all reported using kelp-supplements. According to the supplement labels, one serving would provide 150 µg iodine, while the observed UICs in the two morning spots were 1409 and 1093 µg/L for person a, 1253 and 75 µg/L for person b, and 846 and 598 µg/L for person c. The remaining nine individuals with UIC above 300 µg/day included three children , three adolescents (UIC range 301 to 646 µg/L) and three adults (UIC range from 227 to 776 µg/L). Of these, one adolescent and one adult reported iodine supplement use. In the pregnant women, no participants had excessive iodine intake, i.e., UIC above 500 µg/L. In all participants combined, the median  calculated iodine intake from food was 103  µg/day and the corresponding intake from food and supplements combined was 112  µg/day . The majIn all participants combined, the median  estimated usual iodine intake was 101  µg/day and the mean iodine intake was 119 µg/day. The mean probability of adequate iodine intake was 60% in all participants combined . Childrep < 0.05 for all). Correlations were lower for estimated usual iodine intake than for calculated iodine intake. The results showed moderate to high correlation between calculated iodine intake and UIC. In all participants combined, the correlation with total iodine intake was 0.46 for UIC expressed as µg/L and 0.47 for UIC expressed as µg/g creatinine. The corresponding correlation coefficients in subgroups ranged from 0.35 in adolescents to 0.46 in children, 0.50 in pregnant women, 0.55 in adults, 0.56 in vegans, 0.71 in the elderly, and 0.79 in ovo-lacto vegetarians (p < 0.001). Although iodine-supplement use was more prevalent in women, women had lower UIC than men (p < 0.001). In non-pregnant women of childbearing age (18–47), median (95% CI) UIC was 71  µg/L, while the UIC in adult men was 110  µg/L. UIC did not differ between non-pregnant women of childbearing age and pregnant women (p = 0.33).In all participants combined, UIC was higher in iodine-containing supplement users than non-users (p < 0.01) and lower in the elderly (p = 0.05) and vegans (p < 0.01) than in adults (p = 0.19) or between OL-vegetarians and non-vegetarian adults (p = 0.50).Pairwise comparison of UIC between the non-overlapping population groups showed that median UIC was higher in children (n adults A. Howevep = 0.029), higher in pregnant women than in non-pregnant, non-vegetarian women (p < 0.001), and lower in vegans than in non-vegetarian adults (p < 0.001). There was no difference in iodine intake between adolescents, the elderly or OL-vegetarians and non-vegetarian adults   B. In multiple regression analyses of UIC (log-transformed), calculated iodine intake from food and iodine supplement use as independent variables, with additional adjustment for age, sex and energy intake, explained 40% of the variance in UIC (β = 0.43 for a 10 µg increase in iodine intake and β = 0.51 for iodine supplement use). In multiple quantile regression evaluating change (µg/L) in median UIC we did not include iodine from food, but examined differences in median UIC in supplement vs. non-users, three age groups  vs. adults (reference), female vs. male (reference), smokers vs. non-smokers (reference) and vegans vs. non-vegans (reference). Compared to adults, children (3–9 years) and adolescents (10–17 years) had significantly higher median UIC and the elderly (≥ 65 years) had lower median UIC. Female participants, smokers and vegans had significantly lower UIC than male participants, non-smokers and non-vegans . IncludiRegressing creatinine-adjusted UIC as the dependent variable (urinary iodine as µg/g creatinine rather than µg/L) resulted in comparable coefficients for the differences between iodine-supplement vs. non-supplement users, children vs. adults, and vegans vs. non-vegans. However, the differences in UIC between non-smokers vs. smokers, adults vs. adolescents, and adults vs. elderly disappeared when UIC were adjusted for creatinine (data not shown). Evaluation of food intakes and iodine sources showed that milk, including yoghurt and milk products, was the main source of iodine in all subgroups except vegans , contribThe present study evaluated iodine status in groups defined by age, life stage and vegetarian dietary practice. The results should be interpreted with caution as the study population was not nationally representative and the groups were small. According to the WHO criteria for median UIC , childreOur results add to the evidence that inadequate iodine intake is not confined to low-income countries . InadequWe found that children had the highest median UIC, the highest usual iodine intake and the lowest probability of inadequate iodine intake. Adequate iodine nutrition has been reported in school-age children in Sweden , Portugan = 15–70), the results of all the studies, independent of sample size, corroborate insufficient iodine intake or status in vegans. Vegans clearly rely on iodine supplements to secure recommended iodine