Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion  from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for  Clostridium botulinum type C or D  is a predominantly fatal, rapid multiple system neuropathy of grazing horses with reported incidence rates of 2.1–2.3%  cervical ganglion  from EGS-affected patients and appropriate control cases postmortem. The cranial (superior) cervical ganglion (CCG), which supplies sympathetic innervation to the head and neck, was selected because chromatolysis of a high proportion of CCG neurons is a consistent feature of EGS . Here, This study therefore represents the first application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease). It is also the first to demonstrate correlation and conservation spanning the degenerative molecular cascades from an apparently unrelated condition of large animals to small animal models with altered neuronal vulnerability and a range of human neurological conditions from childhood neurodegenerative conditions such as spinal muscular atrophy through to diseases associated with advancing age such as Alzheimer's. Finally, this study highlights the feasibility and benefits of applying differential proteomics techniques to the investigation of the neurodegenerative processes in diseases of large animals.In accordance with MCP guidelines, more detailed discussion of experimental design and potential limitations can be found in the accompanying Supplementary Discussion file. General methodology is provided below.Tissue samples were collected at necropsy from horses that were euthanized on humane grounds, with the horse owners' consent. The study was approved by the local ethics committee.Supplemental Fig. 1). Due to these considerations and the fact that the initiating insult remains unproven, molecular analyses are currently performed on pooled samples as an attempt to reduce “noise” due to interanimal variability through factors such as individual disease response, age, and breed, among other considerations. EGS was confirmed in all cases by necropsy, including histopathological examination of autonomic ganglia  and six control  mixed-breed and mixed-gender horses within 60 min of euthanasia by administration of barbiturates . CCG wer ganglia . Control ganglia  and fixem Urea, 2 m thiourea, 2% CHAPS, 0.5% SDS, and protease inhibitor mixture (Roche) for proteomic processing (see below). Samples were pooled by condition and manually homogenized in a dounce glass homogenizer. Homogenized samples were sonicated in a cup style sonicator six times for 15 s at power level 7.5 with vortexing for 30 s between each round of sonication. Samples were left on ice for 10 min before being revortexed then centrifuged at 20,000 g for 30 min at 4 °C. The resulting pellet containing proteins insoluble when processed in this manner was stored at −80 °C, and the supernatant was transferred to a fresh 1.5 ml tube to be processed for iTRAQ labeling as previously described  for QFWB (see below) or iTRAQ extraction buffer containing 6 escribed –20.m Urea, 2 m thiourea, 2% CHAPS, 0.5% SDS, and protease inhibitor mixture , (pH 7.4)) before acetone precipitation and labeling for iTRAQ analysis. The Mass spectrometry proteomic data have been deposited to the ProteomeXchange consortium via the PRIDE partner repository with the dataset identifier PXD002956.Protein was extracted in iTRAQ extraction buffer , and 2 μl of reducing agent Tris(2-carboxyethyl)phophine (TCEP)). The samples were allowed to incubate for 1 h at 60 °C prior to protein estimation in triplicate (3 × 1 μl) by microBCA assay . Samples were run in duplicate to utilize all four tags from the 4plex kit and increase peptide identification yield as previously described (Protein extracts (escribed –20.m KH2PO4 in 20% CH3CN (pH 2.7) and buffer B: 500 mm NaCl in 5 mm KH2PO4 in 20% CH3CN (pH 2.7).The flow rate was set to 0.2 ml per minute with a linear gradient from 0 to 50% B over 25 min then a linear gradient from 50 to 100% B over 9 min.Each sample equivalent to 100ug was processed separately using the filter aided sample preparation (FASP) method prior to digestion with trypsin . After digestion, the samples were dried using a SpeedVac concentrator and then resuspended in 25 μl of dissolution buffer as provided in the iTRAQ Reagents Multiplex kit . The samples were then labeled, respectively, according to the protocol provided by the manufacturer (AB Sciex). The four labeled samples  were then pooled together in equal proportions and subsequently dried using a SpeedVac concentrator. The pooled iTRAQ 4plex sample was then desalted using a homemade porous R2 ZipTip column and then fractionated by Strong Cation eXchange (SCX) using a Polysulfoethyl A column  on a Ultimate U3000  hplc system. The following buffer system was used Buffer A: 5 mEach fraction from the SCX fractionation was then dried using a SpeedVac concentrator and stored at −80 °C. Stored SCX fractions of the pooled iTRAQ 4plex sample were then resuspended in 10 μl of 5% formic acid, diluted to 1% formic acid, and then 15 μl aliquots injected onto an Ultimate RSLC nano UHPLC system coupled to a LTQ Orbitrap Velos Pro . The iTRAQ-labeled peptides were injected onto a trapping column  and then separated using a 2 h linear gradient from 2–40% B  on a separation column  at a flow rate of 300 nl/min.The mass spectrometry parameters were set as follows: Fourier transform mass spectrometry (FT-MS) (survey scan) resolution was set at 60,000; the 15 most-intense precursor ions were chosen for fragmentation by high energy collisional-induced dissociation (HCD); the precursor isolation window was set at 1.2 Da; and the ms/ms scan resolution was set to 7,500. Automatic gain control (AGC) values for FT-MS and FT-MS/MS were set at 1e6 and 5e4 ions, respectively. The maximum fill times for FT-MS and FT-MS/MS were set at 500 and 200 ms, respectively.The raw data were extracted using Proteome Discoverer  for both quantitation and for identification by searching against the NCBI mammalia  1,173,629 sequences) and NCBI bacteria  20,989,102 sequences) databases using the Mascot Search Engine . Search parameters included the following: enzyme: trypsin/P; fixed modifications (carbamidomethyl (C)), iTRAQ4plex (N-term); variable modifications: oxidation (M), dioxidation (M), acetyl (N-term), Gln->pyro-Glu (N-term Q), iTRAQ4plex (K), iTRAQ4plex (Y); peptide mass tolerance ± 10 ppm; fragment mass tolerance ± 0.06 Da; maximum of two miss-cleavages. Threshold score/expectation value for accepting individual spectra was based on Mascot ion score threshold (0.05) as the standard ion score threshold specifically calculated by Mascot for each database search. As an indication of identification certainty, the false discovery rate for peptide and protein matches above identity threshold were calculated by Peptide Validator at 1.0% (strict) and 5.0% (relaxed), respectively.Validation of altered expression levels for selected candidate proteins was carried out by quantitative fluorescent Western blotting using CCG protein extracts and by immunohistochemistry on CCG sections. See QFWB was carried out as previously described , 22. Brim citrate buffer (pH 6.0) for 15 min in a pressure cooker. The slides were then left to cool for 20 min. A commercial immunolabeling kit  was used according to the manufacturer's instructions. Slides were rinsed with Tris buffered saline containing 0.5% Tween  and incubated with peroxidase blocking agent  for 10 min. Slides were rinsed in TBST and incubated with murine monoclonal anti-human synaptophysin (Dako M0776) diluted 1 in 20 in TBST for 60 min at 25 °C. TBST replaced primary antibody for negative controls. Slides were rinsed and incubated with horseradish peroxidase-labeled polymer for 30 min then rinsed and incubated with substrate chromogen solution  for 10 min. Slides were rinsed once in distilled water, counterstained with Harris's hematoxylin (1 min), dipped in Scott's tap water substitute, dehydrated, cleared using ethanol then xylene, and mounted under DPX. The intensity of labeling of CCG neurons was assessed blindly. IHC for B-APP, total Tau, and ubiquitin was carried out with antibodies detailed in 2 .Formalin-fixed, paraffin-wax-embedded CCGs were dewaxed and rehydrated. Antigen retrieval was performed by heating sections in 0.1 http://www.ingenuity.com/).To obtain further insight into potential cellular pathways that may be modified as a result of protein changes identified in our experiments, the Ingenuity Pathways Analysis (IPA) application  was used as previously described –20, 23)23). Pathp < .05 was considered to be significant. Individual statistical tests used are detailed in the results section or figure legends as appropriate.Unless otherwise stated, data were collected into Microsoft Excel spread sheets and analyzed using GraphPad Prism software. For all analyses C and 1D and Supplemental Fig. 1), there is likely to be a spectrum of stages of neurodegeneration present, ranging from neurons in the late stages of acute neurodegeneration to those with relatively normal morphology  . At thisFigs. 1. 