Hyponatremia is the most common electrolyte disorder among hospitalized patients. Controversies still exist over the relationship between hyponatremia and outcomes of hospitalized patients.To analyze the association of low serum sodium levels at hospital admission with in-hospital mortality and patient disposition and to compare the distribution of the risk of death associated with hyponatremia across the lifespan of hospitalized patients, we conducted an observational study of 2.3 million patients using data extracted from the Cerner Health Facts database between 2000 and 2014. Logistic regression models were used in the analyses.At hospital admission 14.4% of hospitalized patients had serum sodium levels [Na] <135 mEq/L. In adjusted multinomial logistic regression analysis, we found that the risk of in-hospital mortality significantly increases for [Na] levels < 135 or ≥143 to ≤145 mEq/L compared to the reference interval of 140 to <143 mEq/L (p<0.001). We observed similar trends for the relationship between [Na] levels and discharge to hospice or to a nursing facility. We demonstrated that younger age groups  had a higher risk of in-hospital mortality compared to older age groups  for [Na] levels <130 mEq/L or 143 to ≤145 mEq/L (p<0.001).Hyponatremia is common among hospitalized patients and is significantly associated with in-hospital mortality, discharge to hospice or to a nursing facility. The risk of death and other outcomes was more evident for [Na] <135 mEq/L. The mortality associated with low [Na] was significantly higher in younger versus older patients. Hyponatremia is the most frequent electrolyte disorder among hospitalized patients , 2. It iTo address this gap in our knowledge regarding the cutoff points of hyponatremia associated with poor outcomes of hospitalized patients and the distribution of the risk of death associated with hyponatremia across the lifespan of these patients, we analyzed data on patients hospitalized between January 2000 and November 2014 derived from the Cerner Health Facts database.We conducted an observational study of patients hospitalized between January 2000 and November 2014 using the Cerner Health Facts database. The study was done using the Health Facts database which is a national database that includes de-identified electronic health records (EHR) information from over 600 hospitals and clinics in the United States. The study was reviewed and approved by the University of New Mexico Institutional Review Board (15–531). The IRB waived the requirement for informed consent. We included the index hospital admission defined by the first inpatient encounter of any patient during the study period who met the following inclusion criteria: 1) age ≥ 18 years, and 2) first [Na] drawn during 24 hours before admission. The second inclusion criterion was chosen to reduce the possibility that the [Na] was affected by any treatments or iatrogenic causes after hospital admission.Information was gathered regarding patients’ demographics, co-morbidities, causes for admission, laboratory studies and disposition status at hospital discharge. The co-morbidities were identified using the International Classification of Diseases, 9th Edition, Clinical Modification (ICD-9-CM) codes, while the laboratory tests were identified using Logical Observation Identifiers Names and Codes (LOINC).The Deyo-Charlson Comorbidity Index (Deyo-CCI) was also calculated . Deyo-CCContinuous variables were summarized as means and standard deviations (SD) and were compared using student’s t tests. Categorical variables were summarized as percentages and were compared using chi-square tests.We used logistic regression models to evaluate associations between patient characteristics and hyponatremia status (<135 mEq/L vs. ≥135 mEq/L). The patient characteristics of primary interest included age, gender, race and selected comorbidities and reasons for hospitalization see . In thesth, 27.5th, 50th, 72.5th, and 95th percentiles of the cohort’s [Na] respectively. Second, for simplicity in interpretation, we formed the following eight [Na] categories: 1: < 120; 2: ≥ 120 to < 125; 3: ≥ 125 to < 130; 4: ≥ 130 to < 135; 5: ≥ 135 to < 138; 6: ≥ 138 to < 140; 7: ≥ 143 to ≤ 145 mEq/L and with 0: ≥ 140 to < 143 mEq/L as the reference group. We categorized the [Na] into 5 mEq/L intervals for [Na] between 120 and 135 mEq/L, as in previous studies. Since we had a very large number of patients with [Na] ≥ 135 mEq/L, we used narrower quantile-based intervals for higher [Na] to generate categories comprising of approximately equal numbers of patients in the higher categories.We were interested in evaluating broader associations between [Na] and outcomes. We therefore examined associations between quantitatively measured [Na] and our study outcomes in two ways. First, in order to visualize flexible relationships between [Na] and the outcomes, we modeled the associations using restricted cubic splines with five knots placed at 132, 136, 138, 140, and 143 mEq/L corresponding to the observed 5As there were multiple interrelated and unordered outcomes, we used multinomial logistic regression models to assess associations between [Na] and the outcomes. These multinomial models allowed us to assess associations of each outcome while accounting for the competing risks of the others. In these models, we used “Discharge to home” as the reference outcome. We used the restricted cubic spline terms in these models to generate predicted probabilities across the continuum of [Na], and subsequently used these predictions in order to visualize the patterns of the associations between [Na] and the study outcomes. We used our defined [Na] categories in these models to examine associations between specific [Na] and the outcomes of interest, both with and without accounting for other covariates: age, gender, race, and the selected comorbidities and reasons for hospitalization outlined in We evaluated whether age has a differential effect on the relationships between [Na] and the outcomes. To accomplish this, we first used restricted cubic splines in age stratified multinomial logistic regression models to visualize trends in the predicted probabilities. We subsequently modeled the interaction between the age groups and the [Na] categories using multinomial logistic regression models while adjusting for age, gender, race, and the selected comorbidities and reasons for hospitalization. We also performed a sensitivity analysis after replacing the specific comorbidities with the Deyo-CCI in the covariate list.In addition to examining associations between [Na] levels and the categories of primary outcomes, we also assessed the relationships between [Na] levels and length of hospitalization among those who were discharged to home. We used linear regression approaches to model the number of days of hospitalization with [Na] categories, while adjusting for age, gender, race, and the selected comorbidities and reasons for hospitalization. We additionally tested for the differential effect of age on these associations. In order to meet the model assumptions, we performed analyses after logarithmic transformation of the length of stay variable, and reported results after back-transformation to the original scale. All analyses were performed using Stata version 14.1 .We found 2,698,978 patients who met our inclusion criteria. From this cohort 210,526 patients were lacking a concomitant serum glucose determination and were excluded from the study cohort. Our initial cohort of patients included 2,488,437 patients . The prePatients with hyponatremia were older than patients with normonatremia ([Na] = 135–145 mEq/L), 64.1±18.1 vs. 60.8 ±19.1 years old (p<0.001). Patients with hyponatremia had a higher overall Deyo-CCI score compared with normonatremic patients 1.72±2.25 vs. 1.29±1.81, respectively, (p<0.001). Other pertinent patient characteristics are summarized in We found that hyponatremia increases with increasing age after controlling for gender, race and the selected comorbidities and reasons for hospitalization. The odds ratios (ORs)  of having hyponatremia for ages 45 to <65, 65 to <75 and 75 were 1.21 [1.20–1.23], 1.35 [1.33–1.37] and 1.56 [1.54–1.58], respectively, compared to the reference age group 18 to <45 years old .The crude probability of in-hospital mortality corresponding to [Na] was lowest between 135 and 143 mEq/L and continued to increase above or below this range. The probability of discharge to hospice demonstrated a similar pattern, though less profound. The estimated probability of discharge to nursing facility also demonstrated a similar pattern but started to decrease at [Na] below 115mEq/L. We observed the opposite for discharge to home, such that the probability increased up to [Na] between 135 and 143 mEq/L, then decreased above this range .For the [Na] categories, the crude in-hospital mortality increased as [Na] decreased or increased relative to the 138 to <140 mEq/L interval. This was also true for discharge to hospice and discharge to nursing facility. However, the percentage