intake. However, the form of supplements must be considered and our finding of UIC values above 1000 µg/L in kelp-supplement users highlight that standardization of the iodine content in kelp supplements is problematic. Because there is large variation in the iodine content in different types of kelp, kelp-supplements should be avoided. To the best of our knowledge, this is the first study to evaluate iodine intake in Norwegian vegetarians. The number of people adopting a vegetarian diet is increasing worldwide . We cateComparison of population UIC in spot samples with established UIC cut-offs is the recommended method for evaluation of iodine adequacy at the population level, while no existing biomarker accurately reflects iodine intake at the individual level . As UIC There is diurnal variation in iodine excretion and iodine measured in morning urine samples has lower intra-individual variation than iodine measured in spot urine samples collected at random time points . It mustIodization of salt has successfully reduced iodine deficiency worldwide in the last decades and WHO encourages universal mandatory salt iodization . Public The strengths of this study include two spot urine samples and two detailed 24-h food diaries for all participants. A major limitation of the current study is the relatively small sample size, particularly in the subgroups comprising the elderly, OL- vegetarians and vegans. Another limitation is that participants are not representative of the Norwegian population as they were primarily recruited among employees at two research institutions. The participants therefore are likely to have better socio-economic status (SES) and include fewer smokers than the general population. Given that individuals with high SES are likely to be more health conscious and have better diets than individuals with lower SES, it is not likely that iodine status will be better in the general population than in this study population. However, the results need to be replicated in larger, better designed and nationally representative samples. This cross-sectional study in population groups defined by age, life stage and vegetarian dietary practice, showed that children had median UIC above the WHO cut-off and also had the highest probability of adequate iodine intake according to dietary assessment. However, the elderly, pregnant women, non-pregnant women of childbearing age and vegans had median UIC below the WHO cut-off and had the lowest probability of adequate iodine intake. The current study shows that dietary sources of iodine do not secure adequate iodine intake in all groups of the population, and use of iodine containing supplements is required to meet iodine recommendations in vegans and individuals who cannot meet iodine recommendations from diet alone. However, consideration needs to be given to the form of supplement as kelp supplements can lead to iodine excess. Although the results must be interpreted with caution, this study confirms the need for the monitoring of iodine intake and status in nationally representative samples of the Norwegian population.


Endothelial function (EF) reflects the balance between vasodilatory and vasoconstrictive factors produced by (or acting on) the innermost lining of blood vessels. Endothelial dysfunction, an imbalance between these factors that favors vasoconstriction, has been associated with increased risk for cardiovascular disease. However, the influence of race/ethnicity and glycemic status on association between EF and cardiovascular risk factors remain to be clarified.We assessed EF in relation to glycemia and cardiometabolic profile in African-American (AA) and European-American (EA) offspring of parents with type 2 diabetes (T2D), who are participants in the prospective pathobiology and reversibility of prediabetes in a biracial cohort (PROP-ABC) study. Assessments at enrollment included a 75 g oral glucose tolerance test (OGTT), blood pressure, anthropometry, body composition (DEXA), and lipid profile. Other assessments were insulin sensitivity and resting energy expenditure. EF was measured using flow-mediated vasodilation (EndoPAT 2000) and expressed as reactive hyperemia index (RHI).2. Based on OGTT data, 96 subjects  had prediabetes and 94 subjects were normoglycemic (48 AA and 46 EA). The RHI was lower in AA than EA  and in prediabetic than normoglycemic subjects . Using RHI ≤ 1.68 as diagnostic cut-off, 19% of participants with prediabetes and 10% of normoglycemic participants had endothelial dysfunction (P = 0.04). In univariate models, RHI was positively associated with age and HDL cholesterol levels, and inversely associated with adiposity, diastolic blood pressure, and 2hr plasma glucose. The association between RHI and adiposity was stronger in men than women. The association between RHI and age, glucose and HDL cholesterol displayed marked ethnic disparities.We studied 190 subjects , mean age (±SD) 53.1 ± 9.1 years, and body mass index 30.6 ± 