1E and ).left data column and Supplementary Data File 1). Data were filtered to yield a final list of proteins altered in EGS CCG by only including those proteins identified by at least two unique peptides. Moreover, consistent with previously published methodologies to increase stringency in reporting, a minimum cutoff of 20% change versus controls was used as previously described . We therefore consider this refined list of 506 proteins to be representative of the molecular alterations induced by EGS neurodegenerative processes in CCGs. Due to the limited molecular investigations into EGS to date, the vast majority of proteins identified here with altered expression had not been previously associated with this disease.Using an iTRAQ quantitative proteomic approach, total protein extracts from six EGS patient CCG and six control samples were examined , amyloid beta precursor protein (APP—3.66 EGS/control ratio), and various components of the ubiquitin proteasome system UPS; . PSP was33B; . We can confirm (as demonstrated by QFWB) that both PSP and APP are indeed changed in the direction and to the approximate magnitude indicated by the proteomic data . Interestingly, it was possible to identify differential expression alterations in both mono- and multimeric ubiquitin, confirming that not only are specific UPS components altered but also that ubiquitin oligomerization profiles are different in EGS CCG. Here, we see an increase in “free” monomeric ubiquitin and a corresponding decrease in multimeric (-trimeric) ubiquitin . This QFWB data therefore indicate that the proteomic analysis is likely to be an accurate reflection of the molecular alterations occurring in EGS neural tissue.Having selected candidates for validation, we began verifying the accuracy of the proteomic data by performing QFWB as previously described –21, 23. Fig. 4A). Having identified PSP by proteomic analysis and confirmed its alteration by QFWB, it appears from IHC that the increased expression is predominantly within cell bodies of EGS CCG . APP is a highly conserved protein (C). Interestingly, the APP immunostaining is not purely membrane bound but appears as strong diffuse cytoplasmic staining with “rings” of APP denoting the membrane boundary of the cell. The staining intensity and subcellular distribution of APP suggests that the increase in APP expression reflects either increased protein synthesis and/or failure of APP transport from the perikaryon to the nerve terminus, perhaps correlating with previously reported defects in the Golgi apparatus and axonal transport  and the complex cellular functions of the proteins selected for validation by QFWB, we next sought to examine their expression profiles at the cellular level. Here, we carried out DAB-based IHC with whole plane analysis of reconstructed CCG. Each staining run and subsequent analysis comprises the examination of whole reconstructed sections  from three EGS (two acute and one subacute) and three control ganglia A. Having protein , integra protein . When we protein C. Intereransport .A and 4E) an increase in “free” mono ubiquitin . In neurons, such alterations result in an increase in diffuse ubiquitin staining (cytoplasmic) and a reduction in dense nuclear staining (higher-order oligomers) such as shown in EGS ubiquitin immunostained CCG sections relative to controls (D). Similar accumulations have previously been reported in TG22/UCHL1 overexpressing mice  and a decrease in the appearance of dense nuclear accumulations 62.3 ± 7.2%  in EGS horses compared with controls. This information therefore serves to further verify the robust nature of the proteomic characterization of the molecular alterations taking place in EGS ganglia. Moreover, these data represent the first demonstration that the neurodegenerative processes in EGS share some molecular characteristics described in other neurodegenerative conditions, including humans and their murine models such as the accumulation and/or redistribution of proteins such as APP and ubiquitin.Ubiquitin is a protein with complex roles within neurons. Upon further examination by QFWB A and 4E controls D. Similaing mice . Quantitin silico analysis using IPA software . This application was used to identify direct and indirect molecular interactions, pathway associations, and functional assignments involving the filtered candidate list .Having confirmed the veracity of the proteomic data, we attempted to obtain further insights into potential molecular pathways and processes that may be modified by explorative in silico analysis, using the IPA database (i.e. those that may be contributing to the degenerative responses (conserved in SMA and EGS), those that may be generic responses to a neurodegenerative insult (conserved in Wlds and EGS in direction), and those that may be regulatory in nature (opposite in Wlds and EGS)  . For exa Fig. 2; ) and mem Fig. 2; . This in Fig. 2;  suggestsA, 4D, and 5C), IPA-based pathway analysis highlighted a network that appears likely to represent a molecular system whose perturbation may be actively contributing to EGS-induced neurodegenerative processes . It is therefore likely that Tau alterations are consequential rather than causative. Prion protein has previously been considered as a potential initiator of the disease process in EGS, but this has never been proven, and prion protein was not present in our dataset. However, QFWB of CCG protein isolates from EGS and control horses suggests that there is no disease-associated prion protein (PrPres) present . Of the proposed upstream regulators, only APP appeared in the functional clustering/network analysis of the proteomic data. This further supports the data presented above and strengthens the suggestion that APP may be a key regulatory protein in EGS-related degenerative processes.IPA software now provides the tools to predict potential upstream regulators of the functions that may be perturbed in the system of interest . These phallmark –45. Tau While studies of neurodegenerative diseases utilizing post-mortem patient tissues remain constrained by sample availability, the data presented here represent a combined molecular snapshot of late-stage disease processes from six horses with a spontaneous multiple system neuropathy (EGS) in comparison to six unaffected controls. Importantly, significant perturbations of the cellular proteome are evident even from analysis of a heterogeneous population of EGS animals , suggestAt the stage of disease when CCG samples were collected, histological examination indicates that CCG neurons are undergoing a spectrum of stages of neurodegeneration and that the majority are undergoing acute neurodegeneration. Consequently, the proteome of EGS CCG extracts will likely reflect the overall range of proteins originating from individual neurons at various stages of degeneration. Therefore, proteins representative of nonspecific neuronal death responses, as well as representatives of the processes initiating and regulating the neurodegenerative progression of EGS, will have been identified see . The altTo address the, albeit unlikely, possibility that the extracts from EGS CCG contained a protein neurotoxin derived from a causal bacterium or fungus, iTRAQ data were also run through NCBIs all species and bacterial databases. While Mascot analysis returned identification of 2,301 in the all species database (Supplementary Data File 1), it only identified 37 proteins in the bacterial database (Supplementary Data File 2). However, none of these were known toxins or were considered to be of relevance to the aetiopathogenesis of EGS. It is important to note that this does not preclude EGS being caused by a bacterial or fungal neurotoxic protein. It remains possible that the coverage of the proteome may be insufficient to facilitate toxin identification, a toxin may be present at concentrations below detection limits, or a toxin may not persist within the CCG of EGS horses.Supplemental Tables 1 and 2). Specific ubiquitin cofactors such as UCHL1/PGP 9.5 have previously been reported in association with altered innervation in nasal mucosal . Perturbations of the UPS have been implicated in various human neurodegenerative conditions, including; Alzheimer's, Huntington's, Parkinson's, and motor neuron diseases  . Here, w mucosal  and auto mucosal  in EGS. diseases , 33–36. seases 33, Fig. 5.For example, one of the cellular processes identified as being significantly altered in EGS was synaptic transmission . The dif47i.e. citrate synthase—CS in SMA) or correlate with vulnerability status . In  2 and 3 , APP thein silico analytical techniques to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease). As such, it has facilitated the identification of more than 500 protein alterations in equine grass sickness cranial (superior) cervical ganglion, most of which had not been previously linked with EGS. This is also the first demonstration of correlation and conservation for neurodegenerative molecular cascades spanning from an apparently unrelated large animal neuropathy, small animal models of altered neuronal vulnerability, and a broad range of human neurological conditions. Finally, this study highlights the feasibility and benefits of applying differential proteomics techniques to the investigation of the neurodegenerative processes in diseases of large animals.Reliable markers with potential modulatory ability are urgently required to improve both diagnostic and therapeutic efficacy, and these remain the key challenges for all neurodegenerative disease research. This is the first application of modern proteomic tools and