of discharge to nursing facility decreased for [Na] intervals 120 to <125 mEq/L and <120mEq/L .In the adjusted multinomial logistic regression analysis, we found that the likelihood of in-hospital mortality significantly increased for [Na] < 135 mEq/L or ≥ 143 to ≤145 mEq/L compared to the reference interval of 140 to <143 mEq/L (p<0.001). Patients with [Na] of <120, 120 to <125, 125 to <130, 130 to <135, 135 to <138, 138 to <140 and 143 to ≤145 mEq/L compared with 140 to<143 mEq/L had in-hospital mortality relative risk ratios [95% CI] of 4.85 [4.20–5.60], 3.12 [2.87–3.40], 2.43 [2.32–2.54], 1.45 [1.41–1.49], 0.90 [0.88–0.92], 0.78 [0.76–0.80] and 2.15 [2.08–2.22] respectively  .This strong association with in-hospital mortality continued to rise with the increase in the severity of hyponatremia . PatientThere was a significant association between [Na] categories and length of hospitalization (p<0.001). These overall relationships are shown in . For thoWe observed that for low [Na], the probability of in-hospital mortality was higher for younger age groups compared to older age groups . For disWe obtained similar findings after modeling the interaction between each age group and the [Na] categories . The gloThere were also significant age differences in associations between hospitalization length and [Na] categories (p<0.001) for patients who were discharged to their homes . PatientIn this observational study using a large multicenter database, we were able to demonstrate using the cutoff point of <135 mEq/L that the prevalence of hyponatremia was common at hospital admission (14.4%). We showed that having [Na] < 135 mEq/L or ≥ 143 to ≤145 mEq/L is a significant risk factor for in-hospital mortality and increased discharge to hospice or to a nursing facility. Moreover, we found that younger patients admitted with [Na] <130 mEq/L or ≥143 to ≤ 145 mEq/L had a higher risk of in-hospital mortality compared to older patients.In general, our results corroborate those of several other investigators. For example, Waikar et al  reportedOur results are in accordance to previous studies showing that hyponatremia is an independent risk factor for in-hospital mortality , 8, 14. We documented a significant association between hyponatremia and the increased risk of discharge to hospice or to a nursing facility. Furthermore, we demonstrated that the in-hospital mortality continues to worsen with the increase in the severity of hyponatremia, a conclusion that was also reached by Wald et al . HoweverAlthough in the present study the risk of in-hospital mortality was the lowest in the 138 to <140 mEq/L group, the in-hospital mortality for the 135 to < 138 mEq/L group was still low compared to the other [Na] categories. Our results support the traditional lower cutoff point of normonatremia of 135 mEq/L. Realizing that the risk of in-hospital mortality intensified for values ≥ 143 to ≤145 mEq/L, we support the proposition expressed by Wald et al that the traditional higher cutoff point of normonatremia (≤ 145 mEq/L) needs to be reevaluated.We demonstrated that the likelihood of in-hospital mortality associated with [Na] levels varied between the various age groups. Younger age groups (18 to <45 years and 45 to <65 years) had higher likelihood of in-hospital mortality compared to older age groups  for [Na] <130 mEq/L or ≥143 to ≤ 145 mEq/L. It is the contention of some investigators that elderly patients have a high risk of mortality at hospital admission –17. HoweThe differential association between hyponatremia and mortality in the different age groups has several plausible explanations. It is well known that chronic hyponatremia (>48 hours) is very common in elderly population: chronic hyponatremia is present in 11.3% of elderly people living in the community  and 18% Our study has several strengths. The size of our study allowed us to robustly investigate the association between the different levels of hyponatremia and selected outcomes of hospitalized patients even with very low levels of hyponatremia. For example, for patients with serum sodium levels < 120 mEq/L the number of deaths was 260 out of 2413, which, to our knowledge, is higher than that found in any other published report. Our cohort included a diverse patient population from many hospitals in the United States, hence our results are generalizable to the US population.Our study also had several limitations. First, inherent in our study was the use of ICD-9 codes to identify patients’ comorbidities, which is prone to billing and coding errors. However, the Health Facts data undergo extensive review before being added to the database. Second, the identification of the comorbidities and reason for hospitalization using ICD-9 codes in our study does not account for the severity of these medical conditions. Due to the limitation of our observational study, we were not able to include a disease severity scoring modality such as the Acute Physiology and Chronic Health Evaluation (APACHE) or Sequential Organ Failure Assessment (SOFA). This may have led to unmeasured confounding. However, we were able to adjust for the Deyo-Charlson Comorbidity Index (Deyo-CCI) which has been used previously by studies that demonstrated an independent association between hyponatremia and in-hospital mortality , 8, 14. Hyponatremia is common among hospitalized patients and regardless of the cause, it contributes significantly to the morbidity and mortality of hospitalized patients. It is an important risk factor for increased in-hospital mortality and increased discharge to hospice or to a nursing facility. Moreover, younger patients admitted with hyponatremia had a higher risk of in-hospital mortality compared to older patients. Our study underscores the need for more alertness to the [Na] in the outpatient setting and emphasizes the need for providing equal attention and care to all patients presenting to hospital with hyponatremia. Studies are needed to evaluate the traditional higher cutoff point of normonatremia (≤ 145 mEq/L).S1 TableOR = Odds Ratio; Ref = Reference; DM = Diabetes Milletus; ESRD = End Stage Renal Disease; AIDS = Acquired Immunodeficiency syndrome; SIADH = Syndrome of Inappropriate Antidiuretic Hormone; UTI = Urinary Tract Infection.*Adjusted for age, gender, race and selected comorbidities and reasons for hospitalization.(DOCX)Click here for additional data file.S2 TableCI = confidence interval; Deyo-CCI = Deyo-Charlson Comorbidity Index.a Serum Sodium Levels corrected by adding 1.6 mEq/L for each 100 mg/dL above 100 mg/dL of the concomitantly measured serum glucose levels.bThe adjusted relative risk ratios were derived from a multinomial logistic regression model adjusted for age, gender, race, and the Deyo-CCI .(DOCX)Click here for additional data file.S3 TableCI = confidence interval.aSerum sodium levels corrected by adding 1.6 mEq/L for each 100 mg/dL above 100 mg/dL of the concomitantly measured serum glucose levels.bThe adjusted relative ratios were derived from linear regression model adjusted for age, gender, race, and the selected comorbidities and reasons for hospitalization .(DOCX)Click here for additional data file.S4 TableaThe percentages and the relative risk ratios (RRR) of in-hospital mortality are for each age group with the respective [Na] category.bThe relative risk ratios are derived from a multinomial logistic regression model adjusted for age, gender, race, and the selected comorbidities and reasons for hospitalization. CI = Confidence interval. p<0.001 for all except: c p = 0.45.(DOCX)Click here for additional data file.S5 TableRRR = Relative Risk Ratio.Age0 = Age (≥18 to <45); Age1 = Age (≥45 to <65); Age2 = Age (≥65 to <75); Age3 = Age ≥75.(DOCX)Click here for additional data file.S1 FigThe relative risk ratios were derived from multinomial logistic regression models adjusted for age, gender, race, and Deyo-CCI. Discharge to home and serum sodium levels of (140 to <143 mEq/L) served as referent. Serum sodium levels were corrected by adding 1.6 mEq/L for each 100 mg/dL increase above 100 mg/dL of the concomitantly measured serum glucose levels. Error bars indicated 95% CI. CI = confidence interval; Deyo-CCI = Deyo Charlson Comorbidity Index.(DOCX)Click here for additional data file.S2 FigThese estimated probabilities were derived from a multinomial logistic regression models stratified by age.(DOCX)Click here for additional data file.S3 FigThese estimated probabilities were derived from a multinomial logistic regression models stratified by age.(DOCX)Click here for additional data file.S4 FigThese estimated probabilities were derived from a multinomial logistic regression models stratified by age.(DOCX)Click here for additional data file.S5 FigThese estimated geometric means were derived from a linear regression model on the logarithmic-transformed number of days of hospitalization on [Na] levels with age categories included both as main effects, and in interactions with [Na] levels, while also adjusting for age, gender, race, and Deyo-CCI.(DOCX)Click here for additional data file.


