To evaluate the relationship between retinal vascular caliber and AMD in patients with AIDS.Participants enrolled in the Longitudinal Study of the Ocular Complications of AIDS had retinal photographs taken at enrollment. Retinal vascular caliber  and intermediate-stage AMD were determined from these retinal photographs. Photographs were evaluated by graders at a centralized reading center, using the Age-Related Eye Disease Study grading system for AMD and semiautomated techniques for evaluating retinal vascular caliber.P = 0.009) and mean CRVEs . The unadjusted differences were: CRAE, 4.3 μm  and CRVE, 5.5 μm . After adjustment for age, race/ethnicity, sex, human immunodeficiency syndrome (HIV) transmission category, smoking, enrollment and nadir CD4+ T cells, and enrollment and maximum HIV load, the differences between patients with and without AMD were as follows: CRAE, 5.4 μm  and CRVE, 6.0 μm .Of the 1171 participants evaluated, 110 (9.4%) had AMD and 1061 (90.6%) did not. Compared with participants without AMD, participants with AMD had larger mean CRAEs (151 ± 16 μm versus 147 ± 16 μm; In patients with AIDS, AMD is associated with greater retinal arteriolar and venular calibers, suggesting a role for shared pathogenic mechanisms, such as persistent systemic inflammation. Early-stage AMD consists of small drusen. Intermediate-stage AMD consists of extensive medium-size drusen or any large drusen, with or without pigment changes. Late-stage AMD is defined by the presence of either choroidal neovascularization or geographic atrophy.47 Antiretroviral-treated, immune-restored, human immunodeficiency virus (HIV)-infected persons have a marked reduction in the incidence of opportunistic infections and a substantially increased life span compared with those from the era before modern combination antiretroviral therapy (cART).811 Despite these improvements, antiretroviral-treated, immune-restored, HIV-infected persons have a substantially shortened life span compared with similarly aged, HIV-uninfected peers, which largely is due to an increased risk of non-AIDS diseases associated with aging .1216 This increased risk of age-related diseases suggests that antiretroviral-treated, immune-restored, HIV infection is associated with an “accelerated and/or accentuated aging” phenotype.16 Consistent with this accentuated aging, patients with AIDS have an approximately 4-fold increased age- and sex-adjusted prevalence of intermediate-stage AMD and an approximately 1.75-fold increased race- and sex-adjusted incidence of intermediate-stage AMD when compared with that in HIV-uninfected cohorts.18 Patients with AIDS also have retinal vascular calibers consistent with those of HIV-uninfected persons who are on average 10 years older, again consistent with an accelerated/accentuated aging phenotype.19 Therefore, we investigated the relationship between AMD and retinal vascular caliber in persons with AIDS enrolled in the Longitudinal Study of the Ocular Complications of AIDS (LSOCA).AMD is a leading cause of visual impairment and blindness in persons older than 65 years.21 Enrollment began on September 1, 1998, and was completed on July 31, 2011. All participants had AIDS diagnosed according to the 1993 Centers for Disease Control and Prevention revised criteria for the diagnosis of AIDS.22 Recruitment occurred at 19 clinical centers throughout the United States, typically located in urban centers with a large HIV-infected population.20 Participants with and without ocular opportunistic infections were recruited.LSOCA was a prospective, observational, cohort study of patients with AIDS in the era of modern cART.1721 Laboratory testing at enrollment included hematology and blood chemistry, blood CD4+ T cells, the amount of circulating HIV RNA in the blood (HIV load), and the presence of antibodies to hepatitis C.21At enrollment, all participants gave a detailed medical and HIV-related disease history; relevant findings were confirmed from the medical record. Fifty to 60-degree retinal photograph of the disc and macula were taken at enrollment, as previously described.Approval for the study and its procedures was obtained from the institutional review boards of the individual participating clinical centers and the three resource centers . Written, informed consent was obtained from all participants. The study was conducted in accordance with the principles of the Declaration of Helsinki.18 Digital image grading used software tools to calibrate and locate the grid.18 Approximately 10% of the enrollment photographs were digital. Grading of digital images at a reading center for AMD has been demonstrated to be comparable with that of film images.23 Fundus photographs were graded for the features of intermediate-stage AMD, including the presence, size, and area of drusen, and the presence and area of pigmentary abnormalities. Grading procedures were based on the Age-Related Eye Disease Study system for classifying AMD from retinal photographs.7 Graders were masked as to clinical information. The outcome of interest was the presence of intermediate-stage AMD, defined as at least one large druse  or extensive medium-sized drusen with pigment abnormalities.24 Quality control was provided by a resampling and regrading of 10% of the photographs by the reading center project ophthalmologist (RPD).Photographs were graded at the Studies of the Ocular Complications of AIDS (SOCA) Reading Center at the University of Wisconsin from stereoscopic color photographs of the macula obtained at enrollment. Photographers and camera systems were certified for SOCA photographic procedures by reading center personnel. Images were graded either from 35-mm film, mounted in typical slide mounts and viewed on a light box with a Donaldson 5X stereoscopic viewer, or from digital images displayed on calibrated computer monitors and viewed with a stereoscopic viewer . The Early Treatment Diabetic Retinopathy Grid, measuring 7.2 mm on the retina, was placed on film slides using an acetate overlay sized for the camera type and degree of view.25 Briefly, the six largest arterioles and venules in a ring-shaped area located between 0.5 and 1.0 disc diameter from the optic margin were identified. Computer software measured the caliber of these individual vessels, then combined them into two summary variables for the eye: the projected caliber sizes of the central retinal artery equivalent (CRAE) and of the central retinal vein equivalent (CRVE). Because drusen are difficult to evaluate in the face of extensive retinal necrosis and scarring from cytomegalovirus (CMV) retinitis, eyes with ocular opportunistic infections were not graded, and these participants were not included in the AMD study.26Retinal vascular indices were determined in a semiautomated manner as previously described.2 test for categorical variables, the t-test for normally distributed continuous variables, and the Wilcoxon rank sum test for non-normally distributed continuous variables.27 Multiple linear regression was used to assess relationships of CRAE and CRVE with presence of AMD in three models: unadjusted, adjusted for age, and adjusted for age and other baseline covariates. P values were two-sided and nominal. Statistical analyses were conducted with SAS/STAT, version 9.3  and Stata version 15.0  software packages.The association of the presence of AMD at baseline with characteristics of the study population was assessed using the χOf the 2392 participants enrolled in LSOCA, 535 had an intraocular opportunistic infection (primarily CMV retinitis) and could not be assessed for AMD, leaving 1857 participants. Of these, 1583 were assessed for AMD from the enrollment photographs. Of these 1583 participants, 1171 also were assessed for the retinal vascular caliber from enrollment photographs. The 1171 participants assessed for both AMD and retinal vascular caliber form the study population. Of these 1171 patients, 110 (9.4%) had intermediate-stage AMD, and 1061 (90.6%) did not.+ T cells, nadir CD4+ T cells before enrollment, HIV load at enrollment, maximum HIV load before enrollment, use of cART, and comorbidities. Participants with AMD were older (mean age 47.0 ± 8.3 years) than those without AMD  and appeared to have a greater proportion of participants whose HIV transmission category was either injection drug use or heterosexual transmission  characteristics of the study population and of participants with and without AMD are listed in P < 0.009). The mean . The mean CRAE and CRVE declined with age for participants with and without AMD, but were consistently larger for participants with AMD , and the difference in mean CRVE between those with and without AMD was 6.0 μm .Participants with AMD had larger CRAE and CRVE than participants without AMD , 2. The with AMD . In the 18 In this study, we demonstrate that persons with AIDS with AMD have larger retinal arteriolar and venular calibers than HIV-infected persons without AMD. Previous work in HIV-uninfected populations has demonstrated several risk factors for changes in retinal vascular caliber.2834 Older age is associated with narrower retinal vascular caliber, and hypertension is associated with narrower retinal arterioles; conversely, cigarette smoking is associated with wider retinal arterioles and venules, although some studies have reported a greater effect on venules.2830 Biomarkers of systemic inflammation consistently are associated with wider retinal venules.32 The results of studies of HIV-uninfected persons with AMD have been variable, with one study showing only wider retinal arterioles and another study only wider retinal venules.34 In our study, both retinal arteriolar caliber and venular caliber were greater among persons with AMD. This effect was consistent across age, and adjustments for baseline parameters only increased the estimated difference. Our study did show an age-related decline in both CRAE and CRVE in both persons with and without AMD, as is seen in HIV-uninfected persons, but the CRAE and CRVE consistently were larger among patients with AMD than those without.We previously reported that patients with AIDS had an approximately 4-fold increased prevalence of intermediate-stage AMD and an approximately 1.75-fold increased incidence of intermediate-stage AMD, consistent with the accelerated/accentuated aging phenotype present in antiretroviral-treated, immune-restored, HIV-infected persons.16 Antiretroviral-treated, immune-restored, HIV-infected persons are