Rice (>42 %) was the main food source of Mn. The prevalence of the MetS was 28·0 % (590/2111). Higher Mn intake was associated with a decreased risk of the MetS in men  but an increased risk in women . In addition, Mn intake was inversely associated with abdominal obesity (Ptrend=0·016) and hypertriacylglycerolaemia (Ptrend=0·029) in men, but positively associated with low HDL-cholesterol in both men (Ptrend=0·003) and women (Ptrend<0·001). Our results suggest that higher Mn intakes may be protective against the MetS in men. The inverse association between Mn intake and the MetS in women might be due to the increased risk for low HDL-cholesterol.Animal studies have suggested that Mn might be associated with some components of the metabolic syndrome (MetS). A few epidemiological studies have assessed dietary Mn intake and its association with the risk of the MetS and its components among Chinese adults. In this study, we assessed daily dietary Mn intake and its relationship with MetS risk among Chinese adults in Zhejiang Province using data from the 5th Chinese National Nutrition and Health Survey (2010–2012). A total of 2111 adults were included. Dietary Mn intake was assessed using 3-d 24-h dietary recalls; health-related data were obtained by questionnaire surveys, physical examinations and laboratory assessments. The mean intake of Mn was 6·07 ( A highly significant trend was observed between the number of these risk factors and both CVD and all-cause mortality. In 1988, Reaven found that these factors commonly clustered together and consequently introduced the concept of the metabolic syndrome (MetS) for the first time. The MetS can be diagnosed on the basis of several indicators including waist circumference (WC), serum TAG, serum HDL-cholesterol, systolic blood pressure (SBP) and diastolic blood pressure (DBP), and fasting blood glucose (FBG). As a multiplex risk factor for CVD, the MetS has received much clinical attention in recent years. Although the prevalence of the MetS has reached a plateau in the USA in recent years (from 36·1 % in 2007–2008 to 34·7 % in 2011–2012), its prevalence has increased rapidly in developing countries. In China, the age-standardised prevalence of the MetS increased 4-fold from 5·4 % in 2002 to 21·3 % in 2010. Given its very large population size, development of effective strategies for MetS prevention is of great public health importance in China. A combination of environmental, genetic and lifestyle factors has been found to contribute to the development of the MetS. Recently, dietary intake has attracted much interest, as it offers a potentially modifiable prevention strategy.Abdominal obesity, dyslipidaemia, hypertension and insulin resistance are well-established and strong risk factors of CVD and diabetes1. A h. Insufficient Mn intake may have harmful health effects. Animal studies have found a link between dietary Mn and metabolisms of amino acids, lipids, proteins and carbohydrates, suggesting that dietary Mn intake may be associated with some components of the MetS. It has been suggested that Mn may be essential for maintaining lipoprotein structures because of its high affinity for complexing with the polar heads of phospholipids, thereby stabilising the lipoprotein particle. Mn deficiency-induced oxidative stress accelerates proliferation of the vascular cells, causes blood vessel thickening and narrowing of the internal diameter as well as damages the endothelium, thus increasing vasoconstriction and the risk of hypertension. In addition, impaired superoxide dismutase has also been shown to lead to significant deterioration indices of β-cell function, such as glucose-stimulated insulin secretion.Mn, as an essential mineral, is an enzyme cofactor and a constituent of metalloenzymes. Mn-containing polypeptides such as arginase and Mn-containing superoxide dismutase play important roles in enzyme activities and oxidative stress7. Ins(–). However, thus far, a few studies with larger sample sizes have assessed the source and level of dietary Mn intake among Chinese adults. These data are necessary if appropriate guidelines are to be developed for dietary Mn intake for the Chinese population.Mn is rich in plant foods such as grains and rice, soyabeans, nuts, vegetables, fruits and tea. Previous studies have examined dietary Mn intake in different countries. Intake levels were found to vary substantially, from 1·38 to 11·0 mg/d–18. . A few epidemiological studies have examined the association between Mn intake and MetS risk, and the results have not been consistent. In one study conducted in 550 Chinese adults, compared with participants in the lowest quartile (Mn intake <2·62 mg/d), those in the highest quartile (Mn intake >4·56 mg/d) had a significantly lower risk of the MetS. However, no association was observed among Korean adults with similar quartile cut-offs of Mn intake. More studies with a larger number of participants are needed to clarify this relationship.Insufficient intakes of plant foods have been found to have adverse health effects including obesity, decreased glucose intolerance and cancer,21. The present study aimed to assess dietary intake levels and the main food sources of Mn in a Chinese population, and sought to examine the association between dietary Mn intake and the MetS as well as its components, using data from the 5th Chinese National Nutrition and Health Survey (CNNHS) conducted between 2010 and 2012., which was conducted between 2010 and 2012. In this cross-sectional study, a stratified, multistage probability cluster sampling design was used to recruit participants. In the first stage, six sampling sites  were randomly selected in Zhejiang Province. In the second stage, six communities were randomly selected in each city or county, using a probability–proportional-to-size sampling scheme. In each community, twenty-five households constituted a cluster by geography of household addresses; three clusters were randomly selected, giving a total of seventy-five households. We used 3-d 24-h dietary recalls and FFQ as the major dietary assessment methods for this survey; 3-d 24-h dietary recalls were used in the twenty-five households in the first cluster and the first five households in the second cluster to estimate nutrient intakes and assess the associations with diseases. The remaining households completed FFQ to assess dietary intake in the past 12 months and/or instant food questionnaires. The survey was conducted between August and October in each of the calendar years 2010, 2011 and 2012.The data used in this study were derived from a part of the 5th CNNHS24, whSubjects younger than 18 years of age were excluded from the present analysis. Questionnaire surveys, dietary assessments and physical examinations were conducted among all 2111 participants at a central survey site in their residential area. The questionnaire was administered face-to-face by trained interviewers to collect socio-demographic information including age, sex, area of residence, marital status, occupation, educational status, household income and lifestyle- and health-related information on smoking, alcohol consumption, physical activity, personal and family history of major chronic diseases, and history of medication.China Food Composition (2nd ed.) provided by the Chinese Nutrition Society, which included Mn contents in 1506 foods.Dietary intake was assessed in person by using unannounced 24-h dietary recalls on 3 consecutive days (2 weekdays and 1 weekend day). Participants were asked to recall everything they had eaten in the preceding 24 h, including quantities. The foods included staple foods, snacks, fruits, vegetables, etc. Measuring guides were used to enhance recall and facilitate the estimation of an average portion size. Intakes of condiments were assessed by Weighed Diet Records. The nutrient contents of the diet were analysed, and the daily dietary nutrient intakes, including manganese, were calculated on the basis of the n Society25, whAnthropometric measurements including height, weight, BMI and WC were obtained for each adult participant by trained field workers, following standard protocols. WC was measured to the nearest 0·1 cm at the midpoint between the top of the iliac crest and the lower margin of the last palpable rib in the mid-axillary line using a tape measure. After 5 min of rest in the sitting position, SBP and DBP  were measured using a mercury sphygmomanometer  in the right arm with an appropriately sized cuff, in accordance with the Guidelines for Prevention and Treatment of Hypertension in China (2005); three consecutive readings each separated by 5–10 min were taken, and the mean values of the three measures were used for the analysis.g for 10–15 min. The serum collected in the 2-ml tube was separated for TAG (mmol/l) and HDL-cholesterol (mmol/l) measurements by an automatic biochemical analyser, and 20-μl serum was separated from the 4-ml vacuum tube for FBG measurement. After the 6-ml fasting blood samples were collected, participants took 75 g glucose dissolved in 300 ml water orally within 3 min, and another blood sample was collected into a 2-ml vacuum tube 2 h later (±3 min). An additional 20 μl of serum was separated after centrifugation at 1500 g for 10–15 min for an oral glucose tolerance test. The remaining blood samples were stored at −70°C.Venous blood samples were collected and measured in the local hospitals; 6 ml of blood after fasting for 10–12 h was collected into a 2-ml vacuum, lithium heparin anticoagulant tube and a 4-ml vacuum tube, respectively. Whole blood samples were centrifuged at 1500 The serum levels of TAG were measured using the glycerol phosphate oxidase-phenol amino phenazone (GPO-PAP) method and a Pars Azmoon kit (Pars Azmoon Inc.). Serum HDL-cholesterol levels were tested using the sedimentary method with the same kit using the Liasys autoanalyser device (Liasys). FBG