Background. The clinical diagnosis of pneumonia is sometimes difficult since chest radiographs are often indeterminate. In this study, we aimed to assess whether serum C-reactive protein (CRP) could assist in identifying patients with pneumonia. Methods. For one winter, all consecutive patients with acute respiratory symptoms admitted to the emergency ward of a single center were prospectively enrolled. In addition to chest radiographs, basic laboratory tests, and microbiology, serum levels of CRP were measured at entry. Results. A total of 923 (62.3%) of 1473 patients hospitalized for acute respiratory symptoms were included. Subjects with a final diagnosis of pneumonia had higher serum CRP levels (median 187 mg/L) than those with exacerbations of chronic obstructive pulmonary disease (63 mg/L) or acute bronchitis . CRP was accurate in identifying pneumonia . The multilevel likelihood ratio (LR) for intervals of CRP provided useful information on the posttest probability of having pneumonia. CRP intervals above 200 mg/L were associated with LR+ > 5, for which pneumonia is likely, whereas CRP intervals below 75 mg/L were associated with LR < 0.2, for which pneumonia is unlikely. Conclusion. Serum CRP may be a useful addition for diagnosing pneumonia in hospitalized patients with acute respiratory symptoms. Pneumonia is a leading cause of hospitalization and death in developed countries . HoweverRecent guidelines and review studies have suggested that serum C-reactive protein (CRP) may be helpful in distinguishing pneumonia from other acute respiratory illnesses , 5. NeveA prospective study was therefore conducted in a large and unselected population with the goal of clarifying whether serum CRP could identify patients with pneumonia.A prospective study was performed in a 500-bed university hospital, and patients with the following inclusion criteria were recruited during one winter season (2013-14): (1) adults > 18 years old admitted to the emergency ward, (2) respiratory symptoms  as the main complaint, with or without fever, and (3) disease duration of less than two weeks. The exclusion criteria were (1) a final diagnosis of acute decompensated heart failure, pulmonary embolism, lung cancer, or an upper respiratory infection , (2) severe immunosuppression  or receiving immunosuppressive therapy , and (3) no hospitalization required.Hospitalization was considered necessary if patients met one of the following: (1) need for either respiratory support (Sa 02 < 90% or Pa 02/Fi 02 < 300), mechanical ventilation (respiratory acidosis with pH < 7.30), or vasopressor drugs, (2) worsening of associated comorbidities , (3) inability to take oral drugs, or (4) no response to an initial adequate treatment in the emergency department.The local ethics committee approved this study and written informed consent was obtained from each patient. Streptococcus pneumoniae and Legionella pneumophila antigen detection tests in urine samples from those with pneumonia. Serology was ordered according to the criterion of the attending physician. To stratify severity in pneumonia patients, a validated prediction rule was used, namely, the CRB65 Severity Index [At the initial visit to the emergency department, demographic and basic clinical information was collected from each patient. In addition to routine blood tests, a serum sample was obtained to measure CRP. Microbiological studies included sputum sampling for Gram staining and culture in all patients with LRTI, when possible, as well as blood cultures, andty Index . AntibioBlood samples for CRP were analyzed by a particle-enhanced turbidimetric assay following the manufacturer's instructions . The range of detection for this CRP assay is from 0.2 to 480 mg/L.9 cells), regardless of antibiotic use. For pneumonia, a new infiltrate on the chest radiograph was also required. COPD was defined by postbronchodilator spirometric criteria, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, as FEV1/FVC ratio < 70%. Acute bronchitis was defined as LRTI in the absence of an underlying lung disease (COPD) or focal chest infiltrates on chest X-rays [LRTI was defined by the presence of at least one respiratory symptom  plus at least one finding during auscultation  or one sign of infection , according to the American College of Cardiology/American Heart Association guidelines . Pulmonaχ2 and Fisher's exact tests for categorical variables and the nonparametric Kruskal-Wallis and Mann-Whitney U tests for continuous variables. Sensitivity, specificity, and positive and negative likelihood ratios (LR), with confidence intervals based on exact binomial distribution, were calculated using standard methods. The area under the receiver operating characteristic curve (AUC) was used to establish the optimum cut-off points for CRP and leukocyte counts. Multilevel LRs were calculated as previously described with the use of equally spaced cut-off points [p ≤ 0.05. Calculations were performed with statistical software SPSS version 22.0 .Results are reported as means (SD) or medians (quartiles) as appropriate. Comparisons between groups were performed with f points . StatistA total of 1473 consecutive patients admitted to the emergency ward with acute respiratory symptoms were initially recruited, of whom 550 were excluded because of diagnoses other than LRTI (309), immunosuppressive condition or therapy (52), or no hospitalization requirement (189). Therefore, 923 patients with the final diagnoses of pneumonia (557) or other LRTI (366), namely, acute bronchitis and acute exacerbation of COPD, were included . Streptococcus pneumoniae (118), Haemophilus influenzae (23), Chlamydophila pneumoniae (7), Legionella pneumophila (5), influenza A (5), Pseudomonas aeruginosa (4), Mycobacterium tuberculosis (4), Mycoplasma pneumoniae (2), and one for each of Moraxella catarrhalis, Staphylococcus aureus, Escherichia coli, and Enterococcus faecium. In comparison with other LRTI, patients with pneumonia were younger and had fewer comorbid conditions, higher temperatures, and both higher blood leukocyte counts and serum CRP levels. Additionally, more patients with pneumonia required admission to the intensive care unit, although in-hospital mortality was similar between groups  and microorganisms were found in 171 (30.7%), as follows:n groups .9/L , and serum CRP > 150 mg/L .In a logistic regression model, only 4 variables were independently related to pneumonia diagnosis: under 70 years of age , temperature > 38°C , leukocyte count > 15 × 10p < 0.01). The CRP reached AUC of 0.84 (95% CI 0.82–0.87) to distinguish pneumonia from other LRTI. The operating characteristics of different cut-off serum CRP values are shown in Serum CRP levels according to disease etiologies are shown in p = 0.70).It was also observed that CRP levels were not related to the variable “days of symptoms.” Indeed, median CRP values in patients with ≤2, 3–5, and ≥6 days of symptoms were 107 mg/L (45–196), 127 mg/L (45–214), and 123 mg/L (43–222), respectively , whereas CRP intervals below 75 mg/L were associated with LR < 0.2 (for which pneumonia is unlikely). Between these intervals, the serum CRP did not provide useful clinical information.One drawback of the study was that detailed information on physical signs was not provided. However, previous studies have shown that there are no findings from the history or physical examination capable of confidently ruling in or out the diagnosis of pneumonia . Also, oThe results of our study suggest that the routine use of serum CRP levels in hospitalized patients with acute respiratory symptoms can help clinicians to differentiate pneumonia from other respiratory infections. Indeed, serum CRP levels above 200 mg/L or below 75 mg/L make the diagnosis of pneumonia likely or unlikely, respectively. A further prospective validation of CRP ranges in an independent population is warranted.


This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism.P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta ( Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway. Chronic kidney disease (CKD) is defined by reduced glomerular filtration rate or kidney damage, which affects 10–15% of residents in the US, Europe, and Asia . The impQuercetin is an important dietary flavonoid, which is present in red onions, apples, berries, citrus fruits, tea, red wine, and many Chinese herbs . NumerouTo explore more detailed evidence for the protective effects of quercetin on chronic renal failure, we have designed this study to investigate the effects of quercetin on the development of VC in adenine-induced chronic renal failure rats.All experimental procedures meet all applicable standards for the ethics of experimentation and research integrity and are approved by the Animal Experimentation Ethics Committee of Harbin Medical University. The chronic renal failure model was prepared as described . Briefly −80°C until serum analysis within a short period of time. Moreover, thoracic and abdominal arteries of the rats were obtained and flushed with cold phosphate-buffered saline (PBS). Aorta arch was fixed with 10% phosphate-buffered neutral formalin  for histology and the remaining artery tissue was used for other experiments .At the end of week 6, all rats were sacrificed by bloodletting after an overnight fast and pentobarbital anaesthesia. Blood samples were obtained from the abdominal aorta and centrifuged (3000 rpm for 15 min). Serum was then obtained, aliquoted, and stored atThe serum uric acid (UA) and creatinine (CRE) were determined by a ROCHE Modular P800 Automatic Biochemical Analyzer . Serum superoxide dismutase (SOD), malonaldehyde (MDA), and glutathione peroxidase (GSH-Px) were detected by commercial kits using enzymatic methods .3 and diluted with a blank solution . The calcium was determined by atomic absorption spectrophotometry at 422.7 nm . For the alkaline phosphatase (ALP) activity, about 10 mg (1 cm) abdominal aortic tissue was incubated with isotonic PBS  to prepare tissue homogenates. After centrifugation for 10 min at 4°C, 1600g, the supernatant was collected and ALP activity was determined by the use of an ALP detection kit .Calcium content in the aorta was measured as previously described . Briefly3 for 30 min. Specimens were then counterstained with safranin (red staining) and examined under a light microscope.Von Kossa calcium staining was applied to determine calcium in aorta. A 1 cm segment of aorta was excised and fixed with 10% phosphate-buffered neutral formalin  for 24 h; then samples were dehydrated in a graded series of ethanol solution and embedded in paraffin . SectionFor the hematein-eosin (HE) staining, aortic tissues were placed in formalin for no more than 24 h and then fixed in neutral-buffered formalin solution, dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin. Then, these blocks were sectioned using a microtome, dehydrated in graded alcohol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination.β-actin band intensities and p-p38MAPK to p38MAPK band intensities represented the iNOS or p-p38MAPK protein relative content, respectively, while β-actin or p38MAPK served as the internal reference.INOS, p38MAPK, and p-p38MAPK expressions of aorta were determined by Western blot analysis. Antibodies against iNOS, p38MAPK, and p-p38MAPK were purchased from Cell Signaling . Protein from aorta was extracted with a RIPA lysis buffer, and nuclear protein extracts and total protein extracts were prepared using protein extraction kits (Biotype Institute of Biotechnology). Protein concentrations were determined by the Bradford method . Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The corresponding molecular weight zone of the membranes was blocked with 5% non-fat milk in Tris-buffered saline containing 0.2% Tween 20 for 30 min and then incubated with primary antibody at 4°C overnight and then completely rinsed and incubated with HRP-conjugated secondary antibody at a constant temperature for 2 h. The signal was amplified by color development using the ProtoBlot II AP System with a stabilized substrate . Data are presented as the relative intensity of the protein bands. Experiments were replicated at least three times, and representative blots are shown in the figures. The ratio of iNOS to P < 0.05 considered to be significant. All P values were two-sided. Statistical analysis was carried out using the SPSS software .All data are expressed as mean ± standard deviation (SD). Data were analyzed using one-way ANOVA followed by post hoc test of LSD, with P < 0.05). Treatment with quercetin significantly prevented the increase of phosphorous, UA, and CRE levels and improved status of inflammation compared to those in the adenine diet group (All P < 0.05). However, the level of serum calcium and GSH-Px was not altered.After 6 weeks of treatment, the calcium in the aorta increased significantly in groups of adenine diet and adenine diet with quercetin supplementation compared with the control group, whereas the calcium in the aorta of the adenine diet with quercetin supplementation group was significantly lower than that in the adenine diet group . SimilarSevere calcification developed in the aorta after the treatment and the histopathology study of aorta is shown in β-actin ratio significantly increased in the adenine diet and adenine diet with quercetin supplementation group compared with the control group (P < 0.05). And iNOS/β-actin ratio was lower in the adenine diet with quercetin supplementation group compared with that in the adenine diet group (P < 0.05). p-p38MAPK/p38MAPK ratio significantly increased in the adenine diet and adenine diet with quercetin supplementation group compared with the control group (P < 0.05). And p-p38MAPK/p38MAPK ratio was lower in the adenine diet with quercetin supplementation group compared with that in the adenine diet group (P < 0.05).VC is a basic pathological process generally associated with atherosclerosis, hypertension, and CKD. VC is found in 80% of vascular injuries and 90% of patients with coronary artery disease  and is rThe present study was designed to compare the adenine-induced VC to those VC models fed with quercetin in rats. Our results revealed the significantly lower severity of VC, oxidative stress, and renal function biomarkers in quercetin group at the dose of 25 mg/kg·BW/day, compared with those in the VC group. To the best of our knowledge, this is the first investigation that found quercetin exerted a protective effect on adenine-induced VC through the modulation of oxidative stress and proteins in rats. The average daily intake of flavonoids was estimated below 100 mg and quercetin, present in foods as quercetin glycosides, represents 60–75% of the total dietary flavonols plus flavones intake . This stβ (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-15 (IL-15), and tumor necrosis factor-α (TNF-α) [Quercetin is a versatile molecular. It has been reported as a free radical scavenger and also could modulate the action of several inflammatory cytokines that are of particular concern to transplant recipients, including interleukin-1 (TNF-α) . However (TNF-α)  and warf (TNF-α) . Within  (TNF-α) . Our aniOxidative stress could lead to dysfunction of various organs, including cardiovascular diseases in CKD. Quercetin could scavenge free radicals and inhibit xanthine oxidase and lipid peroxidation. It protects against oxidative damage in renal tubular cells and renal tissues . Our stu2O2 activates the p38MAPK and downstream kinases in vascular smooth muscle cells of rats and human endothelial cells, resulting in heat shock protein 27 phosphorylation. p38MAPK also mediates osteogenic differentiation and calcification of calcifying vascular cells and the effects of oxidative stress in the development of VC [MAPK is involved in the regulation of cell proliferation, development, differentiation, apoptosis, stress, and inflammation. p38MAPK is the critical conductor of the pathway and serves as downstream effectors of ROS . H2O2 acnt of VC . NO is ant of VC . ALP is nt of VC . ALP alsThe present study has some limitations that need to be addressed. First, the anti-VC mechanism of quercetin is unclear. Whether p38MAPK/iNOs pathway is the only target of quercetin is unclear, which should be clarified in in vitro studies in the future. Second, the diverse doses of quercetin were not evaluated in the current study. Third, the results of our study cannot be transferred to human as data of other species is limited; therefore, further investigations are needed using other species or humans. However, these data provide evidence for the involvement of oxidative stress as a potential mechanism of vascular calcification, and quercetin can be treated as a potential clinical drug for prevention of vascular calcification in CKD patients.In conclusion, the results of this study demonstrated, for the first time, that quercetin exerted a protective effect on vascular calcification of adenine-induced chronic renal failure rats through the modulation of oxidative stress and p38MAPK/iNOs pathway. These data provide evidence for the involvement of oxidative stress as a potential mechanism of vascular calcification and for the potential value of clinical application of quercetin to prevent vascular calcification in CKD patients. Further studies on the effects of quercetin with different doses on chronic kidney disease should be considered in humans.


Coridothymus capitatus L., Lavandula stoechas L., Lavandula angustifolia Mill., Mentha spicata L. subsp. condensata, Origanum syriacum L., Rosmarinus officinalis, Salvia fruticosa Miller., Satureja cuneifolia Ten., Satureja thymbra L., Thymbra spicata L., and Vitex agnus-castus L. and study the chemical composition and antimicrobial activity of their essential oils (EOs). Our survey showed that Lamiaceae species are mainly used against gastrointestinal disorders and microbial infections. Chemical analysis of the EOs obtained from these plants allowed us to identify seventy-five compounds describing more than 90% of the relative composition of each EO. Essential oils with high amounts of thymol and carvacrol possessed the strongest antimicrobial activity. As expected, these two compounds demonstrated an interesting antifungal efficacy against the filamentous fungus T. rubrum. Our results confirmed that some of the Lamiaceae species used in Lebanon ethnopharmacological practices as antimicrobial agents do possess antibacterial and antifungal potential consistent with their use in alternative or complementary medicine.Many Lamiaceae species are consumed in the Lebanese cuisine as food or condiment and are largely used in the traditional medicine of Lebanon to treat various diseases, including microbial infections. In this article we report the traditional medicinal uses of eleven Lamiaceae species: The evolution of resistance to currently used antimicrobial compounds is neither a surprising nor a new phenomenon; however, infections are becoming more common, more severe, and more easily transmitted. According to the WHO, many infectious diseases will become untreatable and uncontrollable in the upcoming years . Many au in vitro screening programs based on ethnobotanical approaches proved to be very efficient in validating traditional uses of medical plants and providing new ways in the search for active compounds [Aromatic plants have been recognized since antiquity and widely used as bactericides, fungicides, virucides, antiparasitics, and pesticides. Their properties are mainly attributed to their volatile oils , 6. Inveompounds . Salvia with around 900 species [ Ajuga, Coleus, and Salvia but others are widely used as culinary herbs and spices, such as sage , thyme (Thymus), mint (Mentha), oregano or marjoram (Origanum), rosemary (Rosmarinus), lavender (Lavandula), and basil (Ocimum). Mint and lavender are grown for their oil used in perfumery, cosmetics, pharmaceutical, and food industries as active ingredients or as flavour and