characterized by a state of chronic immune activation with ongoing systemic inflammation.35 Studies in HIV-uninfected persons demonstrate that systemic inflammation is a risk factor for AMD.3639 Therefore, the increased retinal venular caliber in HIV-infected persons with AMD may relate to the common role of systemic inflammation in the pathogenesis of AMD and in altered retinal vascular caliber. Nevertheless, it also is possible that other, unobservable factors are responsible for both the occurrence of AMD and vascular dilation in these patients.Antiretroviral-treated, immune-restored, HIV-infected persons have immunologic changes similar to those seen in HIV-uninfected persons older than 70 years, a phenomenon sometimes termed immunosenescence.21 The nadir CD4+ T cells were very low, even though many participants had substantial immune recovery by enrollment. The effects of such levels of immune deficiency on subsequent immune activation and systemic inflammation may be different from those seen in HIV-infected patients who never reach this level of immune deficiency. As such, the prevalence and incidence of AMD in HIV-infected patients with earlier stages of HIV infection remain uncertain. Nevertheless, because of late diagnosis and difficulties in completely suppressing HIV replication among all patients, many individuals with HIV infection will present with or progress to AIDS,41 so that there remains a population of HIV-infected patients who will progress to AIDS and for whom these results are relevant. Furthermore, the LSOCA cohort is similar to the AIDS epidemic in the United States in terms of demographic and other characteristics,20 suggesting some degree of generalizability to patients with AIDS. Finally, we only could analyze (and adjust for) those factors where we had data from the LSOCA cohort study. As such, it is possible that other, unobservable factors may contribute to both AMD and retinal vascular dilation in this population.Caution should be taken in interpreting our data. Although the AMD lesions seen in the LSOCA population are clinically and photographically identical to those seen in HIV-uninfected populations, we do not have histology to confirm the nature of the lesions. Caution also should be taken in extrapolating our data to all HIV-infected persons. The LSOCA cohort enrolled only persons with AIDS and not earlier stages of HIV infection.3639 as well as the state of chronic immune activation and systemic inflammation among antiretroviral-treated HIV-infected persons,35 suggest a possible common pathogenetic role for inflammation in both of these findings.In sum, LSOCA data demonstrate increased retinal vascular caliber, both CRAE and CRVE, in persons with AIDS and AMD. The relationship of systemic inflammation to AMD in HIV-uninfected persons and to increased retinal venular caliber,


One of the major genetic alterations in pancreatic ductal adenocarcinoma (PDAC) is the point mutation of K-ras gene. Plectin-1 was also recently identified as PDAC specific biomarker. The aim of this study was to investigate the improvement of diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) by using additional K-ras mutation analysis and Plectin-1 staining in patients with pancreatic mass.A total of 85 study patients with pancreatic mass underwent EUS-FNA and the final diagnoses were as follows; PDACs: 70 patients, pancreas neuroendocrine tumor: 4, metastasis to pancreas: 5, autoimmune pancreatitis: 3, chronic pancreatitis: 1, tuberculous lymphadenitis: 1, pseudocyst: 1.Sensitivity, specificity and accuracy of pathologic diagnosis in EUS-FNA specimen were 81%, 80% and 79% accordingly. When we combine K-ras gene mutation analysis with histological assessment, we could get the following results for sensitivity, specificity and accuracy; cytology and K-ras mutation analysis: 93%, 87%, and 92%, cytology, K-ras mutation analysis, and Plectin-1 staining: 96%, 93%, and 95%.Triple combinations of the techniques; cytology, K-ras gene mutation analysis, Plectin-1 staining could increase accuracy in diagnosis of PDACs. Further investigation of using minimal specimens from EUS-FNA may give us insight to understand the biological behavior of PDAC. Many studies have reported that KRAS mutation analysis with EUS-FNA appears to be very accurate at differentiating between benign and malignant pancreatic lesions [Pancreatic cancer remains an incurable and rapidly lethal cancer, with a 5-year survival rate of less than 5% and a median survival of less than 1 year . After h lesions –17. Plec lesions . It was  lesions .The aim of presented work was to investigate techniques using minimal specimen acquired by EUS-FNA in order to achieve accurate diagnosis in patients with suspected PDACs. The key of this study was on finding an ideal combination in optimum results when used in combination with commonly used pathologic readings.Clinical information and laboratory data was reviewed among the 85 study patients Table . The finDNA concentration was 81 ng/uL .Characteristics of the EUS-FNA specimens are depicted in Table Mutation analyses for KRAS codon12/13) were successful in the 60 cases, and 25 cases were excluded from KRAS mutation analysis due to inadequate specimen. Thirty-nine patients showed a specific mutation of KRAS codon12 with a single base change from GGT (Gly) to GAT (Asp). 22 patients showed change from GGT (Gly) to GTT , 4 patients to CGT (Arg), and 9 patients to GCT(Ala). No mutation could be detected in other tissues except 1 ampulla of Vater adenoma high grade and 1 pancreatic neuroendocrine carcinoma  of PDACs   Table .vs those with wild-type KRAS. Figure vs 8.1 months, p = 0.1). Also, there was no statistically significant differences about number of mutation in median OS .Of the 70 patients who were analyzed, there was no statistically significant differences in median overall survival (OS)  as compared with conventional cytology . More accurate diagnosis for pancreatic masses was able with KRAS mutation analysis and Plectin-1 staining. So, we established new helpful platform with EUS-FNA for diagnosis of PDACs. Plectin-1 is known to distinguish malignant intraductal papillary mucinous neoplasm (IPMN) from the benign IPMN, and also distinguish from PanIN I/II to PanIN III and PDACs.[Despite the recent advances in radiology, about 50% of all PDAC patients are found to have metastatic disease, surgical resection can be performed in only about 20%.–17 used nd PDACs., 21 Furtvs those with wild-type KRAS: 18.1 vs 8.1 months, p = 0.1), some studies demonstrated that the presence of KRAS mutations in tumor have a significant worse impact on survival time and response of treatment.[Considering low negative predictive value of EUS-FNA in pancreatic malignancy and possibility of peritoneal seeding, some doctors resected pancreatic masses without EUS-FNA. But, thereatment. It is st3 and/or prothrombin time international normalized ratio > 1.5); 3) resectable pancreatic masses highly suspected of PDACs in radiological imaging; and 4) refusal to participate in this study. Informed consents were obtained before the procedure. Demographic and clinical parameters were acquired from the electronic medical record, and the study protocol was approved by Institution Review Board of Seoul National University Hospital, Seoul National University College of Medicine (IRB NO: H-1202-070-398). All procedures and data acquisition were performed in accordance with STROBE statement.A total of 85 patients with pancreatic mass were consecutively enrolled in this study between June 2011 and July 2012 Fig . The incEUS-FNA specimen was acquired by two experienced endoscopists (Professors: J.K.R. and Y.-T.K.) under the conscious sedation using meperidine and midazolam in pancreatobiliary clinic of GI division at Seoul National University Hospital. After the sedation, pancreatic mass was identified and explored the optimal site for EUS-FNA. The procedure was carried out using a linear array echo endoscope  with the 22G FNA needle . EUS-FNA was performed as follows; after proper targeting to a mass, the FNA needle was passed through the biopsy channel and advanced into the target lesion under EUS guidance. To-and-fro movements were then performed 10 to 20 times with suction using a 10-mL syringe. After retracting the needle, the aspirated contents were expressed onto glass slides with the stylet smeared, and fixed in 99% ethanol. Pancreas mass located head or uncinate process was acquired by transduodenal passes while the body or tail mass was acquired through the puncturing stomach wall. The endoscopists assessed the gross adequacy of the sample, and selected the number of needle passes due to the absence of on-site cytopathologist. Additional needle passes to obtain study samples were not needed.The aspirated specimen was expelled onto a glass slide by reinsertion of the stylet and flushing with air, if needed. Ten to fifteen slides were stained with alcohol and prepared using Papanicolaou & Diff Quick cytological stain to describe the cellularity and to diagnose malignancy in each specimen. There were three experts of pathology who immediately evaluated the specimens. Slides were evaluated using a Y-FL microscope . The pathology reports of EUS-FNA are as follows: (1) positive for malignant cells (2) suspicious for malignant cells (3) atypical cells (4) benign cells (5) inadequate for diagnosis. Hence, the cytological results were interpreted as follows: (i) if the results were positive for malignant cells or suspicious for malignant cells, the masses were considered as malignant; (ii) if the results were atypical cells or inadequate for diagnosis, the masses were considered as malignant when other pathologic diagnosis with surgery, ERCP, or sono-guided/CT-guided biopsy was positive for malignant or clinical and imaging follow-up was consistent with malignancy, such as clinical progression or metastasis; (iii) if the results were atypical cells, benign cells, or inadequate for diagnosis, the masses were considered as benign based on the clinical manifestation . Plectin-1  antibody was used for IHC analyses. To determine intensity of Plectin-1 expression, nerve tissue stained with Plectin-1 was used as a positive control and reference. Nerves were known to have a moderate staining intensity. Staining intensity was recorded by two independent pathologists without giving clinical information and results from Papanicolaou & Diff Quick cytological stain. If there was any case of discrepant result, it was evaluated by a third pathologist. Plectin-1 staining was classified as negative if the staining intensity was weaker than nerves. It was classified as positive if the staining was as least as strong as nerves.The DNA was recovered from FNA cytology slide to detect KRAS mutation. Mutant KRAS was detected using a validated KRAS mutation kit  that identifies seven somatic mutations located in codons 12 and 13  using allele-specific real-time polymerase chain reaction –32. A cep values < 0.05 was considered statistically significant. All statistical analysis was performed using SPSS v.18.0 .Continuous variables were described by mean and standard deviation , and the Wilcoxon rank sum test was done if normality could not be demonstrated. Differences across groups were compared using Student's t or the Mann-Whitney U test for continuous variables and the Fisher's exact test for binary variables. Correlation between continuous variables was assessed using linear regression models. All tests were two-tailed; in order to minimize overall type I error, only select associations were tested for statistical significance. Concerning the diagnosis obtained by EUS-FNA, sensitivity, specificity, and accuracy were calculated. Overall survival was analyzed by the Kaplan-Meier method, with use of one-sided log-rank statistics. The