was measured by the glucose oxidase method using the same auto-analyser device.; the criterion of WC was appropriate for Asians. Participants were diagnosed as having the MetS if they met at least three of the following criteria: (1) abdominal obesity, WC≥90 cm in men and WC≥80 cm in women; (2) hypertriacylglycerolaemia, TAG≥1·7 mmol/l (150 mg/dl) or on drug treatment for elevated TAG; (3) low HDL-cholesterol, HDL-cholesterol<1·03 mmol/l (40 mg/dl) in men and HDL-cholesterol<1·3 mmol/l (50 mg/dl) in women or on drug treatment for reduced HDL-cholesterol; (4) hypertension, SBP/DBP≥130/85 mmHg or on antihypertensive drug treatment in patients with a history of hypertension; and (5) hyperglycaemia, FBG≥5·56 mmol/l (100 mg/dl) or on drug treatment for elevated blood glucose.The MetS was diagnosed according to the American Heart Association and the National Heart, Lung and Blood Institute scientific statement26; tht tests for continuous variables or χ2 tests for categorical variables between participants with and without the MetS by sex. Mn intake was the main exposure of interest. The residual method was used to calculate energy-adjusted Mn intakes, which were the residuals from a linear regression model with total energy intake as the independent variable and absolute Mn intake as the dependent variable. Energy-adjusted Mn values were divided into quartiles on the basis of the distributions among men and women, respectively, with the lowest quartile (Q1) as the reference group. To assess the associations between Mn intake and the risk of the MetS and its components, multivariable logistic regressions were used to estimate OR and 95 % CI. The covariates that were significantly different in the univariate analyses were adjusted in the multivariable logistic models. The adjusted variables were age, area of residence, household income and total energy intake for men and age, area of residence, marital status, educational level, drinking status and intake of supplements, Mg and Zn for women. Linear trends were tested by entering the median values of quartiles as continuous variables in the regression. Multiplicative interactions were assessed by adding the interaction terms (sex×quartiles of Mn intake) and comparing the two models with and without the interaction terms using likelihood ratio test. We also explored the association between Mn intake and MetS risk using other cut-off points – for instance, above and below the minimum recommended Mn intake values.As sex was found to modify the association between Mn intake and the MetS, all analyses were performed for men and women separately. Demographic and lifestyle characteristics, intakes of Mn and other nutrients, and MetS components were compared using independent P value<0·05 was considered statistically significant for the MetS analyses. For components of the MetS, considering that type I error may be inflated by multiple testing, P values were adjusted using the Bonferroni correction by dividing the original 5 % significance level by the number of components (P adjusted=0·05/5=0·01).All analyses were conducted using Statistical Package for the Social Sciences software version 17.0 (SPSS Inc.). All statistical tests were two-tailed, and a sd 14·2) years for men and 52·1 (sd 14·0) years for women (P=0·586). A significant difference in the mean daily intake of Mn was found between men and women (men: 6·07 (sd 2·94) mg/d; women: 5·13 (sd 2·65) mg/d; P=0·003).A total of 2111 adults were included in the analysis, of whom 590 were identified as having the MetS, resulting in a prevalence of 28·0 %. The mean age was 54·2 (P<0·05). In addition, male participants with the MetS had a higher WC, FPG, 2hPG (plasma glucose) and TAG than their counterparts without the MetS. Among women, participants with the MetS were less likely to have higher educational levels, be married and be drinkers, but they had higher levels of FPG, 2hPG, DBP and TAG and were more likely to use supplements ; Ptrend=0·043). Men with the highest intake of Mn had an increased risk of the MetS than their counterparts with the lowest intake ; Ptrend=0·078). A significant interaction was found between Mn intake and sex (Pfor interaction=0·002).Pfor interaction=0·023) and hypertriacylglycerolaemia (Pfor interaction=0·017). A higher Mn intake was marginally associated with lower risks of abdominal obesity (Ptrend=0·016) and hypertriacylglycerolaemia (Ptrend=0·029) among men, but not among women . In addition, Mn intake was significantly and positively associated with low HDL-cholesterol risk for both women (Ptrend=0·003) and men (Ptrend<0·001). No significant association was found between Mn intake and hypertension or hyperglycaemia for both men and women.Significant interactions between Mn intake and sex were found for abdominal obesity .Our results indicated that Mn intake was sufficient and in the safe range in this Chinese adult population in Zhejiang Province, China. The major food source of Mn was rice. High dietary Mn intake was associated with a decreased risk of the MetS, abdominal obesity and hypertriacylglycerolaemia among men, but an increased risk of the MetS among women, and an increased risk of low HDL-cholesterol among both men and women., the minimum safe level of Mn intake is 2 mg/d, the maximum safe level is 11 mg/d and the recommended intake level is 4·5 mg/d in China. The average daily intake of Mn in this study population (men: 6·07 (sd 2·94) mg/d; women: 5·13 (sd 2·65) mg/d) was higher than the recommended intake level but lower than the maximum safe level. Our results indicated that Mn intake was sufficient in this Chinese adult population. Previous studies have shown that the mean intake of Mn varied substantially from 1·38 to 11·0 mg/d across different countries. In general, the mean Mn intake level among participants in our study was similar to those of other Asian populations  but higher than European populations . The different Mn intake levels may be explained by various dietary patterns in different countries and different Mn contents in various types of foods.According to the Chinese Nutrition Society25, th. This is because Mn can be accumulated in rice through incorporation into proteins with superoxide dismutase activity, and Chinese dietary patterns, particularly those in southern China, typically have rice as the staple supplemented by wheat. Zhejiang Province is well known as a centre for rice production, and this may explain why an adequate intake level of Mn was found in this study and also why higher mean intake levels were found in our study population compared with European populations.Rice (>42 %) was the main food source of Mn in this study. Similar results were also observed in a study conducted among Korean children, which found that cereals (57·3 %) contributed the most to Mn intake31. Th. No association was observed in two cross-sectional studies conducted among Korean adults, using data from the Korea National Health and Nutrition Examination Survey. The inverse association observed among men in our study was consistent with the results from another cross-sectional study conducted by Li et al. among 550 Chinese adults. In that study, participants in the highest quartile of Mn intake had a 53 %  lower risk of the MetS than those in the lowest quartile. However, sex difference was not further explored in the study by Li et al. because of the limited number of participants. Differences in CVD or metabolic disease risk by sex have been suggested due to differences in oestrogen and androgen levels, HDL dysfunction and gene–environment interactions. With a larger sample size, we were able to conduct a stratified analysis and found that the association between Mn intake and MetS risk was modified by sex. More research is needed to confirm the potential effect modification by sex observed in our study.Our study found that a higher Mn intake was associated with a decreased risk of the MetS for men but an increased MetS risk for women. To our knowledge, only a few studies have examined the associations between Mn intake and the risk of the MetS and its components,,23, who found no association for obesity, TAG, glucose and HDL-cholesterol, but observed that participants with higher Mn intake had lower BP. However, the findings from our study are consistent with those of previous small intervention trials, in which Mn supplementation has been found to be important in reducing obesity and regulating blood lipid metabolism. A small clinical trial with fourteen adults reported that Mn gluconate supplementation of diets, combined with either calcium phosphate or calcium carbonate, increased faecal excretion of fat, thus decreasing body fat. Another clinical trial found that a Mn-adequate diet of conventional foods could decrease plasma levels of cholesterol in seven men aged 19–22 years. Evidence from animal studies also provides biological support for the potential role of Mn in MetS prevention. Mn supplement or intake could reduce abdominal fat accumulation by decreasing fatty acid synthase and malate dehydrogenase activities in the liver and glycerol in adipose tissue, as well as decrease total cholesterol, LDL-cholesterol and HDL-cholesterol because Mn may be an essential component of the lipoprotein structure. However, to our knowledge, no study has examined the different effects of Mn intake on the MetS and its components by sex. Given the biological evidence and the inconsistent results of the cross-sectional studies, more studies, particularly large cohort studies with long-time