6.8 kg/mIn our biracial cohort comprising offspring of parents with T2D, prediabetes increased the risk of endothelial dysfunction. However, the association between EF and cardiometabolic risk factors was significantly modified by ethnicity and gender. Our findings support current understanding of endothelial dysfunction as an early sensitive indicator of cardiometabolic risk. Endothelium, the largest end organ in the body by surface area, is the target of numerous noxious stimuli, including proinflammatory cytokines, oxidative stress, shear stress from blood pressure, glycemia, and lipidemia, among others . EndotheHowever, the association between endothelial dysfunction and prediabetes has not been as well documented as that between endothelial dysfunction and established diabetes. Moreover, the influence of race/ethnicity on any association between endothelial dysfunction and prediabetes merits investigation. Indeed, the few studies on EF in prediabetic individuals are limited by lack of demographic diversity among participants , 8. The The pathobiology and reversibility of prediabetes in a biracial cohort (PROP-ABC) study is a prospective study of non-diabetic AA and EA offspring of parents with T2D. The cohort comprises participants with normal glucose values and those with recently recognized prediabetes. Data from our biracial PROP-ABC participants has enabled us to investigate the influence of ethnicity on EF before the development of diabetes and to document evidence of endothelial dysfunction in prediabetes. Studies have shown that lifestyle intervention (and certain medications) could prevent or delay the development of T2D among people with prediabetes –14. ThusWe studied AA and EA male and female offspring of parents with T2D, who are participants in the prospective PROP-ABC study. The latter is a continuation of the pathobiology of prediabetes in a biracial cohort (POP-ABC) , 16.Briefly, the POP-ABC study was a longitudinal study in which normoglycemic AA and EA offspring of individuals with T2DM were followed for the occurrence of incident prediabetes, defined as impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) using American Diabetes Association criteria . The preThe procedures at enrollment included a 75-g oral glucose tolerance test (OGTT), anthropometric and blood pressure measurements, lipid profile, and blood biochemistries. Blood pressure was recorded in the seated position, using an automated sphygmomanometer ; the average of two readings was used for calculations. The ankle-to-brachial blood pressure index (ABI) was calculated as the ratio of the mean systolic blood pressure measured over the posterior tibial artery in both lower extremities to the systolic blood pressure measured in the brachial vessel of the right arm. The average of two measurements taken from each location, 10 min apart, was used for calculating the ratio. Body weight (in light outdoor clothing) was measured in duplicate on a calibrated balance beam scale . Standing height (without shoes) was determined in duplicate with a standard stadiometer. The body mass index (BMI) was calculated as weight in kilogram divided by the square of the height in meters. Waist circumference was determined to the nearest 0.1 cm at the midpoint between the highest point of the iliac crest and the lowest costal margin in the mid-axillary line, using a Gulick II tape measure. Total and trunk fat mass was measured using DEXA. Insulin sensitivity (Si-clamp) was measured using the hyperinsulinemic euglycemic clamp, as previously described . In brieEndothelial function was assessed using the well-validated EndoPAT 2000 device  and expressed as reactive hyperemia index (RHI) , 20. Part-tests for discrete variables and χ2 test for ordinal variables. General linear regression models were used to compare anthropometric and cardiometabolic data in AAs and EAs and between prediabetic and normoglycemic subjects. The relationship between EF (RHI) and cardiometabolic markers  was analyzed using linear regression and Pearson correlation coefficients. The level of significance was set at P < 0.05.All statistical analyses were conducted using SAS statistical software, version 9.3 . Data were reported as means ± SD unless otherwise specified. Differences between groups were analyzed using unpaired P = 0.36); the mean BMI 31.6 ± 7.00 kg/m2 in Progressors and 29.1 ± 6.33 kg/m2 in Non-progressors (P = 0.003).We studied 190 subjects  who had complete results for EF. Of the 190 subjects, 96 (52 AA and 44 EA) were participants who developed incident prediabetes during follow-up (Progressors) and 94 (48 AA and 46 EA) were age-, gender-, and ethnicity-matched participants who maintained normoglycemia (Non-progressors). The mean age (±SD) was 53.1 ± 8.64 years in Progressors and 52.5 ± 9.56 years in Non-progressors (P < 0.0001) and insulin sensitivity lower  in AA than EA participants, but there was no difference in fasting plasma glucose (FPG), 2-hour plasma