SiR is involved in oxidative stress response is largely unknown. Here, we show that SiR functions in methyl viologen (MV)-induced oxidative stress in Arabidopsis. The transcript levels of SiR were higher in leaves, immature siliques, and roots and were markedly and rapidly up-regulated by MV exposure. The SiR knock-down transgenic lines had about 60% residual transcripts and were more susceptible than wild-type when exposed to oxidative stress. The severe damage phenotypes of the SiR-impaired lines were accompanied by increases of hydrogen peroxide (H2O2), malondialdehyde (MDA), and sulfite accumulations, but less amounts of glutathione (GSH). Interestingly, application of exogenous GSH effectively rescued corresponding MV hypersensitivity in SiR-impaired plants. qRT-PCR analysis revealed that there was significantly increased expression of several sulfite metabolism-related genes in SiR-impaired lines. Noticeably, enhanced transcripts of the three APR genes were quite evident in SiR-impaired plants; suggesting that the increased sulfite in the SiR-impaired plants could be a result of the reduced SiR coupled to enhanced APR expression during oxidative stress. Together, our results indicate that SiR is involved in oxidative stress tolerance possibly by maintaining sulfite homeostasis, regulating GSH levels, and modulating sulfite metabolism-related gene expression in Arabidopsis. SiR could be exploited for engineering environmental stress-tolerant plants in molecular breeding of crops.As an essential enzyme in the sulfate assimilation reductive pathway, sulfite reductase (SiR) plays important roles in diverse metabolic processes such as sulfur homeostasis and cysteine metabolism. However, whether plant  The assimilatory reduction of inorganic sulfate into organic sulfur compounds proceeds via a highly coordinated mechanism in higher plants . First, SiR exists as a single copy in the genome of Arabidopsis and is localized exclusively in the plastids (Arabidopsis or tomato plants against sulfite toxicity (Plant SiR is a soluble protein containing one (4Fe-4S) cluster and one siroheme that catalyzes the six-electron reduction of sulfite to sulfide . SiR exiplastids . SiR actplastids . It has toxicity . Furthertoxicity .SiR functions in methyl viologen (MV)-induced oxidative stress in Arabidopsis.Environmental stresses including abiotic and biotic stress provoke cellular redox imbalances and generate excessive reactive oxygen species (ROS) such as hydroxyl radicals and superoxide ions, which result in oxidative stress in plants . When plArabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type in this study. The wild type and RNAi transgenic seeds were surface sterilized and germinated on plates containing 1/2 Murashige and Skoog (MS) medium. Seeds were stratified at 4°C in darkness for 3 days and then transferred to a growth chamber at 22°C with a 16-h-light/8-h-dark photoperiod. After 1 week, the seedlings were transferred to sterilized low-nutrient soil to obtain fully grown plants. Plants were grown in a growth room at approximately 22°C, 70–80% relative humidity, a photoperiod of 16 h/8 h (day/night) and light intensity of 200 μmol m-2 s-1, as described before  in response to MV treatment. Total RNA extraction, first-strand cDNA synthesis and qRT-PCR with gene-specific primers  construct, a 369-bp-length fragment of Agrobacterium tumefaciens (strain GV3101) and then transformed into Arabidopsis (Col-0) via the floral dip method (Arabidopsis SiR-modified lines that contained single-site transgene insertions were identified and maintained growth to set seeds until T3 generation. Homozygous T3 lines were used for further experiments.The binary construct was introduced into p method . Transfo2O-treated controls. The whole experiment was repeated three times. The remaining chlorophyll content of WT and RNAi lines was determined 3 days after treatment. Leaf damage level (LDL) was determined as the ratio of damaged area divided by the whole leaf area. The relative damage level (%) was calculated as the mean of LDL of five leaves from each plant.The WT and transgenic lines  were cultured in 1/2 MS medium under a 16 h light/8 h dark cycle at 22°C for 1 week, and then the plants were transplanted into small pots with soil  and grown for 4 weeks under standard growth conditions. Five-week-old WT and RNAi lines were sprayed with 20 μM of MV. Three replicates each consisting of two pots of seedlings from each line were included for both MV treatment and H2O2 content was assayed according to our previously used method . The SiR transcript levels were significantly high in leaves and immature siliques . In contrast, SiR transcripts were low in stems. The relative expression in leaves was three times greater than that in the stems .The transcriptional pattern of SiR transcript levels in Arabidopsis plants in response to oxidative stress was performed by qRT-PCR . The transcript levels of SiR were increased rapidly after 1 h, and reached a maximal level at 12 h (about sixfold increase in transcripts) during 48 h period of MV treatment . In contrast, no significant difference was observed under control conditions . These data suggest that SiR could be involved in oxidative stress response.Time-course analysis of SiR in oxidative stress tolerance, transgenic Arabidopsis plants under-expressing SiR were generated . Due to early seedling lethal caused by SiR inhibition . Accordingly, these RNAi lines showed significantly reduced levels of SiR activity . However, the RNAi line Ri-11 showed severe growth retardation and could not be used for screening homozygous line because of strong suppression in SiR expression . Among the remaining four lines, three homozygous lines  had 55–70% of the wild type levels, which were chosen for further experiments.To further investigate the role of hibition , only fihibition . qRT-PCRSiR under-expressing Arabidopsis under MV-induced oxidative stress, T3 seeds of the three homozygous RNAi lines were directly sown in soil. 20 μM of MV was applied to 5-week-old seedlings by spraying directly onto the leaves. Three days after MV spraying, the RNAi transgenic plants showed relative higher necrosis and wilting than the wild-type plants . Accordingly, the mean LDL of the RNAi lines was about twofold higher than that in the WT  and the remaining chlorophyll content in the RNAi lines was significantly lower than that in WT plants . These results demonstrate that impairment of SiR in Arabidopsis plants decreases tolerance to MV-induced oxidative stress.To characterize the performance of SiR RNAi lines prompted us to detect the differences in lipid peroxidation. Malondialdehyde (MDA), a product of lipid peroxidation was measured between the WT and RNAi plants 3 days after MV treatment. The MDA levels in the RNAi lines  were significantly higher than in the WT (only 80% increase), suggesting that the RNAi plants suffered more membrane damage than the wild type . By contrast, in the presence of GSH, the MDA levels in the RNAi lines were only 70% increase on average after MV exposure .Reduced oxidative tolerance in SiR knockdown plants had higher MDA levels under oxidative stress, implying that they may be subjected to more serious oxidative damage than the WT. Therefore, it was of interest to detect ROS accumulation in the WT and RNAi lines during oxidative stress. Quantitative determination of H2O2 accumulation was performed in 3-day MV-treated leaves along with controls from RNAi and WT plants. As shown in Figure 4B, H2O2 content increased in both WT and transgenic lines after oxidative stress. However, RNAi lines accumulated higher levels of H2O2 (200% increase averagely) relative to WT (130% increase) after MV treatment . By contrast, in the presence of GSH, there was no significant difference in H2O2 content between RNAi and WT plants after MV exposure . These physiological indices demonstrated that higher ROS accumulation and lipid peroxidation in the RNAi transgenic lines may be correlated to their decreased tolerance to oxidative stress and was attenuated by GSH.The in planta changes in the levels of substrate and products upon MV exposure, sulfite and S-metabolite GSH contents were determined in treated and control leaves from wild-type and SiR under-expressing plants. The effect of MV on total sulfite levels in these SiR under-expressing lines resulted in relatively bigger increases of 61–73% . By contrast, the sulfite content in the leaves of WT plants only increases by 36% upon MV exposure . In addition, under control conditions, sulfite levels in the RNAi lines had no significant increase compared to the wild-type plants .To monitor Figure 5B). Noticeably, under control conditions, total GSH levels in these RNAi lines had significant reductions compared to the wild-type plants . These results demonstrated that reduced SiR expression resulted in excess sulfite accumulation and insufficient biosynthesis of GSH, indicating that SiR was involved in MV-induced oxidative stress tolerance largely attributable to GSH levels in plants.For changes in the GSH content, a significant increase was detected in the wild-type