follow-up and full adjustment of confounders, are needed to further confirm the association between Mn intake and the risk of the MetS and its components, particularly for males and females.For MetS components, we found a significant inverse association between Mn intake and the risk of abdominal obesity and hypertriacylglycerolaemia for men, but a positive association with low HDL-cholesterol, and no association with hypertension and hyperglycaemia for both men and women. Our results differ from those obtained by Choi & Kim22, whA major strength of this study is the relatively large number of participants to explore the different associations among males and females, which has not been assessed in previous studies. The stratified, multistage probability cluster sampling method ensures the representativeness of the study sample. The use of the 3-d 24-h recall dietary assessment method provided detailed dietary intake information to allow for an accurate estimation of Mn intake. Finally, the diagnosis of the MetS and its components was based on objective physical and laboratory measurements, which reduced the likelihood of misclassification.This study has several limitations. First, it was a cross-sectional study, making it difficult to establish a cause-and-effect relationship. Second, only confounders such as age, sex, residential area, smoking status, alcohol intake and total energy intake were adjusted for in the multivariable analyses. Other potential confounders such as physical activity were not adjusted for. Third, although 24-h dietary recalls are widely used as a standard dietary assessment method, using biomarkers may provide more objective measurements of the biologically available and active levels of Mn. However, limited funding prevented us from measuring blood levels of Mn.In conclusion, this study found that rice was the main food source of Mn among Chinese adults in Zhejiang Province, China. A higher Mn intake (e.g. >5·12 mg/d) was associated with a reduced risk of the MetS and its two components – abdominal obesity and hypertriacylglycerolaemia – among men, whereas a positive association was observed between Mn intake (e.g. >4·26 mg/d) and the risk of the MetS and low HDL-cholesterol among women. As diet is potentially modifiable, increasing Mn intake while maintaining a safe level may suggest a potential strategy for the prevention of some components of the MetS for men.


Diseases were associated with socioeconomic status and age of patients and with serotype of organism. Salmonella infection, Shiga toxin–producing Escherichia coli infection, and hemolytic uremic syndrome by census tract–level socioeconomic status (SES) in the Connecticut Foodborne Diseases Active Surveillance Network site for 2000–2011. Addresses of case-patients were geocoded to census tracts and linked to census tract–level SES data. Higher census tract–level SES was associated with Shiga toxin–producing Escherichia coli, regardless of serotype; hemolytic uremic syndrome; salmonellosis in persons ≥5 years of age; and some Salmonella serotypes. A reverse association was found for salmonellosis in children <5 years of age and for 1 Salmonella serotype. These findings will inform education and prevention efforts as well as further research.Foodborne pathogens cause >9 million illnesses annually. Food safety efforts address the entire food chain, but an essential strategy for preventing foodborne disease is educating consumers and food preparers. To better understand the epidemiology of foodborne disease and to direct prevention efforts, we examined incidence of  Salmonella and Shiga toxin–producing Escherichia coli (STEC) are leading bacterial causes of foodborne illness in the United States and result in a combined estimated 1.2 million cases of gastrointestinal illness, ≈22,000 hospitalizations, and 400 deaths per year  measures. Surveillance data usually include street addresses of residences of persons diagnosed with foodborne infections, making use of ABSES possible. Census tract–level poverty, in particular, is a validated ABSES measure recommended by the Public Health Disparities Geocoding Project on the basis of a series of exhaustive studies  and of STEC  for 2000–2011 by census tract–level SES and to assess whether findings changed over time. Our goal was to help direct public health educational efforts to decrease illness from these foodborne pathogens.Our study sought to describe the incidence of Salmonella and STEC infection with onset during 2000–2011. Salmonella, Escherichia coli O157:H7 gastroenteritis, Shiga toxin–related disease, and HUS are reportable by physicians and laboratories in Connecticut. All isolates of Salmonella and E. coli O157:H7 and broths from positive Shiga toxin test results are sent to the Connecticut Department of Public Health (DPH) Laboratory for confirmation, isolation (Shiga toxin–positive broths), and serotyping  in Connecticut for all incident cases of >20%  by using Topologically Integrated Geographic Encoding and Referencing shape files from the US Census Bureau and either a US street locator or a North American address locator. If automatic ArcGIS settings were unsuccessful, interactive geocoding was performed. Case-patients whose addresses were geocoded were matched to census tracts by using a spatial join in ArcGIS: those for 2000–2005 were spatially joined to census tracts by using the 2000 Census; those for 2006–2011 were joined to census tract designations from the 2010 Census. Census tract-specific SES data for percentage of the population living below the federal poverty line were taken from the 2000 Census for case-patients for 2000–2005  were calculated for age-adjusted rates for SES categories by using the <5% poverty group as the reference. IRRs were calculated separately for all Salmonella, STEC, and HUS case-patients and for the leading Salmonella serotypes and O157 and non-O157.Statistical analysis was limited to case-patients whose address could be geocoded and successfully linked to a census tract. During 2000 and 2010, data were missing for 7 (<1%) census tracts, so they could not be assigned to a poverty category, but no case-patients resided in these tracts. All case-patients were aggregated into 3 age categories on the basis of similar age-specific incidence rates: 0–4, 5–9, and Salmonella, STEC, and HUS incidence and census tract–level SES were examined by using χ2 tests for trend to test the statistical significance of the gradients among the 4 categories. All analyses were conducted by using SAS 9.3  except χ2 tests for trend, which were calculated by using Epi Info 2000 .Associations between >20% below the federal poverty level, was the smallest for both periods .Overall, the SES category with the most persons in Connecticut was the highest SES group, <5% below the poverty line (55.2% of the population during 2000–2005 and 48.9% during 2006–2011). The lowest SES group, Salmonella infection confirmed by culture were reported to the Connecticut DPH and FoodNET. Of these, 5,204 (94.9%) were matched to a census tract. Case-patients that could not be matched did not differ from matched case-patients by age group or sex; however, a higher percentage of case-patients could not be matched during the earlier years of surveillance than in the later years . Serotype information was available for all but 1 case-patient. The most frequently observed serotypes were Enteriditis , Typhimirium and its variants , Newport , and Heidelberg . The overall Salmonella IR for 2000–2011 was 12.43 cases per 100,000 person-years. Younger children had the highest IRs; children 0–4 years of age had a 3.47-fold higher rate and children 5–9 years of age a 1.46-fold higher rate of Salmonella infection than those >10 years of age  was 5.6% higher than for the first 6 years . No statistical differences were found in Salmonella incidence by sex.During 2000–2011, a total of 5,484 case-patients with s of age . The SalSalmonella incidence for all 12 years of data combined ; the lowest IRs occurred in the 2 highest SES groups (5%–9.9% = 33.46 and <5% = 34.63)  .Salmonella incidence with higher SES was amplified after age-adjustment; the lowest SES group had a 0.86-fold lower IRR than the highest SES group , Newport , and Montevideo , a reverse gradient of higher incidence and lower census tract–level SES was found for serotype Heidelberg . No other serotype had an association with census tract–level SES, including Typhimurium, the second most common serotype.The overall association of higher >18 years; children 0–4 years of age had a 4.81-fold higher rate of STEC than those >18 years of age, and those 5–17 years of age had a 3.96-fold higher rate of STEC than those >18 years of age. The STEC IR in the last 6 years of the study period (2006–2011) was 16% lower than in the first 6 years . Female patients across all age groups had a 1.31-fold higher IR for STEC than male patients (p<0.001).During 2000–2011, a total of 764 case-patients with STEC confirmed by culture were reported to the Connecticut DPH and FoodNET. Of these, 744 (97.4%) were matched to a census tract. Those that could not be matched did not differ from matched cases. The most frequently observed serotypes were STEC O157 , O103 , O111 , O26 , and O45 . The overall STEC IR for 2000–2011 was 1.78 case-patients/100,000 person-years. Younger children had higher IRs than persons >5 years of age  and lower for children >4 years of age  different Salmonella serotypes had different associations with census tract SES, with serotypes Enteriditis, Newport, and