glucose (2hrPG), or REE between the two groups . In contrast, progressors to prediabetes had significantly lower RHI than Non-progressors who maintained normoglycemic during longitudinal follow-up  (Table P = 0.04).Endothelial function assessed by flow-mediated vasodilation (RHI) was similar in men and women, and tended to be lower in AA than EA subjects, although the difference was of marginal statistical significance  Table . Using tr = 0.21, P = 0.005) and inversely associated with weight , BMI , diastolic blood pressure , and 2hrPG  in the entire study population. The association between RHI and body weight was stronger in men  than women  ; total fat mass ; and trunk fat mass .In linear regression models, RHI was positively associated with age . Similarly, we found significant Black–White differences in the strength of association between EF and 2hrPG  and HDL cholesterol levels   in AA and 0.1 (P = 0.40) in EA  in AA and 0.09 (P = 0.43) in EA.The association between EF (RHI) and age seemed stronger in AA than EA subjects . In this study, we observed that the association between EF and adiposity measures was much stronger in men than women. Our findings are consistent with a recent report by Dass et al. that brachial artery flow-mediated dilation was significantly, inversely associated with total and central adiposity measures in men but not in women, among 108 healthy AA nonsmokers aged 18–45 years . As we f–8. In thP = 0.05). Other workers have reported significant ethnic disparities in EF, driven in part by greater adiposity and cardiometabolic risk factors in AAs  (We observed no significant correlation between EF (RHI) and insulin sensitivity in our EA or AA participants. As insulin-induced nitric oxide production from the endothelium increases blood flow and glucose uptake in skeletal muscle, a direct correlation between insulin sensitivity and EF can be expected . However= 0.001) . Thus, t= 0.001) –51. AlsoThe strengths of our study include the large sample size, the largely operator-independent procedure for assessing EF, and utilization of a biracial cohort. Also, the study design of the parent POP-ABC study allowed us to study subjects with incident prediabetes detected during longitudinal follow-up. The limitations of our study include the lack of serial data on temporal changes in EF and our inability to account for behavioral and environmental factors that could affect EF. Despite these limitations, our findings are concordant with previous observations regarding correlates of endothelial dysfunction. Moreover, our study extends previous reports by demonstrating, in a biracial cohort comprising AA and EA offspring of parents with T2D, that prediabetes increased the risk of endothelial dysfunction. In conclusion, we have shown that endothelial dysfunction is a sensitive indicator of recent transition from normoglycemia to prediabetes status, thus reinforcing current understanding of the cardiovascular risk associated with prediabetes.The authors’ individual contributions included the following: SD-J as the principal investigator, developed the study concept and design, and wrote the manuscript. IO collected the data, conducted the statistical analysis, drafted, reviewed, and revised the manuscript. NU collected the data, reviewed, and revised the manuscript. HM collected the data, reviewed, and revised the manuscript. SE reviewed and revised the manuscript. JW conducted statistical analysis, reviewed, and revised the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Octreotide (OCT) has been successfully used for treatment of acromegaly and neuroendocrine tumors for more than 30 years. However, long-term safety of OCT has not been documented in placebo-controlled setting. This present analysis pooled safety data from two similarly-designed, randomized, and placebo-controlled studies to evaluate long-term safety of long-acting OCT ; targeted post-hoc analyzes focused on cardiac, hepatic, and renal safety.Two studies  were conducted in patients with diabetic retinopathy . In this analysis, patients were stratified based on baseline glomerular filtration rate. Hepatic, cardiac, and renal adverse events (AEs) were identified by standardized MedDRA queries.Median duration of exposure was >3.5 years. Most common AEs reported with OCT were diarrhea, cholelithiasis, hypoglycemia, nasopharyngitis, and hypertension. Incidence of cardiac events (QT prolongation and arrhythmia) with OCT20 and OCT30 were comparable to placebo . For ECG findings, changes in QTcF were similar in treatment groups, and outliers did not exceed 480 ms. Incidence of cardiac ischemia was lower with OCT than placebo . Incidence of liver-related AEs was higher with OCT30 than placebo ; incidences were comparable with OCT20 and placebo . Overall incidences of renal AEs were comparable between treatment groups . Drug-related SAEs were reported more frequently with OCT ; predominantly gallbladder-related, GI-related, and hypoglycemia.The