plants (60% increase), but not in the three RNAi lines . To examine this directly, 1-week-old seedlings from WT and SiR mutants (Ri-4 and Ri-6) were treated with MV or MV plus GSH for a week. MV exposure caused marked reductions in chlorophyll and survival rates that were alleviated in the presence of GSH in both wild-type and RNAi lines . Interestingly, the WT plants exhibited the lowest chlorophyll degradation and the highest survival of seedlings . This observation indicates that GSH may play a protective role in MV-induced oxidative stress.The MV-induced loss of chlorophyll and accelerated cellular damage in SiR under-expression plants by qRT-PCR. After MV exposure for 12 h, the transcripts of all the genes except for the SQS1 displayed similarly increasing trends between the WT and RNAi plants . In particular, enhanced expression of the APR transcripts was quite evident in SiR-impaired plants compared with those in wild-type plants. Moreover, the transcripts of the APR1 were significantly up-regulated even under normal conditions . This suggests that the increased sulfite in the SiR-impaired plants could be a result of the reduced SiR coupled to enhanced APR expression during control and oxidative stress.The transcripts of sulfite metabolism-related enzymes sulfite oxidase (SO), UDP- sulfoquinovose synthase (SQS1), and adenosine-5’-phosphosulfate reductase  were monitored upon MV exposure in the wild-type and As a key enzyme in the sulfate assimilation pathway, SiR is not only involved in plant growth and development, but in stress response , 2014. OSiR, there exists one copy in Arabidopsis genome and two copies in the genomes of rice and poplar . This indicates that SiR is evolutionarily conserved in plant species. SiR was found to be expressed in nearly all tissue types in Arabidopsis , indicating this gene may be constitutively expressed during both vegetative and reproductive growth. Besides leaves, higher transcript levels of the SiR were observed in siliques; suggesting that SiR-dependent sulfate assimilation may be involved in sulfur-containing protein biosynthesis during seed development in Arabidopsis. Interestingly, relative higher levels of SiR transcripts were also found in roots, indicating that SiR may play a role in response to nutritional stress such as sulfate deficiency in roots. This notion was supported by the clue that SiR mRNA levels were up-regulated when Arabidopsis plants were subjected to sulfate starvation (SiR was confirmed to be involved in oxidative stress response in Arabidopsis (This study). Taken these findings together, it is strongly evidenced that plant SiR is functionally divergent.For the d poplar . A phyloarvation . Severalarvation  and partarvation , 2014; AAPR1, APR2, and APR3) were significantly up-regulated in the SiR-impaired lines during MV-induced oxidative stress compared to those in the WT plants . Moreover, sulfite content significantly increased in the leaves of the SiR knock-down lines . These results suggest that toxic sulfite levels were increasingly accumulated in plant cells during oxidative stress because of enhanced APR expression and impaired SiR. This observation could be supported by the evidence that SiR mutant RNAi plants were severely suffered by SO2 stress due to excess sulfite accumulation , indicating that sulfolipid synthesis in plastids may not be a predominant pathway for sulfite homeostasis during oxidative stress. Although transcript levels of SO were up-regulated upon MV exposure, SiR-impaired plants showed more severe damage than the WT. These results demonstrate that SiR is indispensable for maintaining sulfite homeostasis during oxidative stress. Further work is needed to dissect the SiR-dependent and SO-dependent sulfite homeostasis networks in SiR and SO mutants using transcriptome, proteome, or metabolome approaches.SO, SiR, SQS1, and APR are key enzymes that catalyze the diversion of sulfite to other assimilatory pathways . In thismulation . BesidesMethyl viologen  is one of the most widely used herbicides in agriculture. It rapidly enters leaves and then chloroplasts, where it disrupts PSI electron transport, reducing oxygen to ROS. Thus, it can lead to cell toxicity, membrane peroxidation and even cell death. Sulfite is reduced by SiR to form sulfide, which is then incorporated into Cys and other sulfur-containing compounds such as GSH. As a major organic thiol-containing metabolite, GSH has an important role in maintaining redox homeostasis .SiR knock-down transgenic lines . Further investigations showed that a significant increase in the GSH content was detected in the WT, but not in the SiR under-expressing lines . This indicates that amounts of GSH were influenced by the SiR levels and in return, GSH levels affected oxidative stress response of the SiR-impaired plants directly. In addition, the accumulation of hydrogen peroxide in these RNAi lines may also be a consequence of the lower GSH levels, which weakened the antioxidant capacity. In support of this viewpoint, In this study, higher hydrogen peroxide and MDA levels, which are hallmarks of oxidative stress were detected upon MV exposure in the SiR-regulated sulfate reductive pathway involved in MV-induced oxidative stress response using mutants of sulfite metabolism-related genes in Arabidopsis.In summary, SiR can protect plants from oxidative stress, and has a potential for application to economically important crop plants. Future work will be needed to dissect the mechanisms in detail by which the ZX designed the research. MW, YJ, ZXu, and ZX performed research and conducted data analyses. ZX wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


American Indian and Alaska Native (AI/AN) youth face multiple health challenges compared to other racial/ethnic groups, which could potentially be ameliorated by the dissemination of evidence-based adolescent health promotion programs. Previous studies have indicated that limited trained personnel, cultural barriers, and geographic isolation may hinder the reach and implementation of evidence-based health promotion programs among AI/AN youth. Although Internet access is variable in AI/AN communities across the United States, it is swiftly and steadily improving, and it may provide a viable strategy to disseminate evidence-based health promotion programs to this underserved population.We explored the potential of using the Internet to disseminate evidence-based health promotion programs on multiple health topics to AI/AN youth living in diverse communities across 3 geographically dispersed regions of the United States. Specifically, we assessed the Internet’s potential to increase the reach and implementation of evidence-based health promotion programs for AI/AN youth, and to engage AI/AN youth.This randomized controlled trial was conducted in 25 participating sites in Alaska, Arizona, and the Pacific Northwest. Predominantly AI/AN youth, aged 12-14 years, accessed 6 evidence-based health promotion programs delivered via the Internet, which focused on sexual health, hearing loss, alcohol use, tobacco use, drug use, and nutrition and physical activity. Adult site coordinators completed computer-based education inventory surveys, connectivity and bandwidth testing to assess parameters related to program reach , and implementation logs to assess barriers to implementation (program errors and delivery issues). We assessed youths’ perceptions of program engagement via ratings on ease of use, understandability, credibility, likeability, perceived impact, and motivational appeal, using previously established measures.Sites had sufficient computer access and Internet connectivity to implement the 6 programs with adequate fidelity; however, variable bandwidth  and technical issues led some sites to access programs via back-up modalities . The number of youth providing engagement ratings varied by program . Across programs, youth rated the programs as easy to use (68-91%), trustworthy (61-89%), likeable (59-87%), and impactful (63-91%). Most youth understood the words in the programs (60-83%), although some needed hints to complete the programs (16-49%). Overall, 37-66% of the participants would recommend the programs to a classmate, and 62-87% found the programs enjoyable when compared to other school lessons.Findings demonstrate the potential of the Internet to enhance the reach and implementation of evidence-based health promotion programs, and to engage AI/AN youth. Provision of back-up modalities is recommended to address possible connectivity or technical issues. The dissemination of Internet-based health promotion programs may be a promising strategy to address health disparities for this underserved population.Clinicaltrials.gov NCT01303575; https://clinicaltrials.gov/ct2/show/NCT01303575 (Archived by WebCite at http://www.webcitation.org/6m7DO4g7c) As of 2012, an estimated 5.2 million individuals in the United States identified as American Indian and Alaska Native (AI/AN) alone or combined with other races, comprising 2% of the nation’s population ; howeverAI/AN youth also face disparities related to obesity and diabetes. Previous studies have indicated that 20-30% of AI/AN children are obese , comparereach to their intended audience, be implemented as intended in the real world, and adequately engage their audience [These health disparities could potentially be ameliorated by the dissemination and implementation of evidence-based adolescent health promotion programs. The United States Department of Health and Human Services’ Office of Adolescent Health, Substance Abuse and Mental Health Services Administration, and Centers for Disease Control and Prevention (CDC) have identified adolescent health promotion programs proven to impact behavior change across various health domains, including sexual health, violence and substance use prevention, physical activity, and nutrition -14. Howeaudience .Limited data exist regarding the reach and implementation of evidence-based health promotion programs among AI/AN youth. The Bureau of Indian Education (BIE) supports 183 primary and secondary schools in 23 states, serving approximately 50,000 students. A school health assessment of BIE schools conducted in New Mexico in 2006 reported that 38 of 39 schools (97%) provided health education and/or health promotion activities, and 67% used a comprehensive health education curriculum . CurricuTechnology-based programs may offer a viable strategy to increase the reach and implementation