Montevideo associated with high census-tract SES, serotype Heidelberg associated with low census-tract SES, and serotype Typhimurium having no association. These findings provide additional information about the epidemiology of these foodborne diseases and should inform efforts to reduce their incidence.Few studies are available that examine the relationship between foodborne disease incidence and socioeconomic status. Our study showed the following key findings: 1) STEC disease, whether caused by O157 or non-O157 serotypes or whether manifested as HUS, was uniformly associated with high census tract SES; 2) salmonellosis in persons 5–9 and Salmonella incidence increased with higher education and income levels , compared with persons in lower SES census tracts. A commonly proposed reason is that those living in areas with higher census tract–level SES might have increased access to care and might be more likely to submit specimens, regardless of disease severity, whereas those in lower socioeconomic groups might seek care or diagnostic testing only when illness is serious or prolonged , and eating at restaurants (Salmonella infection. In addition, all persons with S. enterica ser. Heidelberg infection were more likely to live in a low SES census tract. These findings are novel: the Michigan study did not look at age, and no reported studies in the United States have systematically examined the relationship between census tract–level SES and Salmonella serotype incidence. However, the previously published Connecticut campylobacteriosis study had similar findings: children living in low SES census tracts had the highest incidence (Salmonella serotypes are associated with different food items (S. enterica ser. Heidelberg has been associated with the consumption of eggs and poultry (Salmonella prevalence in 6 commodities at point of processing found high prevalence of S. enterica ser. Heidelberg in chicken and turkey (In contrast to the findings in adults, children <5 years of age who live in low SES census tracts were more likely than those living in high SES census tracts to have Salmonella infection and about what actions they can take to reduce risk. Education campaigns about high-risk foods other than meat and about the importance of properly handling produce could be run in publications with a higher SES target audience. Regarding research, several considerations need to be explored further. Whether our findings in Connecticut are representative of STEC and salmonellosis incidence nationwide is unknown. The specific reasons behind SES-related risk for foodborne illness still need to be made clear, including for each of the leading Salmonella serotypes. For young children with salmonellosis, potential intervention points to reduce exposure inside and outside the home need to be identified. It remains unclear why children living in areas of higher SES are more at risk for STEC infection and HUS than children living in lower SES areas. These unanswered questions need to be investigated so that effective consumer-level interventions can be developed.Our findings have several implications for risk communication and research. Efforts could be made to increase awareness among persons in high SES groups about their relatively high risk for STEC and ,This study has several limitations. First, it was limited to data from Connecticut, and findings might not be generalizable to other states. Second, SES information was unavailable for 7 census tracts; however, they accounted for <1% of all census tracts in Connecticut. Third, although the data came from active surveillance and are therefore complete within the context of laboratory-confirmed disease, not all persons with gastrointestinal illness seek medical care or have diagnostic testing (Salmonella serotype-specific incidence by census tract–level SES. The findings suggest direction for risk communication efforts and for additional studies to explain the differences and to facilitate additional education and outreach activities.Public health infrastructure objective 7.3 of Healthy People 2020 stresses the need to “increase the proportion of population-based Healthy People 2020 objectives for which national data are available by socioeconomic status” (


Acute kidney injury (AKI) is associated with extended hospital stay, a high risk of progressive chronic kidney diseases, and increased mortality. Patients undergoing radical prostatectomy are at increased risk of AKI because of intraoperative bleeding, obstructive uropathy, older age, and preexisting chronic kidney disease. In particular, robot-assisted laparoscopic radical prostatectomy (RALP), which is in increasing demand as an alternative surgical option for retropubic radical prostatectomy (RRP), is associated with postoperative renal dysfunction because pneumoperitoneum during RALP can decrease cardiac output and renal perfusion. The objective of this study was to compare the incidence of postoperative AKI between RRP and RALP.We included 1340 patients who underwent RRP (n = 370) or RALP (n = 970) between 2013 and 2014. Demographics, cancer-related data, and perioperative laboratory data were evaluated. Postoperative AKI was determined according to the Kidney Disease: Improving Global Outcomes criteria. Operation and anesthesia time, estimated blood loss, amounts of administered fluids and transfused packed red blood cells, and the lengths of the postoperative intensive care unit and hospital stays were evaluated. Propensity score matching analysis was performed to reduce the influence of possible confounding variables and adjust for intergroup differences between the RRP and RALP groups.P < 0.001). However, the estimated blood loss and amount of administered fluids in RALP were significantly lower than in RRP (both P < 0.001). Also, RALP demonstrated a significantly lower incidence of transfusion and smaller amount of transfused packed red blood cells than RRP (both P < 0.001). Importantly, the incidence of AKI in RALP was significantly lower than in RRP . Furthermore, the length of hospital stay in RALP was also significantly shorter (P < 0.001).After performing 1:1 propensity score matching, the RRP and RALP groups included 307 patients, respectively. The operation time and anesthesia time in RALP were significantly longer than in the RRP group (both The incidence of AKI after RALP is significantly lower than after RRP. RALP can therefore be a better surgical option than RRP in terms of decreasing the frequency of postoperative AKI. RALP has been known to be related to lower blood loss and blood transfusion rates and shorter hospital stays in comparison with RRP.10 However, RALP requires a longer operation time and results in worse physiological changes due to pneumoperitoneum and the steep Trendelenburg position in comparison with RRP.12 The glomerular filtration rate, renal blood flow, and urine output can thereby decrease with intraperitoneal carbon dioxide insufflation during RALP.15Radical prostatectomy is a standard surgical treatment for clinically localized prostate cancer.17 However, there have been no comparable studies to date on the evaluation of AKI between RRP and RALP. Therefore, we aimed in our current study to compare the incidence of postoperative AKI based on Kidney Disease: Improving Global Outcomes (KDIGO) criteria between RRP and RALP using propensity score matching analysis.Acute kidney injury (AKI) is increasingly recognized as a serious postoperative complication and is linked to increased health costs and adverse outcomes including progression to chronic kidney disease and death.Following approval by the institutional review board of Asan Medical Center, the records of all patients who underwent RRP or RALP at Asan Medical Center, Seoul, Republic of Korea between January 2013 and December 2014 were searched. Of the 1376 searched patients, we excluded those who underwent additional procedures (n = 8) or had any history of chronic kidney disease (n = 28). A final cohort of 1340 patients was included in the present study  and colloid (Volulyte). Systolic arterial blood pressure was maintained at 90 mm Hg or more during surgery. If systolic arterial blood pressure was less than 80 mm Hg, vasoactive drugs  were administered. The hemoglobin concentration was maintained at 7 g/dL or more; if the hemoglobin concentration was less than 7 g/dL, a packed red blood cell transfusion was planned.18 For RRP, a lower midline abdominal incision was made and the endopelvic fascia was opened from the base of the prostate to the apex. For RALP, pneumoperitoneum was established using a Veress needle, and 6 trocars were inserted. For RALP, the prostate was dissected using the antegrade approach. In both surgeries, bilateral pelvic lymph node dissection was performed, and the neurovascular bundles were spared for all potent patients. After the surgical specimen was removed, vesicourethral anastomosis was performed using a 20-Fr urethral catheter.The key procedures for RRP and RALP were performed according to the standard protocols of our institution.