results from these long-term placebo-controlled studies confirm the established safety profile of long-acting OCT, in particular low risk of cardiac, hepatic and renal toxicity in a high-risk population. This present analysis pooled the safety data of these two studies to evaluate the overall long-term safety and also in the sub-populations defined by the baseline level of renal function. In addition, the targeted post-hoc analyses also focused on cardiac, hepatic, and renal safety.N = 195 on OCT20, N = 197 on OCT30, and N = 193 on placebo) and 313 patients in study 2 .Patient population and study design were similar for both studies that differed only in the recruitment region and number of investigational treatment arms . The studies enrolled male and female patients with type 1 diabetes or type 2 diabetes, irrespective of insulin dependence, between 18 and 70 years with an HbA1c < 13.0%. Patients had moderately severe or severe non-proliferative diabetic retinopathy or low risk proliferative diabetic retinopathy. The main exclusion criteria were a history of brittle diabetes or hypoglycemia unawareness, symptomatic gallstones without a cholecystectomy, a positive pregnancy test, or prior treatment with octreotide or another SSA. Overall, 898 patients were randomized, 585 in study 1  and for study 2 consisted of 308 patients . The safety assessments included laboratory data, electrocardiogram (ECG), vital signs, and gallbladder ultrasound. Single 12-lead ECGs were collected at baseline and at the end of treatment. During the study, ECGs were performed if they were clinically indicated by the patient’s symptomatology (as per investigator opinion). All ECGs were assessed centrally by an ECG core laboratory retrospectively for central blinded assessment. For this present analysis, data from the two studies were pooled. Adverse events of special interest  were identified by standardized MedDRA queries (SMQs) based on MedDRA version 18.0, and multiple AEs within the same SMQ were counted only once per patient.All analyses were based on the safety analyzable population (SAF), defined as patients who received at least one dose of trial medication and for whom one or more post-baseline safety assessments were obtained. The safety population for study 1 consisted of 578 patients (2), and for impaired renal function (baseline eGFR < 60 mL/min/1.73 m2)  ≥ 60 mL/min/1.73 mBaseline values were defined as the last non-missing value prior to the first dose of study treatment. Adverse event summaries include treatment-emergent AEs, occurring up to and including 90 days after the date of last study medication. For the AEs of special interest, relative risk  estimates and 95% confidence intervals (CIs) were calculated using Mantel-Haenszel method and adjusted for study as stratification factor. Pairwise comparisons of OCT30 vs. placebo and OCT20 vs. placebo for each SMQ were performed overall and in the sub-population based on baseline renal status. For the ECG analysis, the ECG recorded at the end of study visit (EOS) was considered as the last on-treatment ECG. The mean, central tendency change for the ECG parameters from baseline to EOS, as well as the number and percentages of ECG events of interest  were summarized.2 overall), male (68%), and Caucasian , and between 51 and 65 years. The demographics and baseline characteristics were mostly similar between the two studies and treatment groups . Hypertension was the most common comorbidity in all treatment groups. A summary of the baseline characteristics is presented in Table All patients had a history of diabetes for ≥10 years , and current diabetic retinopathy. The majority of patients were overweight .Overall, the most common AEs reported in the OCT20, OCT30, and placebo groups, respectively were diarrhea ; cholelithiasis ; hypoglycemia ; nasopharyngitis ; and hypertension  patients on OCT20, 13.8% (48/348) patients on OCT30, and 8.1% (28/347) of patients receiving placebo. Adverse events (suspected to be study drug related) led to discontinuation in 8.9% (17/191) of patients on OCT 20, 16.7% (58/348) of patients on OCT30, and 2.6% (9/347) of patients on placebo. Adverse events (not suspected to be study drug related) led to discontinuation in 10.5% (20/191) of patients on OCT20, 6.9% (24/348) of patients on OCT30, and 8.1% (28/347) of patients on placebo. Neoplasms  were reported in a higher number of patients in the placebo groups in both the studies .The rate for hyperglycemia was comparable between octreotide treatment groups and placebo ; while the OCT30 group experienced higher rates of hypoglycemia compared with OCT20 and placebo groups . However, events related to diabetic neuropathy were numerically more frequent in the OCT20 group compared with the OCT30 and placebo groups ; while peripheral neuropathy and paresthesia were more frequent in the placebo group compared with the octreotide groups . Hypercholesterolemia was less prevalent with octreotide than