of evidence-based health promotion programs in this underserved population . FurtherRecently, Internet-based sexual health promotion programs have been specifically developed or adapted for AI/AN youth, with high satisfaction ratings reported ,24. The This study presents a secondary analysis of data collected during the implementation phase of a randomized controlled trial  that assessed the effectiveness of Native It’s Your Game  relative to a comparison suite of 5 evidence-based Internet-based health promotion programs, across 3 geographically dispersed regions in the United States. A detailed description of the adaptation process for Native IYG is provided in a supplemental file . PrimaryParticipants were primarily self-identified AI/AN youth aged 12-14 years and adult site coordinators  that were recruited from 25 study sites. The sites were located in 13 urban and 12 rural/tribal settings in Alaska, Arizona, and the Pacific Northwest , and comprised schools, tribal community centers, after school programs, and summer youth programs. Given the importance of confidentiality when partnering with AI/AN communities, specific tribal names have been withheld. The study was approved by the Alaska Area Institutional Review Board (IRB), the Portland Area Indian Health Service IRB, the University of Texas Health Science Center at Houston  IRB, and 16 tribal organizations .Study activities were coordinated regionally by 3 organizations that collectively serve 295 federally recognized AI/AN tribes. Regional staff used convenience sampling to recruit tribal communities that were interested in participating in an early adolescent sexual health study. Regional staff sent flyers to schools, tribal community centers, after school programs, and summer camp programs, and advertised on organizational websites, social media outlets, and/or via newsletters. Participating sites were randomized to a treatment condition  or a comparison condition (n=11) featuring a suite of 5 evidence-based Internet-based health promotion programs that were not focused on sexual health.Site coordinators at each study site completed the Collaborative Institutional Training Initiative Program’s online certification in human subject research and a live webinar (tailored for treatment or control conditions) coordinated by UTHealth research staff, which explained intervention content and protocols for logging-in youth, documentation, and maintaining confidentiality.It’s Your Game-Tech (IYG-Tech) [Native IYG is a 13-lesson, multimedia sexual health education curriculum for AI/AN youth (aged 12-14 years). The curriculum was adapted from an Internet-based curriculum for urban middle schools titled YG-Tech) . LessonsYG-Tech) .The 5 evidence-based, Internet-based programs that comprised the comparison suite addressed hearing loss (Dangerous Decibels), alcohol use (N-Squad), tobacco use , drug use (Reconstructors), and physical activity and nutrition (The Quest to Lava Mountain). Each program has undergone usability testing with non-AI/AN youth as part of its own formative development, and has demonstrated efficacy to positively impact health behaviors and/or related psychosocial factors in other adolescent populations. The Dangerous Decibels Virtual Exhibit was developed by the Oregon Museum of Science and Industry as an online component of a public health campaign to reduce the incidence and prevalence of noise-induced hearing loss and tinnitus (ringing in the ear) by improving knowledge, attitudes, and protective behaviors of school-aged children . N-SquadSite coordinators logged participants onto the programs on desktop or laptop computers located in quiet locations . Sites with insufficient bandwidth to accommodate simultaneous Internet access by multiple users were provided with uploadable versions of their respective programs on a Universal Serial Bus (USB) drive or digital video disc (DVD).Prior to implementation, site coordinators completed a computer-based education inventory survey  and connEase of use was based on how easy it was to use the program, using a Likert-type 3-point scale (very easy to kind of hard). Understandability was based on whether youth understood the words used and if they needed hints from an adult to play the game, using a Likert-type 3-point scale . Credibility was based on youths’ perceptions of content correctness, using a Likert-type 3-point scale  and trustworthiness . Likeability was based on how much youth liked the program activities, using a Likert-type 5-point scale (dislike a lot to like a lot). Perceived impact was based on whether youth thought the program would help them make healthy decisions, using a Likert-type 3-point scale . Motivational appeal was based on whether youth would play more program lessons if available, if they would recommend the program to classmates, and whether the program was as much or more fun than other lessons at school, including other health lessons, computer-based lessons, and their favorite video game, using a Likert-type 3-point scale .We used Likert-type scales adapted from previous studies to assess youths’ perceptions of engagement -42. EaseRatings were collected via an Internet-based Qualtrics usability survey administered at the completion of each program. Demographic characteristics  were collected during the study’s baseline survey, using an Internet-based Qualtrics self-report survey . Upon enWe used descriptive statistics  to summarize data on reach parameters  and implementation . Regarding youth engagement, for each parameter we calculated the percent of youth who rated each program favorably, and the range of ratings across all 6 programs, from lowest to highest.Twenty-four site coordinators  provided complete or partial information related to Internet reach parameters. Eighteen computer labs, one classroom, and one after school classroom across the 3 regions were primarily composed of personal computers  and Mac computers , were mostly wired , and most had access to the Internet . Primary Web browsers included Chrome , Safari , Internet Explorer , Firefox , and Mozilla . Download speeds ranged from 0.24 megabits per second (Mbps) to 93.5 Mbps .At some sites, the method of program delivery changed during implementation. Treatment sites commenced the study accessing Native IYG as an online streaming program (n=12) or as an uploadable program from a USB drive (n=2). During the study, several sites that initially accessed Native IYG as an online streaming program converted to uploading Native IYG from a USB drive (n=4) due to the inability of local bandwidth to accommodate larger Native IYG video files while providing simultaneous streaming for multiple youth.Comparison condition sites commenced the study by accessing the suite of health promotion programs via online streaming (n=8) or a combination of online streaming and an uploadable program from DVDs (n=2). Information regarding program access was missing from one site. During the study, most sites continued to access these programs via Internet connections (n=8).The most commonly reported problems that were documented by site coordinators during implementation included frozen screens (4/6 programs), activities taking a long time to load (3/6 programs)—both of which were related to multiple simultaneous users—and trouble navigating the programs (3/6 programs).During implementation, 387 youth received Native IYG, of whom 191 (49%) provided feedback; 136 youth received the comparison suite of programs, of whom 108 (79%) completed at least one feedback survey. Across programs, participants were 48-60% female, with a mean age of 13.1-13.3 years, and 85-90% self-identified as AI/AN .correct (58-90%) and trustworthy (61-89%), and 63-91% reported that the programs would help them make better choices. Regarding likeability, 59-87% of youth liked the programs a little or a lot. In terms of motivational appeal, 35-63% of youth reported that they would play more lessons from each program, if available; 37-66% would recommend the programs to a classmate. Although fewer youth considered the programs as much or more fun than their favorite video game (25-61%), youth stated that the programs were as much or more fun than other lessons at school (62-87%), other health lessons at school (61-79%), and other computer-based lessons at school  and the majority understood the words in the programs (60-83%). However, some participants needed adult assistance or hints to complete the programs (16-49%). Over half of the youth reported that the program content was credible, rating it  57-85%; .AI/AN youth face multiple health challenges compared to youth of other racial/ethnic groups. Viable program delivery strategies that overcome limited personnel with training in health education, cultural barriers, and geographic isolation  are needed to increase the reach and implementation fidelity of evidence-based adolescent health promotion programs in tribal communities. We examined the potential of using the Internet to deliver 6 evidence-based health promotion programs to AI/AN youth living in urban and rural settings in 3 geographically diverse regions of the United States. The health topics addressed included sexual health, hearing loss, substance use, physical activity, and nutrition. Despite variability in connectivity and bandwidth, most sites were able to access the programs via the Internet. However, technical and connectivity issues led some sites to access the programs via back-up modalities . Practitioners interested in implementing Internet-based programs in tribal communities are advised to provide contingency plans as back-ups to technical failures. We also recommend conducting bandwidth assessments, especially when dealing with multiple simultaneous users, prior to implementing an Internet-based program.Adult site coordinators from each tribal community  facilitated youths’ access to the Internet-based programs. Regarding implementation fidelity and program errors, site coordinators reported issues with frozen screens (4/6 programs), activities taking a long time to load (3/6 programs), and trouble navigating the programs (3/6 programs). Given that 16-49% of youth needed assistance to complete the programs, some adult oversight is recommended when implementing Internet-based programs for youth. Although the site coordinators received webinar training prior to program implementation, the training focused primarily on research study-specific protocols . The training related