−1.154 × age−0.203 × [0.742 if female] × [1.210 if African-American].19We collected information regarding the baseline characteristics and laboratory, intraoperative, and postoperative data from the computerized patient record system at our institution . The baseline characteristics included age, height, weight, body mass index, comorbidities , and the use of prescribed medications . Cardiac disease included ischemic heart disease and heart failure. Heart failure was defined as a history of any type of heart failure that was diagnosed by a cardiologist regardless of medication or decreased ejection fraction . Cerebrovascular disease was defined as a history of carotid artery stent or angioplasty, transient ischemic attack, stroke, or cerebral hemorrhagic event. Data on the status of patient's cancer including prostate-specific antigen (PSA) level and Gleason score were collected. The estimated glomerular filtration rate (eGFR), hematocrit, albumin, uric acid, and C-reactive protein levels were collected as preoperative laboratory data. eGFR was calculated using the 4-variable  Modification of Diet in Renal Disease Study equation: eGFR = 186 × serum creatinine20Intraoperative data included operation time, anesthesia time, estimated blood loss, volume of administered fluids, volume of transfused packed red blood cells, and the use of vasoactive drugs. The operation time was defined as the time between first incision and the end of the operation. Anesthesia time was defined as the time from anesthesia induction to tracheal extubation. Estimated blood loss was evaluated by the amount of lost red cell mass, which was calculated using the perioperative change in the hematocrit and transfused red cell mass using the following equation: lost red cell mass (mL) = patient's estimated blood volume (mL) × (preoperative hematocrit in % − postoperative hematocrit in %) + (transfused packed red blood cell in units × 250 (mL) × 0.6).21 However, in the present study, the urine output criterion was not included due to the inconsistency in urine output measurement. The secondary endpoints included the lengths of postoperative intensive care unit and hospital stay.The primary endpoint of this study was the comparison of the incidences of AKI based on the KDIGO criteria between RRP and RALP. According to KDIGO criteria, AKI is defined as an increase in serum creatinine by 0.3 mg/dL or more within 48 hours or an increase in the serum creatinine by 1.5 times or more within the prior 7 days.t test or Mann–Whitney U test for continuous variables, as appropriate. Data are presented as the mean ± standard deviation, or number (percentage), as appropriate. We performed 1:1 propensity score matching analysis to reduce the influence of possible confounding variables and adjust intergroup differences between RRP and RALP groups. To determine the propensity score, a multiple logistic regression model was run using the following 17 variables: age, height, weight, body mass index, hypertension, diabetes mellitus, cardiac disease, cerebrovascular disease, taking beta-blockers or nonsteroidal anti-inflammatory drugs, PSA level, Gleason score, preoperative eGFR, hematocrit, albumin, uric acid, and C-reactive protein .Before propensity score matching, we compared data between the RRP and RALP groups using the Chi-square test or Fisher exact test for categorical variables and the student P < 0.001) (Table P < 0.001) (Table P < 0.001) (Table P = 0.044) . However, there were no significant differences in the lengths of stay in the intensive care unit between the groups.A total of 1340 patients who underwent RRP (n = 370) or RALP (n = 970) were included in the current analyses. Age, height, weight, presence of cardiac disease, PSA, preoperative hematocrit, and albumin level demonstrated statistically significant differences between RRP and RALP groups (Table 1) Table . However1) Table . Also, t1) Table . Importa) Figure . FurtherIn the present study, we found that the incidence of AKI after RALP was significantly lower than after RRP. The amounts of intraoperative blood loss and transfused packed red blood cells in RALP were also significantly lower, and the duration of hospital stay was significantly shorter in comparison with RRP.22 Nevertheless, the exact incidence of AKI after radical prostatectomy using validated criteria have never been determined. Our present study provides the first information on the incidence of AKI after radical prostatectomy according to the KDIGO criteria, which can detect even acute subclinical increases in serum creatinine or decreases in eGFR after surgery.Postoperative AKI is associated with increased costs, morbidity, and mortality and can increase the risk of progressive chronic kidney disease. Patients undergoing radical prostatectomy are at increased risk for AKI because of the common occurrences such as obstructive uropathy, older age, and preexistent chronic kidney disease, as well as intraoperative bleeding.15 These physiologic changes consequently stimulate the renin-angiotensin system and further decreases renal blood flow.24 All of these can contribute to the impairment of renal function. However, previous studies on this issue have reported that postoperative renal function is unaltered after RALP, even under using a pressure of 20 mm Hg for pneumoperitoneum.26 However, these earlier reports analyzed the change in the creatinine clearance or the value of eGFR to measure the differences between preoperative and postoperative renal function, instead of using validated criteria. Also, those studies analyzed only patients that underwent RALP, so the outcomes were not comparable to patients who underwent RRP. In our present study, we used the KDIGO criteria to define AKI, and its incidence between RRP and RALP groups were compared using propensity score matching analysis to reduce the influence of confounding variables and adjust intergroup differences between groups. Therefore, we believe that our present results are highly reliable for the evaluation of AKI after radical prostatectomy.The results of our current analyses showed a postoperative AKI incidence of 5.5% after RALP and 10.4% after RRP. RALP often requires pneumoperitoneum with an intra-abdominal pressure of more than 15 mm Hg for better visualization of the surgical field and continues for more than 3 hours. Direct compression of the intra-abdominal vessels and renal parenchyma by pneumoperitoneum can decrease cardiac output, renal blood flow, the glomerular filtration rate, and urine output.10 RALP demonstrated lower blood loss and blood transfusion rate in comparison with RRP. The steep Trendelenburg position for RALP under pneumoperitoneum improves visualization, and thus bleeding from the dorsal vein complex during surgery can be more easily controlled.25 Furthermore, the tamponade effect by pneumoperitoneum also contributes to reduced blood loss.27 Generally, anemia and blood transfusion are well-known important risk factors of renal injury after cardiac surgery.31 The pathogenesis is unclear, but several mechanisms have been suggested: high vulnerability to hypoxic injury to the kidney and iron-mediated oxidative kidney injury.35 A previous study of 8799 patients who underwent lower-extremity revascularization to investigate the effects of blood transfusion demonstrated that intraoperative blood transfusion was associated with renal failure, as well as morbidity and mortality.36 Another study of 1034 cardiac surgery patients reported that the patients who received a nonleukoreduced red blood cell transfusion were at a higher risk of acute kidney injury and in-hospital mortality than the patients who received leukoreduced red blood cell transfusion.37 Another retrospective study of trauma patients demonstrated that the transfusion of red blood cell stored for more than 14 days was associated with increased renal dysfunction and mortality.38 In our current study, the higher incidence of AKI after RRP may result from the decreases in cardiac output and renal perfusion, diminished oxygen delivery, and increased oxidative stress to the kidney that are associated with a larger amount of blood loss during RRP. Furthermore, the higher incidence of red blood cell transfusion during RRP may also be responsible for the higher incidence of AKI.There are several comparative studies between RRP and RALP in terms of surgical, oncological, and functional outcomes. In line with a previous report,39 In our current study, none of the patients with postoperative AKI previously had rhabdomyolysis or urinary tract obstruction after RRP or RALP.Rhabdomyolysis and urinary tract obstruction can occur after robotic or nonrobotic radical prostatectomy and may be associated with development of AKI. Rhabdomyolysis can lead to glomerular filtration rate impairment. However, prostatectomy-related rhabdomyolysis is a very rare complication, and its incidence is reported to be 0.08%.41 Thus, we performed propensity score matching analysis for 17 confounding variables to minimize these biases. In addition, there are many difficulties in performing a randomized controlled trial to compare RALP and RRP because most patients are unwilling to accept the idea of randomization to a particular surgical treatment. Thus, propensity score matching analysis can be a reliable second-best strategy for comparing RALP and RRP.The inevitable limitation of our current study comes from its retrospective design. Many confounders such as age, body mass index, comorbidities, and preoperative anemia may affect the accurate evaluation of the incidence of postoperative AKI. Also, previous studies show that cancer characteristics  might predict the risk of complications.In conclusion, postoperative AKI occurs at a lower incidence after RALP than RRP. This result provides valuable information on the additional benefit of RALP, which has many well-known advantages in comparison with RRP. Accordingly, RALP can be a better surgical option in terms of decreasing postoperative AKI than RRP.