fragrance. Medicinal properties of the Lamiaceae species are often attributed to their high content of volatile compounds.Lamiaceae (formerly known as Labiatae) is a large plant family of mostly shrubs and herbs. It is the largest family of the order Lamiales with 236 genera and more than 7,000 species, the largest genus being species . Many ar Origanum syriacum, Satureja thymbra, and Thymbra spicata associated with a mixture of Rhus coriaria L. (sumac) and sesame seeds are the main ingredients of a very popular Lebanese pizza called “manakeesh.” Others, like Rosmarinus officinalis, Coridothymus capitatus, and Salvia fruticosa, are eaten as salads and the leaves of Thymus and Origanum species are mixed with traditional Lebanese fresh cheese called “Shanklish” for their aromatic and antiparasitic properties.The Lamiaceae family is particularly well represented in Lebanon, where 136 species belonging to 29 genera have been inventoried . Many La Lavandula, Melissa, Mentha, Origanum, Rosmarinus, Salvia, Satureja, and Thymus. These plants are highly aromatic due to the presence of external glandular structures that produce volatile oil [Indigenous Lamiaceae are also frequently used in Lebanon for medical purposes and are marketed by herbalists. These include the generatile oil  and theitile oil , 13, 14.However, there was no previous study documenting the folk medicinal usage of the Lamiaceae species in Lebanon. We have conducted a large-scale survey on the traditional medicinal uses of Lamiaceae species in different regions of Lebanon. A part of this work has already appeared in a congress report .Coridothymus capitatus L. Reichenb. Fil., Lavandula stoechas L., Lavandula angustifolia Mill., Mentha spicata L. subsp. condensata, Origanum syriacum L., Rosmarinus officinalis L., Salvia fruticosa Miller., Satureja cuneifolia Ten., Satureja thymbra L., Thymbra spicata L., and Vitex agnus-castus L.), (2) to investigate the chemical composition of the EOs extracted from these species, (3) to evaluate the antimicrobial activity of these EOs and their major constituents against opportunistic human pathogens, and (4) to correlate in vitro results with the ethnopharmacological uses of these plants.The main objectives of the present study were (1) to report on the traditional uses of eleven Lamiaceae species most used in the Lebanese folk medicine  of the six governorates (“mohaafazah”) of Lebanon .Ethnobotanical and ethnomedicinal information was obtained between the years 2002 and 2008 from 325 interviewees from 223 villages covering all the districts of Lebanon. Most of the participants interviewed were herbalists (“Attarin” or “dabbous”), shepherds, farmers, folk healers, or older experienced people and midwives (“daye”) between 40 and 70 years old. The people interviewed declared that their knowledge about the traditional medicine was transmitted mainly orally from older generations. Interviewees were accompanied to the field individually where they would point out the herbs that have been using to cure the mentioned disease. This was also to confirm plant identification (vernacular names can be different in different regions of Lebanon) and obtain fresh samples for herbarium voucher. When the fertile part was not available, the plant was visited again at the appropriate time to obtain a fertile sample.The obtained information was cross-checked with that of other informants, and also, after a week, the interview was repeated with the same person. Only medical usages of plants given at least by three separate informants have been reported in this investigation. It was verified that each informant is able to recognize the mentioned plant species in the wild.The questionnaire form was based on the botanical and ethnopharmacognosic survey of traditional medicine plants, suggested by WHO . The colPlant material was collected in several locations throughout Lebanon and voucher specimens were deposited at the Herbarium of Botany of the U.S.E.K., Lebanon. Specimens of the Lamiaceae species were collected as described in http://ww2.bgbm.org/mcl/). We followed the new phylogenetic classification APG II [The plants have been identified based on the “Nouvelle flore du Liban et de la Syrie” (Mouterde)  and the n APG II  in orderThe essential oils (EOs) were obtained by hydrodistillation performed for 3 h using a Clevenger-type apparatus according to the European Pharmacopoeia . Yields µm film thickness or a fused silica HP Innowax polyethylene glycol capillary column . Helium was the carrier gas (0.7 mL/min). The column temperature was initially set to 35°C before being gradually increased to 85°C at 5°C/min, held for 20 min at 85°C, raised to 300°C at 10°C/min, and finally held for 5 min at 300°C. Diluted 1 µL samples  were injected at 250°C manually and in the splitless mode. Flame ionisation detection (FID) was performed at 310°C.Analytical gas chromatography was carried out on a Thermo Electron Corporation gas chromatograph fitted with a DB-5 MS capillary column (30 m × 0.25 mm) with 0.1 µL of each oil sample. A fused silica capillary column DB-5 MS  or a fused silica HP Innowax polyethylene glycol capillary column  was used. Helium was the carrier gas (0.7 mL/min). The oven temperature program was identical to that described in The GC/MS analyses were performed using an Agilent 6890 gas chromatograph coupled with 5975 Mass Detector. The 7683B autosampler injected 1  n-alkanes (C8 to C24) analysed under the same operating conditions. Further identification was obtained by comparing their mass spectra on both columns with those provided in the NIST and Wiley 275 libraries, our homemade library constructed with pure compounds, and EOs of known composition or with mass spectra from the literature [Most constituents were identified by gas chromatography by comparing their retention indices (RI) with those from the literature , 20 or wterature , 21. The Escherichia coli ATCC 25922, the Gram (+) bacterial strain Staphylococcus aureus ATCC 29213, the yeast Candida albicans ATCC 10231, and a clinical isolate of the dermatophyte Trichophyton rubrum SNB-TR [The antimicrobial activity of the essential oils was investigated against the Gram (−) bacterial strainμg/mL. The microplates were incubated at 37°C for 24 h for bacteria, 48 h for yeasts, and 5 days for dermatophytes. The minimal inhibitory concentrations (MIC) refer to the lowest concentrations preventing visible microbial growth (μg/mL) were used as reference antibiotics, while itraconazole (16—0.03 μg/mL) and fluconazole (64—0.125 μg/mL) were used as positive controls for the antifungal assays. The antimicrobial standards were purchased from Molekula, Dorset, UK, and the pure terpenes from Sigma-Aldrich, Lebanon.The antimicrobial activity of the EOs was measured using a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines –26. The l growth . Oxacill Origanum syriacum and Salvia fruticosa are the two most cited plants. It should be noted that the same vernacular name is sometimes used for different species. For example, the appellation “Za'atar” is used for Satureja thymbra, Thymbra spicata, Coridothymus capitatus, and Origanum syriacum.This investigation showed that Lamiaceae species and especially the eleven ones reported in this study are still frequently used in Lebanon as herbal remedies . OriganuThe plant parts used most frequently are the flowering tops and the leaves (34.6% each), followed by the stems (11.5%), flowers (7.7%), whole plant, fruits, and seeds (3.8% each) . per os or internally (71.8%) although the majority of the EOs are applied externally (57.1%). The main modes of preparation are infusion (40.0%), decoction (17.5%), EO application (17.5%), and food consumption (15.0%)  . The conAmong the multiple medical usages of these plants, the eleven Lamiaceae species are mainly used to treat gastrointestinal disorders and microbial infections . Almost  Rosmarinus officinalis and Origanum syriacum was mentioned by the largest number of districts , while the medicinal use of Coridothymus capitatus, Vitex agnus-castus, and Salvia fruticosa was localized in certain regions of Lebanon . Thymol and carvacrol were present in a high relative proportion in several EOs of these Lebanese chemotypes. Thymol was the major constituent of Origanum syriacum (74.4%), Satureja thymbra (44.5%), and Coridothymus capitatus (29.3%) EOs, while carvacrol was highly abundant in the EOs of Satureja cuneifolia (69.5%), Thymbra spicata (64.0%), and Coridothymus capitatus (29.3%). All of these species were also rich in p-cymene andγ-terpinene. The oils of Salvia fruticosa, Rosmarinus officinalis, and Vitex agnus-castus were essentially composed of eucalyptol representing 57.3%, 21.5%, and 20.5% of the total oils, respectively. Pulegone was the main component of the EO of Mentha spicata accounting for 78.7% of the oil. Wild and cultivated lavender EOs were significantly different; Lavandula stoechas (wild lavender) was mainly composed of α-fenchone (26.2%) while linalool (45.8%) was the major constituent of Lavandula angustifolia (cultivated lavender).The yields  obtained by hydrodistillation ranged from 0.3% fromtal oils . It was  Vitex agnus-castus, Lavandula stoechas, and Salvia fruticosa have a very similar chemical composition compared to other countries [ Lavandula angustifolia and Coridothymus capitatus Lebanese EOs are different than those from other regions. According to our study, Lavandula angustifolia EO contains a lower relative proportion of linalyl acetate [ Coridothymus capitatus is richer in thymol [ Rosmarinus officinalis is similar to that reported in Greece [ Satureja thymbra [ Origanum syriacum [ Thymbra spicata [ Satureja cuneifolia [ Mentha spicata oil reported in our study belongs to the pulegone chemotype, while a carvone chemotype has also been described in other regions [The EOs ofountries –48. Lava acetate  and Corin thymol , 51. Then Greece  but is dn Greece , 54. Bot thymbra , 56 and syriacum  or those spicata , 58 and neifolia , 60. Lik regions , 62.