to actual program implementation, such as reviewing specific program content, was limited to approximately one hour, indicating that a broad range of personnel  may be able to implement Internet-based health promotion programs.as engaging as their favorite video game , over 60% stated that the programs were as much or more fun than other lessons at school. These findings indicate that Internet-based health promotion programs are engaging to AI/AN youth in the diverse settings of both rural (reservation and village) and urban locations. These results also support previous studies indicating that Internet-based health promotion programs provide an engaging educational format for Millennium learners. Internet-based programs are uniquely positioned to allow for: the provision of motivational learning experiences delivered via video, animated characters, and gaming formats; the provision of role modeling activities that influence normative perceptions and skills; confidential and personalized presentation of sensitive and potentially embarrassing issues ; and intervention messages that are tailored to specific user characteristics [Overall, AI/AN youth rated the programs favorably. Although fewer youth rated the programs as being eristics ,28. ThesIt is worth noting that youth rated Native IYG most favorably across all programs on 10 of the 13 usability parameters, possibly because this program was specifically adapted for AI/AN youth. This finding aligns with previous studies that point to the value of cultural tailoring . Incorpoas much or more fun than other lessons at school) suggest that the programs would be engaging for AI/AN youth in school, after school, or in community-based settings.Across all programs, we experienced attrition in the number of youth who received the programs compared to those who completed a feedback survey. This attrition may have been due to respondent burden or fatigue in completing surveys after each program. However, the relatively high motivational appeal ratings across programs , as they do not require statistical significance to determine major usability problems . In contDespite these strengths, several limitations should be noted. First, the sample was restricted to early adolescent AI/AN youth, aged 12-14 years; thus, findings may not apply to older AI/AN youth. Second, social desirability may have biased youths’ ratings of the programs; however, the fact that some items scored lower than others  suggests that youth answered honestly, based on their experience with each program. Third, the intervention program, Native IYG, was specifically adapted for AI/AN youth. In contrast, no attempt was made to adapt the comparison group programs for AI/AN youth; this factor may have negatively impacted program ratings relative to Native IYG. Finally, although the use of percent ranges to evaluate engagement is sufficient for this kind of exploratory trial, more thorough quantitative analyses, such as appropriately powered randomized pilot tests of the programs’ impact on behavioral determinants or randomized controlled efficacy field trials, are needed to determine long-term behavioral impact.Despite these limitations, this study demonstrates the potential of using the Internet to disseminate evidence-based health promotion programs to AI/AN youth across 3 separate geographic regions. These findings may have broader implications for understanding the degree to which Internet-based programs may enhance the reach and implementation of evidence-based health promotion programs in tribal communities, and provide an educational format that is engaging for AI/AN youth.


Background: Laparoscopy is rapidly replacing laparotomy in the field of gynecologic surgery. Generally, there are limited data concerning the utility of single-incision laparoscopic surgery (SILS) in gynecologic surgery. Specifically, in Saudi Arabia, a third-world country, data are further limited; only one related study has been conducted so far. The purpose of this study is to retrospectively report our single-institutional experience of SILS in terms of feasibility, safety and perioperative outcomes in the management of various gynecologic conditions.Methods: The study took place at the Women’s Specialized Hospital, King Fahad Medical City, Riyadh, Saudi Arabia. From January 2012 to May 2016, all gynecologic patients who underwent SILS procedures were analyzed for pre-, intra- and post-operative details. SILS was performed using a single multi-port trocar and standard laparoscopic instruments.Results: A total of 54 patients underwent 66 SILS procedures. The median age and body mass index (BMI) were 36 years and 28.2 kg/m2, respectively. Fourteen patients (26%) had ≥ 1 previous abdominal and/or pelvic surgeries. Twenty-four patients (44.4%) were nulliparous. The three most commonly performed SILS procedures were unilateral salpingo-oophorectomy (45.5%) and unilateral ovarian cystectomy (27.3%) and adhesiolysis (6.1%). The median operative time, estimated blood loss and hospital stay were 74 min, 50 ml and 1 day, respectively. Three patients required conversion to laparotomy, as follows: unidentified non-stopping bleeding source (n=1) and endometriosis stage IV resulting in difficult dissection (n=2). One patient developed post-operative incisional hernia that was treated surgically. The median patients’ post-operative pain  within 4 hours was 2. At 4-week post-operatively, the median wound scar length (measured at outpatient clinic) was 2 cm.Conclusions: SILS is feasible, safe and associated with acceptable clinical and surgical outcomes. As opposed to laparotomy, laparoscopy provides plentiful benefits. Such benefits comprise: reduced postoperative pain, faster recovery to previous performance status, shorter hospital stay, better cosmesis, lower cost, reduced morbidity/mortality and overall improved surgical outcomes4.Minimally invasive surgery (laparoscopy) is rapidly replacing laparotomy in the field of gynecologic surgery1. SILS has been demonstrated in retrospective and prospective studies to be feasible, safe and reproducible in managing various gynecologic conditions ranging from simple procedures 9 to highly complicated ones 20.One of the most notable advances in laparoscopy is the introduction of single-incision laparoscopic surgery (SILS). In contrast to the conventional laparoscopy that is regularly executed by using a total of three to five small incisions (5–20 mm each), SILS is performed by using a single small incision of the umbilicus to completely accomplish the laparoscopic surgical procedures21. Specifically, in Saudi Arabia, a third-world country, data are further limited; only one study about SILS in gynecologic surgery has been conducted so far22.Generally, there are limited data concerning the utility of SILS in gynecologic surgeryThe purpose of this study is to retrospectively report our single-institutional experience of SILS  in the management of various gynecologic conditions.The study protocol was approved by the Institutional Review Board (IRB) of King Fahad Medical City [ID: 15-477].2, acceptable preoperative performance status and laboratory profile, technically resectable early stage adnexal or endometrial lesions, and signed written informed consent by patients after being well-informed about the risks/benefits of SILS.This was a retrospective study from January 2012 to May 2016 which took place at the Women’s Specialized Hospital, King Fahad Medical City, Riyadh, Saudi Arabia — a tertiary healthcare institution. At our institution, as of January 2012, SILS has been the standard management option for gynecologic patients who met the inclusion criteria, namely: age less than 90 years, body mass index (BMI) less than 50.0 kg/mFrom January 2012 to May 2016, the medical records of all gynecologic patients who underwent SILS procedures for gynecologic conditions were retrospectively analyzed for pre-, intra- and post-operative details. Pre-operative details comprised: age, BMI, previous abdominal and/or pelvic surgeries, concomitant co-morbidities and parity. Intra-operative details comprised: type of procedures performed, conversion to conventional laparotomy, operative time (OT), estimated blood loss (EBL), lesion size and intra-operative morbidity/mortality. OT was defined as the interval from the initial umbilical skin incision to its closure. Post-operative details comprised: lesion pathology, patients’ self-reported recommendation (yes/no) of SILS to others, hospital stay, and post-SILS wound size, morbidity, mortality and pain. The post-SILS wound size was measured at 4 weeks at the outpatient clinic. Post-SILS morbidity/mortality were defined as any surgery-related complications/death up to 24 weeks postoperatively. The self-reported scores for post-operative pain were documented within 4 hours postoperatively by the operating surgeon/ward nurse using the Wong-Baker FACES Foundation pain rating scale.TM device  was introduced into the peritoneal cavity using packing forceps. The device has one gas inlet and 3-access ports (two 5-mm ports and one 12-mm port). Then, pneumoperitoneum was accomplished using carbon dioxide (CO2) through the gas inlet valve. Various laparoscopic instruments (rigid 0-degree and prebent) were used as deemed appropriate by the operating surgeon to avoid instrumental clashing and improve the operation field. The resected specimens were removed using the 12-mm port and endobag to allow for intact removal of the surgical specimen. The rectus sheath was closed with number 0 maxon sutures and skin closed with 3-0 vicryl sutures. In case of failure of SILS, the procedure was converted to either conventional laparoscopy or laparotomy.All SILS procedures were primarily performed by a single surgeon from Section of Gynecologic Oncology, Department of Obstetrics and Gynecology. Procedures were performed under general anesthesia. Patients were placed in supine/lithotomy position, prepped and draped according to the hospital protocol. A 15 mm transverse intra-umbilical incision was made and skin edges were grabbed with Allis clamps. Afterwards blunt dissection was performed to create a 15 mm opening into the peritoneum. Then, Medtronic SILSAll patients were followed up for at least 6 months post-operatively at the outpatient clinic.The descriptive raw data are reported inDescriptive Raw DataPre-operative, intra-operative and post-operative detailsClick here for additional data file.Copyright: © 2017 Salamah K et