with placebo . The mean standard deviation [SD] change in weight (kg) from baseline to last value on study was higher in the placebo group vs. the octreotide treatment groups .The incidence of cardiac, renal, and hepatic AEs, by SMQ, is summarized in Table Adverse events related to cardiac arrhythmias or QT prolongations were comparable across treatment groups Table . For theEvents potentially related to Torsades de pointes  were similar between the OCT30 and placebo treatment groups. In total, three instances of sudden death occurred during the trial period: OCT20 = no cases; OCT30 = 2 cases; placebo = 1 case. All three patients  had pre-existing hypertension in addition to diabetes and died at home while sleeping. No autopsy reports were available. The investigators assessed the deaths as not related to study treatment.The overall incidence of AEs related to cardiac ischemia  and increased blood pressure  were lower with octreotide than for placebo.The mean baseline eGFR was evenly distributed across treatment groups . There was a gradual decline in eGFR over the treatment period and the trend was similar across all the 3 treatment groups. The overall incidences of renal AEs (identified by SMQ search) were comparable between treatment groups . The most commonly reported event term identified by renal SMQ search was renal failure .The incidence of AEs identified using the four liver SMQs was higher in the OCT30 group than placebo . Comparable incidences in liver-related AEs were observed in the OCT20 and placebo groups .The most common event was hepatic steatosis, occurring in 5.2% (10/191) patients and 8.0% (28/348) patients in the OCT20 and OCT30 groups, respectively, vs. 4.9% (17/347) patients receiving placebo. The only other AE occurring in >1% of the patients in any treatment group was hepatomegaly .The incidences of death for any cause during the study ranged from 4 to 6.3% between groups . In the events with a reported fatal outcome, cardiac disorder was identified as the most common cause with incidences of 4.2% (8/191) on OCT20, 2% (7/348) on OCT30 and 2% (7/347) on placebo, respectively.Overall, the frequency of serious adverse events (SAEs) was similar between treatment groups, but the SAEs suspected to be related to study drug were reported more frequently in the active treatment groups . The most common SAEs suspected to be drug-related  were cholelithiasis ; hypoglycemia ; cholecystitis , diarrhea , abdominal pain , bile duct stone , and cholecystitis chronic . Only one case of cardiac SAE was suspected to be study drug-related (OCT30 group = 0.3% [1/348]) and the respective incidences of cardiac SAEs that were not suspected to be study drug related were as follows: OCT20 = 9.4% (18/191); OCT30 = 9.2% (32/348); placebo = 14.7% (51/347). Of note, the renal and urinary disorders related SAEs were not suspected to be study drug-related .The safety profile of octreotide is well established , 16–21. Based on the RR analysis, the long-term use of octreotide was not associated with an increased cardiac risk in these patients regardless of the presence of other risk factors. The incidences of cardiac events (QT prolongation and arrhythmia) for the OCT20 and OCT30 groups were comparable to placebo . However, the incidences of cardiac ischemic events were lower in the octreotide treated patients and the risk of these events decreased upon octreotide treatment compared with placebo . It is noteworthy that fewer coronary artery disease events occurred on treatment with octreotide than placebo. No SAEs reported in the SOC of cardiac disorders were suspected to be related to octreotide.n = 6; cholestasis, n = 3; hepatic cirrhosis, n = 2; ascites, n = 2; portal hypertensive gastropathy, n = 1; hepatic encephalopathy, n = 1; hepatitis, n = 1; non-alcoholic steatohepatitis, n = 1) occurred at very low numbers, and no clinically relevant imbalance was seen between octreotide and placebo groups.The frequency of liver-related AEs was higher in patients receiving octreotide (OCT20 and OCT30) than in the placebo group. As one might expect, the overall prevalence of hepatic steatosis in this study was high, as this condition is associated with diabetes –24. In tThe overall renal function profile was similar for both studies. The RR of renal events in both the octreotide treatment groups was not significant compared with the placebo treatment group. This was also apparent from the post-baseline change in eGFR values; indicating that the effect of disease on the renal function surpassed any treatment effect. This is consistent with data from the literature indicating that octreotide has no detrimental effect on renal hemodynamics and tubular function –27.In summary, the results from these unique long-term placebo-controlled studies confirm the established safety profile of long-acting octreotide in patients with diabetic retinopathy, and also in particular demonstrate the low risk of hepatic, cardiac, and renal toxicity.