µg/mL or below [ T. rubrum SNB-TR1 was the most sensitive, followed by the Gram (−) bacterium S. aureus and the yeast C. albicans, whereas the Gram (+) bacterium E. coli was more resistant. The oil of Coridothymus capitatus was the only EO to possess a significant antibacterial activity against E. coli with a MIC value of 128 µg/mL. This EO is rich in both thymol and carvacrol . Overall, plants EOs with high relative amounts of thymol and/or carvacrol were the most active against S. aureus, T. rubrum, and C. albicans with MIC values in the range of 64–128 µg/mL.The minimum inhibitory concentrations (MICs) of the Lamiaceae essential oils are presented in or below . The cli S. aureus and T. rubrum. Indeed, among the tested terpenes, only thymol and carvacrol showed significant antibacterial activity against S. aureus (MIC 128 µg/mL). Against T. rubrum, these two compounds were also very active (MIC 32 µg/mL), followed by p-cymene (MIC 64 µg/mL). The antimicrobial potential of a combination of thymol and carvacrol in equal proportions was also measured but no synergistic effect was detected. The MIC of the combination of these two compounds was identical to that of each separate compound. The other important constituents, that is, camphor, eucalyptol, linalool, γ-terpinene, and α-fenchone, did not show any significant antimicrobial activity . Our results corroborate the antimicrobial indications for. rubrum .In conclusion, the ethnobotanical and ethnopharmacological survey of the Lamiaceae plants confirmed that their medical values are widely acknowledged among herbalists and rural communities and that this family of plants is still frequently used in the traditional medicine of Lebanon to treat many ailments, including microbial infections. in vitro antimicrobial activity of some Lebanese Lamiaceae EOs against human pathogens. The antimicrobial potential of these EOs originates from their high content in either thymol or carvacrol. These results validate the traditional antimicrobial use of Lamiaceae and lead us to believe that the use of the most active ones of these plants (or their EOs) can be promoted for the treatment of mycoses under topic applications. It should be noted that Lamiaceae herbs have high consumption in the traditional medicine in Lebanon. This might indicate innocuousness [This study highlights theuousness  although


Mononuclear cells were mildly increased in the cerebrospinal fluid (CSF). Brain magnetic resonance imaging (MRI) showed high intensity along sulci of the frontal and parietal lobes. Brain biopsy revealed lymphocyte and plasma cell infiltration in the meninges, confirming the diagnosis of rheumatoid meningitis. After steroid pulse therapy, seizures resolved and clinical findings improved. When etanercept was replaced by tocilizumab, rheumatoid meningitis did not recur. Although TNF- The common neurological complications of rheumatoid arthritis are peripheral neuropathies, such as carpal tunnel syndrome and myelopathy due to atlantoaxial subluxation. It is very rare to find complications involving pathologies inside the cranium. In the central nervous system, rheumatoid arthritis targets the meninges, resulting in rheumatoid meningitis . We repoA 65-year-old man came to our clinic in September 2012 with a complaint of transient loss of consciousness. His medical history included prostatomegaly, hypertension, and idiopathic thrombocytopenic purpura. He had been diagnosed as having rheumatoid arthritis in February 2012, which was well controlled by methotrexate (12 mg/week), etanercept (50 mg/week), and prednisolone (2 mg/day). There was no notable family history. He was neurologically intact. There were no abnormal findings on brain computerized tomography (CT) and MRI. Electroencephalography (EEG) showed intermittent bursts of bilateral delta activity and infrequent high-amplitude sharp waves predominantly in the frontal regions. Carbamazepine (200 mg/day) was administered for suspected epilepsy, which was later changed to sodium valproate (200 mg/day) due to the occurrence of rash.After this episode, he showed three transient episodes such as loss of consciousness, generalized tonic convulsion followed by consciousness disturbance, and dysarthria associated with left leg weakness during the following 6 months. Each episode led to hospital admission, but the patient had no neurological symptoms when he was investigated. Brain MRI showed abnormal signals and contrast enhancement in sulci of the left frontal and parietal lobes, but no definite epileptiform activity was found on EEG. CSF revealed nonspecific mild pleocytosis. At this point, chronic meningitis was diagnosed, but the cause was not confirmed.μL), without elevated white blood cell count. On biochemical examination, hepatic enzymes and kidney function were found to be normal, and there were no findings suggesting inflammation. CK was mildly elevated (346 IU/L). Tumor markers were all normal, and sIL2R was mildly elevated at 555 U/mL (reference range: 145–519). Immunological examination showed 12 IU/mL for RF , 80 times for ANA , 297 U/mL for SS-A antibodies , and 18.6 U/mL for SS-B antibodies . PR3-ANCA and MPO-ANCA were normal, and anti-CCP antibodies were 275 U/mL . CSF examination showed cell count of 12/μL (mononuclear cells: 12/μL), protein levels of 32 mg/dL, and glucose levels of 55 mg/dL (blood glucose 70 mg/dL). IgG index was 1.7, and cytodiagnosis was negative. CSF culture and PCR for acid-fast bacterium were negative.He was admitted for the fourth time in June 2013, because of a seizure continuing for several minutes and persistent disturbance of consciousness. The patient was slightly disorientated. Examinations of the cranial nerves and motor and sensory systems and deep tendon reflexes and coordination were normal. Serological examination showed that blood platelet count was decreased  was substituted for sodium valproate, because the patient had another seizure lasting for one hour. New findings on brain CT, MRI, or EEG were not observed at that time.A brain biopsy from the lesion in the right frontal lobe showed invasion of lymphocytes and plasma cells into the superficial brain layers, resulting in necrosis of the brain surface . The lymBased on the pathological findings, we diagnosed the patient as having rheumatoid meningitis and performed 2 courses of steroid pulse therapy (methylprednisolone 1000 mg/day × 3 days). After the steroid pulse therapy, seizures disappeared and cell counts in the CSF returned to normal. On brain MRI, abnormal signals in the sulci improved , and theThere are no specific biomarkers for rheumatoid meningitis, and brain MRI and biopsy are essential for its diagnosis . On braiα inhibitors (α inhibitor treatment, which improved with discontinuation of the treatment and steroid pulse therapy. It is possible that TNF-α inhibitors induce rheumatoid meningitis. It has been pointed out that TNF-α inhibitors can create rheumatoid nodules in a variety of tissues, probably through multiple mechanisms, including modifications of the expression of other cytokines [To our knowledge, there have been only four case reports of rheumatoid meningitis during treatment with TNF-hibitors . Huys etytokines , 11. Anoytokines . It is pFurthermore, it is widely known that methotrexate can cause aseptic meningitis, particularly in intrathecal administration . In the α inhibitors, if seizures and disruption in consciousness occur, biopsy and steroid therapy should be considered immediately, as it is possible that the TNF- α inhibitors can induce rheumatoid meningitis.Even when the rheumatoid arthritis is well controlled by TNF-


Proteobacteria (42.35%), Actinobacteria (15.61%), Acidobacteria (13.32%), Bacteroidetes (8.43%), and Gemmatimonadetes (6.0%). The dominant fungi phyla were Ascomycota (40.39%), Basidiomycota (38.01%), and Zygomycota (16.86%). The results of Canonical Correspondence Analysis (CCA) and Redundancy Analysis (RDA) based on land-use types displayed groups according to the land-use types. Furthermore, the bacterial communities were mainly organized by soil organic carbon (SOC). The fungal communities were mainly related to available phosphorus (P). The results suggested that the changes of land use type generated changes in soil chemical properties, controlling the composition of microbial community in the semiarid Loess Plateau region. The microbial community could be an indicator for soil quality with respect to ecological restoration.Similar land-use types usually have similar soil properties, and, most likely, similar microbial communities. Here, we assessed whether land-use types or soil chemical properties are the primary drivers of soil microbial community composition, and how changes in one part of the ecosystem affect another. We applied Ion Torrent sequencing to the bacterial and fungal communities of five different land-use (vegetation) types in the Loess Plateau of China. We found that the overall trend of soil quality was natural forest > plantation > bare land. Dominant bacterial phyla consisted of  Land-use change is one of the main factors affecting the biodiversity and functioning of terrestrial ecosystems32, even in the winter1415To remedy adverse impacts to ecological environments, vegetation restoration has been conducted on the Loess Plateau of China, leading to changes in land-use patterns and above- and below-ground environments. These fluctuations, in turn, have profound indirect and direct effects on soil microorganisms691In this study, we used the Ion Torrent sequencing approach to explore the vast uncultivated soil microbes that are directly related to the effects of land-use change on soil chemical properties. This study investigated the effects of (1) land-use types, (2) seasonality, and (3) soil chemical properties on fungal and bacterial communities in five different land-use types including natural and plantation forests, shrub land, and bare land. We aimed to understand whether land-use