al.2017Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).2 (range: 18.9–44.1). A total of 14 patients (26%) had ≥ 1 previous abdominal and/or pelvic surgeries. Regarding parity, 24 (44%), 2 (4%) and 28 (52%) patients were nulliparous (para 0), primiparous (para 1) and multiparous (para 2+), respectively.A total of 54 patients underwent SILS procedures. Characteristics of the patients are depicted inThe intra-operative details of SILS are portrayed inThe post-operative details of SILS are shown in1. However, there are insufficient data concerning the feasibility, safety and perioperative outcomes of SILS in gynecologic surgery in Saudi Arabia.SILS is one of the cutting-edge developments in the field of minimally invasive surgeryTo the best of our knowledge, this is the second ever study in Saudi Arabia to report the single-institutional experience of SILS in gynecologic surgery. Moreover, it is among the first studies originating from third-world countries that examined the efficacy of SILS in gynecologic surgery. Our study demonstrated that SILS is feasible, safe and associated with acceptable clinical and surgical outcomes. Nearly all patients (98.1%) agreed to recommend SILS to other patients.et al.22. They reported their single-center experience of SILS in the management of benign salpingo-ovarian pathologies. They had 80 patients and a total of 104 performed procedures. They concluded that SILS in the management of benign salpingo-ovarian conditions was generally feasible, potentially safe, and associated with satisfactory operative and postoperative outcomes. As opposed to the Al-Badawiet al. study that included only adnexectomy procedures22, our study included far more complex (non-adnexectomy) procedures, such as: SILS laparoscopic-assisted vaginal hysterectomy , and SILS total laparoscopic hysterectomy . Several studies showed that SILS approach can be used to successfully perform both TLH and LAVH procedures20.The only study of SILS in Saudi Arabia was reported by Al-BadawiIn our study, 3 patients required conversion to laparotomy for several reasons: unidentified non-stopping bleeding source (n=1) and endometriosis stage IV resulting in difficult dissection (n=2). Patient safety should never be recklessly put in danger at any given time. Therefore, conversion from SILS to laparotomy —whenever at the best interests of patients— is more significant than mere cosmetic concerns. Also, such conversion from SILS to laparotomy should not be prematurely judged as a surgical failure or an incompetence of the operating surgeon to perform SILS procedures.23. It has been recommended that patients with BMI less than 28 kg/m2 are regarded as suitable patients for SILS24. In our study, more than 50% of patients had BMI of more than 28 kg/m2 and none experienced eventful surgical courses.Patients with high BMI are regarded as a challenging group for SILS procedures due to the anticipated high intraperitoneal fat contents which can cause port access difficulties and garble the field visualization23. In our study, around 11.1% of patients had 2 or more previous surgeries; however, none of them required conversion to laparotomy.Moreover, patients with previous abdominal and/or pelvic surgeries are regarded as a challenging group for SILS procedures due to the anticipated dense surgery-related adhesions and difficult dissection25. Numerous studies showed feasibility, safety and reproducibility of the SILS for management of precancerous pathologies and select early-stage ovarian and uterine malignancies13. Thus, it can be concluded that SILS should not be avoided in the management of patients with early-stage gynecologic malignancy. In our study, 10 patients had malignant pathologies and successfully underwent SILS procedures without proof of port-site metastasis at a median follow up of 29 months (range: 23–37).Furthermore, port-site metastasis in malignant gynecologic oncology is sometimes worrisome to both patients and laparoscopists. However, the frequency of port-site metastasis following laparoscopic procedures in women with malignant gynecologic pathology is very low (less than 1%)2, less than 3 previous surgeries (irrespective of laparoscopic and/or laparotomic), presence of native umbilicus and early-stage gynecologic malignancy.In brief, apposite selection of patients for SILS is of great significance. According to our study, our recommendations for patients who may be eligible candidates for SILS procedures comprise: BMI less than 40 kg/m26. Measures to prevail these downsides are continuously in progress and comprise the introduction of flexible and prebent laparoscopic instruments of different lengths22.SILS is not without its technical challenges which are well-documented in literature. Such challenges generally comprise: the limited triangulation and retraction capacities as well as the camera/laparoscopic instruments conflict that can distort proper surgical field exposure27. Many measures have been advocated to improve the learning curve, for example: watching live/recorded procedures, virtual simulation and hands-on practice on animals28. There are serious ongoing efforts to implement the above-mentioned measures at our institution.Similar to conventional laparoscopy, SILS is a surgical procedure that necessitates a great deal of fine-motor hand dexterity. Two studies documented that around 10 to 15 procedures were required to perform SILS adroitly which was reflected on reduced operating timeOur future research directions include: 1) the learning curve of SILS at our institution, and 2) comparison between SILS and conventional laparoscopy with regard to management of various gynecologic pathologies.Our study has several limitations and comprise: retrospective study design, lack of control group , relatively small sample size, single-institutional experience and patients subjective self-reported scores for postoperative pain.This is the second single-institutional experience of SILS in gynecologic surgery from Saudi Arabia. Our study demonstrated that SILS is feasible, safe and associated with acceptable clinical and surgical outcomes. SILS is an operator-dependent procedure and requires advanced surgical training.The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Salamah K et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Descriptive Raw Data- Pre-operative, intra-operative and post-operative details10.5256/f1000research.12545.d17645329 et al. The authors reported their single-center experience of single-incision laparoscopic surgery (SILS) in the field of gynecologic surgery from a tertiary healthcare center in Saudi Arabia. The topic is of great significance since this study is only the second ever from Saudi Arabia, and will add valuable data to the limited regional peer-reviewed literature.With great interest I read the paper by SalamahIntroduction: The authors introduced the topic appropriately, defined terminology of SILS, and highlighted the significance of the study thereby clearly mentioned the objectives of study.Methods: The ethical approval for the study was obtained. Appropriate descriptions of the study design/setting, research subjects and surgical procedure are provided. The study was descriptive, retrospective, and one-group of patients. Only simple statistical tests were carried out, i.e., calculations of numbers/percentages, SDs and ranges.Results: Overall, the results section reads well reporting the major findings. Results were presented in simple tables and divided into pre-, intra- and post-operative categories. There are some repetitions between the text and the tables which makes easier to understand the findings. In Table 2, perhaps the authors should correct the “Type of Surgery” data: i.e., Laparoscopy=51, Laparotomy=3  and appropriate percentages.Discussion: It is succinct and properly reflecting on the study’s main findings without unnecessary details. The significance of study was re-highlighted. Brief comparisons with the the existing peer-reviewed literature were made with regards to aspects pertaining to the pre-operative details such as BMI and previous surgery. Most importantly, the study findings were directly compared-and-contrasted to the only study from Saudi Arabia  which is a plausible and appreciated move. Study’s future directions and limitations are adequately pinpointed.Conclusion: Reads very well and reflects on the study’s main conclusions.Overall Comments: The manuscript is very well-written in terms of English language, grammar, word counts, clarity, logical sequence, citations, and appropriate references cited. The strong points of paper are: 1) being the second in Saudi Arabia, which highlights the significance of the study to warrant additional peer-reviewed publication based on scientific soundness, despite no novelty; and 2) the study is of great importance in terms of regional interests as it enriches the scarce limited peer-reviewed literature on the topic. The study weakness mainly revolve around the methodology part as this is a mere descriptive retrospective study without a control-group and detailed statistical calculations, which makes it a “less” strong evidence; however, the study limitations are duly acknowledged by the authors. I highly recommend the authors to pursue future studies proposed in their manuscript, as these directions are going to have more impact and contribute substantially to the body of existing of peer-reviewed literature regionally and internationally. More strong-evidence-based study design should be considered too, such as prospective and controlled-study designs. A cost-effective study comparing conventional vs. SILS in gynecologic surgery indications may be an interesting topic, especially from a third-world country with relatively constrained healthcare economy.Final Remarks and Decision: According to the scope/policy of the F1000 Research Open-Access journal, the study is “scientifically sound” and “suitable” irrespective of the perceived level of novelty. Therefore, I approve the study for publication in its current draft.Conflict of Interest: I report no conflict of interest pertaining to this manuscript.