Aedes aegypti and Ae. albopictus mosquitoes, causes debilitating disease characterized by an acute febrile phase and chronic joint pain. Chikungunya has recently spread to the island of St. Martin and subsequently throughout the Americas. The disease is now affecting 42 countries and territories throughout the Americas. While chikungunya is mainly a tropical disease, the recent introduction and subsequent spread of Ae. albopictus into temperate regions has increased the threat of chikungunya outbreaks beyond the tropics. Given that there are currently no vaccines or treatments for chikungunya, vector control remains the primary measure to curtail transmission. To investigate the effectiveness of a containment strategy that combines disease surveillance, localized vector control and transmission reduction measures, we developed a model of chikungunya transmission dynamics within a large residential neighborhood, explicitly accounting for human and mosquito movement. Our findings indicate that prompt targeted vector control efforts combined with measures to reduce transmission from symptomatic cases to mosquitoes may be highly effective approaches for controlling outbreaks of chikungunya, provided that sufficient detection of chikungunya cases can be achieved.Chikungunya, a re-emerging arbovirus transmitted to humans by  Ae. aegyptiAe. albopictus into temperate regions, combined with the recent evolution of chikungunya towards elevated transmissibility in Ae. albopictus, has exacerbated the risk of temperate chikungunya outbreaks8Ae. aegypti - transmitted chikungunya was initially introduced on the island of St. Martin from which it disseminated throughout the Caribbean, threatening both mainland Latin America, where Ae. aegypti mosquito is widespread, and the southern United States, where both Ae. aegypti and Ae. albopictus are prevalent111213Since the 1953 identification of chikungunya in Tanzania2346Ae. aegypti and Ae. albopictus are endemic811233Chikungunya is characterized by the sudden onset of fever and joint pain, which are often incapacitating171819Ae. aegypti and Ae. albopictus, we evaluated the effectiveness of intervention strategies for mitigating the spread of a chikungunya outbreak. In contrast to the contention that little can be done to contain chikungunyaTo assess the effectiveness of interventions against chikungunya, we developed a stochastic vector-borne transmission model for the spread of chikungunya within a residential neighborhood. Our model takes into account the impact of household transmission of chikungunya as well as human and mosquito movement between houses. We parameterized the model using epidemiological, entomological, clinical, and human movement data. For both To simulate viral transmission in a residential neighborhood, we developed a household-level spatial stochastic model of chikungunya transmission, explicitly modeling individual human and mosquito movement and exposure. We ran our continuous-time model for one year using the Gibson-Bruck adaptation of the Gillespie algorithmWe ran our model on a two-dimensional square lattice with 20 × 20 sites, representing a neighborhood of 400 houses populated with four residents per house27i was assigned a number of contact houses ni sampled from an exponential distribution with mean of 6.26 i spends a fraction ni contact houses, chosen randomly and each assigned an exponentially distributed weight τij and normalized such that We assumed that humans divide their time between their homes and other houses, with the likelihood of the latter depending on the distance of other residences away from their homes, as informed by an empirical studyj to humans from house i was modeled as c is the biting rate, j, i, Nhi is the number of humans in house i, and τij is the proportion of time spent in house j by the individual from house i. After an incubation period ranging from two to six days, exposed humans become pre-symptomatically infectious for two days32431κ of infectious humans become symptomatic while the rest remain asymptomatic.Individuals were designated as susceptible, exposed, infected (pre-symptomatic), infected (70–90% of whom were asymptomatic), or recovered from chikungunya, at rates parameterized from epidemiological, clinical and entomological studies . The traWe parameterized the daily rate of mosquito movement between houses from a field study  as a measure of the likelihood of disease outbreak and intensity of spread through the population. To calculate R0, we simulated the number of secondary cases of human infections that resulted from mosquitoes that were directly infected by an initial human case in a population of fully susceptible humans and mosquitoes. By simulating 10,000 iterations of our stochastic model, we generated a probabilistic distribution of secondary cases likely to arise from a single infection, with mean R0 41Ae. aegypti and Ae. albopictus by fitting the simulated distribution of R0 against empirical estimates from previous and current chikungunya outbreaks31R0 values were estimated for the strain of chikungunya transmitted by Ae. albopictus during the 2004–2005 epidemic in Réunion IslandAe. aegypti for the 2005–2006 epidemic in IndiaWe evaluated the effectiveness of containment intervention strategies for mitigating the spread of an emerging chikungunya outbreak. We used the basic reproduction number , efficacies of vector control for killing adults mosquitoes (ranging from 10 to 90%), and sensitivities of surveillance measures for detecting new cases of symptomatic human infection (low (30% efficacy), intermediate (50% efficacy), high (80% efficacy)). A flowchart outlines the steps of our analysis  of insecticides in perimeter radius around houses with detected cases, indoor residual spraying2245345analysis .Ae. aegypti, the transmission parameter R0 equals 2, 0.14 when R0 equals 4 and 0.24 when R0 equals 6. For Ae. albopictus, the transmission parameter R0 equals 2, 0.22 for R0 equals 4 and 0.36 for R0 equals 6. We validated the time series dynamics of our model against empirical data from the 2013–2014 chikungunya outbreak in Dominica , the combination of disease surveillance measures with at least 80% probability of detecting new cases, efficacious transmission reduction measures for symptomatic individuals with at least 80% reduction of contact between infected individuals and mosquitoes, and vector control measures with at least 50% efficacy in killing adult mosquitoes was found to reduce the epidemic attack ratio from 20%, without control, to less than 1% (R0 = 6), the combined intervention was found to reduce the epidemic attack ratio from 60% down below 20% .