types and/or soil chemical properties are the primary drivers of soil microbial community structure.Quercus liaotungensis), the typical climax forest type, (2) oriental arborvitae  which occurs as a small stand, (3) shrub land including species of early lilac (Syringa oblata), rose (Rosa hugonis), and Caragana microphylla, (4) black locust (Robinia pseudoacacia), a fast-growing introduced species which contributes to major plantations in the regionThe study was conducted at Mt. Gonglushan in the southern suburb of Yan’an City, Shaanxi Province, in the central part of Loess Plateau . This area is a fragile semiarid ecosystem and it contains one of the largest global loess areas. The site is also in the ecological transition zone between forest and forest-steppe ecosystems. Indigenous forests are present in sites with relatively little human disturbance. The mean annual precipitation and air temperature were 504.7 mm and 10.1 °C, respectively, based on a 40-year average (1971–2010) recorded by the city meteorological stationSoil sampling was conducted in August 2013 and February 2014, capturing the growing season and the dormant period, respectively. In this region, the plant growing period contains months from April to October, with June–August being the summer and December–February being the winter. The land-use types and their seasonal abbreviations in this study are as follows: oak forest (WO and SO), oriental arborvitae forest (WA and SA), black locust plantation (WL and SL), shrub land (WS and SS), and bare land (WB and SB) in winter and summer, respectively. In each land-use type, three soil samples were collected from locations approximately 50 m apart, generally on upper, middle, and lower locations of the land slope. At each location, a mixture of the top 20 cm of soil was collected from five sampling points along a “z”-type curve, and a 1 kg sample was obtained by the quartering method. After the roots and debris were removed, the samples were brought to the laboratory and preserved in two conditions: (1) stored at −40 °C in the refrigerator for molecular analysis and (2) sieved through 2 mm mesh and kept field-moist in a cooler at 4 °C® DNA Isolation Kit , according to the supplier’s recommended protocol.For each location, genomic DNA was extracted from a 0.3 g soil sample using the PowerSoilFor bacteria, the V4 region in 16 S rRNA gene was examined after the Polymerase Chain Reaction (PCR) with primers of S-D-Arch-0519-a-S-15 (CAGCMGCCGCGGTAA) and S-D-Bact-0785-a-A-21 (GACTACHVGGGTATCTAATCC)The PCR for bacteria was performed using a Veriti thermal cycler (Applied Biosystems) with initial denaturation at 94 °C for 3 min, 35 cycles of 30 s at 94 °C for denaturation, 30 s at 50 °C for annealing, and 1 min at 68 °C for elongation. The thermal profile of the first PCR for fungi was initial denaturation at 94 °C for 3 min, 20 cycles of 30 s at 94 °C for denaturation, 30 s at 48 °C for annealing, and 1 min at 68 °C for elongation. The second PCR was performed using 2.0 μL of the first PCR product diluted 10 times as the temperate DNA with the following thermal conditions: initial denaturation at 94 °C for 3 min, 35 cycles of 30 s at 94 °C for denaturation, 30 s at 50 °C for annealing, and 1 min at 68 °C for elongation. The PCR was performed twice for each sample and the amplicons were visualized on 0.7% agarose gels stained with SYBR Green I .The successful amplicons were pooled, precipitated, resuspended with isopropanol, and then cleaned using the QIAquick Gel Extraction (Qiagen) and the Agencourt AMPure XP , according to the manufacturer’s instructions. Quantitate purified each PCR product with the Qubit dsDNA HS Assay Kit on a Qubit fluorometer 2.0 . The library mix was then prepared with an equal amount of DNA from each barcode sample. Size distributions and DNA concentrations were measured by Agilent 2100  and diluted to 12 pM before the amplification and purification using Ion OneTouch 2 system with Ion PGM temperate OT2 400 Kit . Sequencing was performed using Ion PGM sequencer with Ion PGM sequencing 400 Kit and a 316 V2 tip .Sequence data was generated in a standard flowgram format (sff) of each barcode sample. First, the primer sequence was eliminated using Cutadapt ver.1.3To assess microbial diversity among samples in a comparable manner, a normalized dataset was used for subsequent analyses, and the lowest sequence number for all sample subsets  was randomly selected. The diversity and evenness indices (Shannon and Simpson) were calculated at genetic distances (D) of 0.03. Shannon emphasizes the richness component of diversity, and Simpson emphasizes the evenness component. For both bacteria and fungi, there were only a few sequences that could not be identified to the phylum level. However, rarefaction analysis showed that all rarefaction curves were approaching smoothness with the increase of read number. It is, therefore, reasonable to infer that the obtained sequences covered the majority of the entire microbial community.P < 0.05). To determine the relationships between soil chemical properties and diversity indices, Pearson correlations were calculated. In order to test a significant shift in microbial community composition based on land-use types, Adonis was used. The length of gradient was calculated firstly with the Detrended Correspondence Analysis (DCA) to determine which model would be most suitable for correlation analysisA non-parametric  test was applied to determine whether there were significant differences in diversity indices among land use types , Actinobacteria (15.61%), Acidobacteria (13.32%), Bacteroidetes (8.43%), and Gemmatimonadetes (6.0%)  existed in most soils, and 29 other rare phyla were also distinguished. Alphaproteobacteria was the most abundant Proteobacteria, followed by Deltaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Thermoleophilia, Actinobacteria, and Acidimicrobiia were the most abundant groups of Actinobacteria. The relative abundance of Proteobacteria was greater in natural vegetation  than it was in the bare land and black locust; Acidobacteria showed a reverse trend. The dominant phyla were the same across land-use types, but their relative abundances were different.The dominant bacterial phyla contained s (6.0%) . In addiP < 0.01). Soil chemical properties account for variation in bacterial community. Each index of soil chemical properties showed significant association with bacterial community composition (P < 0.001). The main factor for grouping the bare land sites was pH with higher abundance. SOC, TN, available P, and C/N tended to have lower values and were negatively associated with the bare land but positively associated with the oak forest sites, while the black locust sites were grouped with higher available K. In total, SOC exerted the largest effect on the distribution of bacterial communities.The results of the RDA of soil chemical properties and bacterial community composition showed that groups according to land-use types . Bare laP < 0.05) .For bacterial, there were diversity (Shannon) and evenness (Simpson) differences between black locust and bare land in the summer, but others has no difference ( < 0.05) . The divAscomycota (40.39%), Basidiomycota (38.01%), and Zygomycota (16.86%). Sordariomycetes, Dothideomycetes, and Lecanoromycetes were the most abundant groups of Ascomycota. Agaricomycetes and Tremellomycetes were the most abundant groups of Basidiomycota. Most of the Zygomycota were incertae. Ascomycota was the most abundant in black locust, and bare land (but summer bare land samples), but was least abundant in the oak forest. The oak forest had the most Basidiomycota, while the black locust had the least. There were no differences in fungi diversity indices between land-use types (The majority of dominant fungi phyla  belongedse types  and no cP < 0.01). Meanwhile, soil chemical properties exerted a significant effect on the composition of fungal community. SOC, total N, available K and P showed significant association with fungal community composition (P < 0.001), and the significance of soil pH and C/N is 0.01. The main grouping factor for the bare land type was pH, and the oak forest type was clustered in the area with high SOC, TN, P, and C/N. Available K showed a positive association and higher abundance in the black locust. In total, available P content was the main factor influencing the fungi community distribution.From the CCA with all the soil chemical properties, the fungal community under the similar land use types typically clustered together . The barA previous study showed that similar land-use types have similar soil chemical propertiesProteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes. This is different from the results of Xiong, et al.Bacteroidetes and Firmicutes were dominant in alkaline lake sediments.Similar to the results of early surveys6et al.Acidobacteria increased with soil pH. However, other studies showed that the relative abundance of Acidobacteria was typically negatively correlated with soil pH3536Acidobacteria only started to decrease markedly when the pH was below 5.53536Microbial community structure was driven by both land-use type and soil chemical properties. The importance of soil chemical properties in shaping microbial communities has been established by a number of studies. As general rule, soil pH plays a key role in controlling the distribution of microbial communities343537Proteobacteria, Acidobacteria, and Actinobacteria) were greatly responsive to SOC content. The results obtained in this study are in line with the general framework that uses the terms copiotroph and oligotroph to describe microbial communities with ecological attributes typical of r- and K- strategists, respectivelyProteobacteria increased with SOC and were the most abundant in SOC-rich oak forest soils. On the contrary, there were many more oligotrophic groups of Acidobacteria, which is expected to be the first bacteria to colonize nutrient-poor