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Small descriptive study about SILS. Based on the presets data, it is difficult to comment about the safety of this approach for TLH or LAVH as the number is very small and there is no informations about the indications of the hysterectomies. Overall promising results and worth to publish .I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The relation between arrhythmias and stress is known. The aim of our current study was to elucidate whether plasma levels of previously described stress parameters are altered in highly symptomatic patients with atrial fibrillation (AF) per se and in patients undergoing ablation therapy by pulmonary vein isolation (PVI).96 patients with AF undergoing PVI were recruited. Plasma levels of Endothelin-1 (ET-1), MCP-1 and Chromogranin-A (CGA) were measured before and three months after ablation completed with clinical follow-up with respect to AF recurrence. Additionally, we examined 40 healthy age- and sex-matched volunteers as a reference.Symptomatic AF patients showed increased levels of ET-1 compared to healthy controls . Baseline levels of ET-1 were higher in patients presenting with AF after PVI . The temporal comparison revealed decreased ET-1 levels in patients without  and unchanged ET-1 levels in patients with AF after PVI. Baseline MCP-1 was increased in AF patients vs. controls . Both groups, with and without AF after PVI, showed an increase of MCP-1 compared to baseline . CGA was lower in AF patients compared to healthy controls . Over time patients without AF after PVI showed an increase of CGA . No change was observed in patients with AF after PVI.Our study demonstrated dysregulated levels of ET-1, MCP-1 and CGA in symptomatic AF patients. We could demonstrate an association between ET-1 to presence or absence of AF. Furthermore, we could show that a decrease of ET-1 as well as an increase of CGA after PVI, representing a trend towards control cohort levels, were both associated with restoration of sinus rhythm. These results provide new insights into the role of stress-related biomarkers in AF and AF treatment by ablation therapy. Atrial fibrillation (AF) is the most common arrhythmia in clinical practice affecting more than six million Europeans ,2. AF isEndothelin-1 (ET-1) is produced by endothelial cells, smooth muscle cells, monocytes and macrophages after stimulation by catecholamines, cortisol, hypoxia, or interleukins . IncreasSimilar findings have been described for monocyte chemotactic protein-1 (MCP-1) ,11. MCP-Chromogranin A (CGA) is an essential part of secretory vesicle in endocrine cells, neurons and neuroendocrine cells. As a part of secretory vesicles CGA is released with the content of the vesicle ,15. The The aim of the present study was to evaluate a potential association of the circulating Endothelin-1, MCP-1, and CGA with AF in highly symptomatic patients as well as to investigate changes of these markers in AF patients undergoing ablation therapy with respect to AF recurrence.To test this, we measured the plasma levels of Endothelin-1, MCP-1, and CGA in AF patients undergoing PVI before and three months after ablation and compared these to healthy controls.96 consecutive patients undergoing PVI in the University Hospital Grosshadern were analysed in our study. To compare baseline levels, 40 age and sex matched patients without any history of atrial fibrillation or cardiovascular disease were selected as “healthy volunteers”. The analysed specimens were collected in a general biobanking effort that was continuously approved by the local ethics committee of the University of Munich and the Ethics Commission of the State Chamber of Medicine in Bavaria, Germany over the last years .Clinical evaluation of every patient was performed before and 3 months after PVI including medical history, EHRA score, physical examination, blood collection, echocardiography, ECG, and Holter ECG. AF freedom was assessed by 24 hour/7 day Holter ECG or clinical AF-related symptoms  three month after PVI. AF recurrence was defined as any atrial tachyarrhythmia lasting >30 seconds documented by ECG .Blood was collected via a direct venous puncture into 9 ml EDTA tubes (Sarstedt Monovette) and was processed for plasma isolation within 4 hours of collection. Blood was processed by spinning at 4000 rpm for 20 minutes at room temperature. Plasma was carefully transferred to a fresh RNAse/DNAse free tube and stored at -80°C.Ablation was performed using a standardized clinical protocol  as previously described . In brieThe plasma concentrations of Endothelin-1, MCP-1, and CGA were measured by a duoset enzyme linked immunosorbent assay (ELISA) .Statistical analysis was performed with SPSS 21. For not normally distributed variables we performed Wilcoxon Test for paired variables and Mann-Whitney-U test for non-paired variables. For dichotomous variables we used Fisher-Yates test. All values mentioned in the results section are medians unless otherwise listed. In addition, linear regression analysis adjusting for age, sex, diabetes, creatinine, hypertension and coronary artery disease was performed for Entothelin-1 baseline levels. Results are presented using box-plots showing median as well as interquartile ranges. A p value lower than 0.05 was considered statistically significant.The study was performed on blood samples that were collected in a general biobanking effort that was continuously approved by the hospital’s ethics committee of the University of Munich (LMU) and the Ethics Commission of the State Chamber of Medicine in Bavaria (BLAEK), Germany over the last years . All procedures were performed according to the 1975 Declaration of Helsinki. Every participant provided written informed consent before participation, all samples were anonymized and processed according to the guidelines the local ethics committee of the University of Munich (LMU), Germany.In total, we included 96 AF patients in our study . Within For a further analysis we separated our AF patient cohort into two groups: 1) patients with recurrence of AF and 2) patients without recurrence of AF three months after ablation . Both grTo evaluate Endothelin-1 (ET-1) as a potential biomarker for AF we measured ET-1 plasma levels at baseline in patients with AF in comparison to healthy volunteers without AF. Patients with AF showed significantly higher levels of ET-1 compared to age- and sex-matched healthy volunteers without history of AF  Patients with AF show marked differences with increased levels of ET-1 and MCP-1 and reduced levels of CGA compared to healthy controls; (2) in AF patients higher ET-1 levels were associated with AF recurrence three months after PVI; (3) freedom from AF after ablation was associated with a significant decrease of ET-1 and a significant increase of CGA three months after ablation compared to baseline.Our study shows that patients with AF have higher levels of Endothelin-1 compared to healthy controls without AF. This finding supports our hypothesis of a stress associated arrhythmia. However, previous reports about the relationship between ET-1 and AF are inconsistent. Some studies demonstrated elevated levels of ET-1 and proposed ET-1 being involved in remodelling processes and atrial dilatation . In contET-1 has been shown to be associated with atrial structural  and elecClinical trials have shown increased serum levels of MCP-1 in stress related cardiovascular disease . In our Circulating levels of CGA are influenced by cardiovascular diseases, which are also known risk factors of AF such as hypertension, heart failure, or acute coronary syndrome ,30–32.Nevertheless, no data is available on CGA in the context of AF. In our study we measured lower levels of CGA in patients with AF, a finding that does not directly support our hypothesis as AF being stress-related or vice versa. Given that the adrenergic system has huge impact on the development of AF , we expeCurrent success rates for PVI are reported with 60–70% after one year ,46. GeneA main limitation of our study is the short follow-up period of three months. In our study about 80% of our patients were still in sinus rhythm three months after ablation and a longer follow-up period might identify more patients with later AF recurrence. Our study results therefore only allow to draw conclusions regarding a short-term recurrence of AF or restoration of sinus rhythm but not a long-term ablation success.To detect AF recurrence we used 24h Holter ECG recordings and patients’ reports on AF related symptoms as recommended by current guidelines. However, using those assessments we cannot detect AF recurrence in 100%, especially in case of asymptomatic episodes. The only alternative are implantable loop recorders that cannot be implanted routinely in all patients at the moment. Nevertheless, we think that this approach is acceptable for the purpose of our study, to evaluate stress biomarkers in the context of AF symptoms.Furthermore, our study evaluated a heterogeneous population including patients with paroxysmal as well as persistent AF. Due to small sample sizes  no separate analysis was possible. As a consequence we cannot rule out different biomarker patterns between these two entities of AF.Further studies are necessary to put our findings in the context of a long-term follow-up and to explain the underlying pathophysiology. As an independent replication of our findings in a larger cohort is needed, our current study has to be called hypothesis generating potentially guiding strategies for future studies.Nevertheless, to the best of our knowledge our study is the first to provide insights into the relationship and the absolute values of these stress markers to the presence or absence of AF before and after an ablation therapy as well as in comparison to a healthy control cohort.In this study we could provide evidence that established stress-related biomarkers ET-1, MCP-1, CGA were differentially regulated among patients with AF compared to healthy controls. Furthermore, the investigated biomarker levels before and three months after an ablation therapy revealed a direct association of ET-1 levels with respect to presence or absence of AF, and an inverse relationship of CGA levels regarding the presence of AF. These data provide new insight for the role of stress-related biomarkers in AF pathophysiology as well as potential biomarker for AF ablation therapy. However, larger, randomized, controlled clinical trials are necessary to finally prove clinical significance of those biomarkers and potentially allow to identify subgroups of patients at higher risk that might benefit from alternative ablation strategies like additional ganglionated plexi modification.S1 TableGFR, glomerular filtration rate; LA, left atrium.(XLSX)Click here for additional data file.