substrates in areas with low resource availabilityAcidobacteria are less abundant and Proteobacteria are more abundant in soils with high resource availability31Proteobacteria/Acidobacteria ratio reflected the soil chemical properties, with lower ratios occurring in oligotrophic soils. Our study further supports this finding because we found the highest ratios in the oak forest site and the lowest ratios in bare land and black locust forest. Therefore, it is likely that land-use and soil nutrient conditions are the major explanatory variables of bacterial community structure. In addition, bacterial community structure can also reflect the soil nutrient status in Loess Plateau, China.The dominant bacteria . Zygomycota, as saprotrophic species, are responsible for decomposing plant litter, alter soil chemical properties43Leckie While seasonal changes in microbial community structure have been noted53555759et al.Jangid, In conclusion, similar land-use types usually have similar soil chemical properties and, similar microbial communities in a specific area. Our results support previous studies that soil chemical properties and land use types have crucial influence on the composition and divertity of microbial community. The changes of land use type generated soil chemical properties, both of which played a significant role in controling the composition of microbial community. In turn, the microbial community composition might be an indicator of changes in soil chemical properties or responses to land use types after ecological restoration on the Loess Plateau.How to cite this article: Tian, Q. et al. Land-use types and soil chemical properties influence soil microbial communities in the semiarid Loess Plateau region in China. Sci. Rep.7, 45289; doi: 10.1038/srep45289 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Multiple studies have reported evidence of an inverse association between parity and bladder cancer risk. However, a comprehensive and quantitative assessment of this association has never been conducted. We conducted this study to clarify this issue.Systematic search of PubMed and Embase was performed to identify all the studies. Studies were selected based on strict screening with inclusion and exclusion criteria. Summary relative risks (RR) with 95% confidence intervals (CI) were calculated by using a fixed-effect model, and the generalized least squares trend estimation was employed to compute study-specific RR and 95% CI per live birth increase. Heterogeneity and publication bias were also evaluated.Twelve studies  were eligible in this meta-analysis. The pooled RR of bladder cancer for parous versus nulliparous women was 0.76 (95% CI: 0.70–0.82). Results were similar in the studies that adjusted for BMI, cigarette smoking , and age . The dose-response meta-analysis showed a lower bladder cancer risk  for each live birth increase in parous women. No evidence of publication bias or significant heterogeneity was detected in the above-mentioned analyses.The finding from current meta-analysis suggest that parity may be related to decreased risk of bladder cancer.The online version of this article (doi:10.1186/s12885-016-3023-5) contains supplementary material, which is available to authorized users. Bladder cancer (BC) is one of the most common genitourinary malignancies in the United States, with an estimated 76, 960 newly diagnosed cases and 16 390 deaths in 2016 [Although the exact biological mechanisms for the gender disparity in BC development and progression are not well established, some studies have suggested that sex hormones and their receptors may contribute to this difference –5. DurinTherefore, we find it necessary to further assess the relationship between parity and BC risk by conducting a meta-analysis of current epidemiologic studies and providing a quantitative dose-response analysis.This study protocol was approved by the institutional review board at the West China School of Medicine before initiation and don’t need ethical standard statement.We searched online databases MEDLINE and EMBASE for the studies assessing the relationship of parity and BC risk before Sep 2016, using the following terms with every possible combination considered: parity, pregnancy, reproductivity, fertility, bladder, genitourinary tract, cancer, carcinoma, tumor. We searched the references of all eligible publications to identify additional relevant publications. Studies were included if they met the following criteria: (1) the study had a prospective or case-control design; (2) the study evaluated the relationship between parity and BC risk; (3) data were provided or would allow the calculation of relative risk (RR) or odds ratio (OR) and a 95% confidence intervals (CI). Exclusion criteria were as follows: (1) was not involved with the associations between parity and BC risk; (2) non-human studies, reviews, and comments; (3) studies not reporting primary outcomes; (4) studies based on overlapping patients. When duplicate publications were identified or data in separate publications originated from the same cohort, we used the publication with the largest number of cases and the most applicable data.Data were independently extracted from the included studies by two investigators (YJB and XMW), and disagreement was settled by discussion with the third investigator. The following data were extracted from each study: first author’ name, publication year, country of study, participant demographics, sample size, duration of follow-up or study period, parity number categories, corresponding OR/RR (with their 95% CIs) for each category, and variables adjusted for in the analysis.The methodological quality assessment was conducted for each of the included studies using the Newcastle-Ottawa Scale quality assessment  on three2 statistics [P < 0.10 as evidence of heterogeneity. An I2 score exceeding 50% is considered to indicate the presence of heterogeneity. The random effect model was used to provide summary estimations if there was heterogeneity, or else the fixed effect model was used. Heterogeneity was explored by stratified analysis. Publication bias was explored using Begg’s and Egger’s test [P < 0.05 indicated the existence of publication bias.Given that bladder cancer is a rare disease, the OR was assumed to be the same as the RR and we therefore report all effect sizes as RR for simplicity. We calculated summary RRs and 95% CIs associated with parity and BC risk, if the study considered nulliparous as a reference. Between-study heterogeneity was evaluated with Q and Iatistics . For ther’s test , 17 and P value for nonlinearity was computed by testing the null hypothesis that the coefficient of the second spline was equal to zero. Statistical analyses were performed with Stata version 12.0 software .We carried out a dose-response meta-analysis using the method proposed by Greenland et al.  and OrsiThe article selection process is shown in Fig. 2 = 0%, p = 0.795, Fig. 2 = 0%, p = 0.562). For women who have ever smoked, the summary RR was 0.90  for ever versus nulliparous, without heterogeneity .All included studies in present study investigated the association between ever parity and BC risk. The summary multivariable-adjusted RR of BC for ever versus nulliparous was 0.76 , without heterogeneity (I2 = 17.7% (P = 0.295). The second group (3–4 births) contained seven reports, compared with nulliparous, the overall RR of BC was 0.79 (95% CI 0.68–0.91) with I2 = 0% (P = 0.997). The third group (≥5 births) contained six reports, compared with nulliparous, the overall RR of BC was 0.76 (95% CI 0.66–0.88) with I2 = 0% (P = 0.994).Six cohort and six case-control studies investigated possible association between parity and BC risk. The results of the effects of different parity number on BC risk are presented in Table 2 = 12.79%, P = 0.7832). Compared with nulliparous individuals, the pooled RRs (95% CI) of BC were 0.84 (0.76–0.94), 0.76 (0.67–0.87), 0.74 (0.65–0.85), 0.75 (0.66–0.85), and 0.76 (0.65–0.88) for 1, 2, 3, 4, and 5 live births, respectively (Fig. Dose-response from nine studies , 20–23 sNo evidence of a significant publication bias was observed in our analyses as assessed using the Egger’s test or Begg’s test and no asymmetry was seen in the funnel plots of included studies.To avoid the influence of cigarette smoking, we evaluated the influence of smoking status by adjustment in the eight reports that considered smoking. However, when data were stratified by adjustment for smoking, we did not find a significant difference between summary RRs that were adjusted and those that were not adjusted for smoking Table . MoreoveNumerous epidemiological studies have focused on BC risk factors in order to explain the gender disparity in BC incidence, while there is also a great interest in identifying factors that affect the risk of BC among women. Although previous studies have reported on the associations between parity and BC, to the best of our knowledge, no other earlier studies have clearly documented a dose-response pattern between parity number and BC risk. In present study, we conducted a dose-response meta-analysis to characterize the association between parity and BC risk. Findings from the present study indicated that parous was significantly inversely associated with BC risk compared with nulliparous, especially for non-smokers. Overall, the risk of BC decreased by 24% for women who had given birth and 53% for those who had never smoked and were ever parous.Although the exact mechanisms underlying this inverse association between parity and BC risk are not completely established, several potential mechanisms have been proposed. First, during pregnancy, estrogen and progesterone levels increase drastically and induce substantial alterations to the structure, function, and histology of the bladder , possiblSecond, smoking habits may have influenced the incidence of BC in women with parity. It is well known that prenatal cigarette smoking exposure may cause detrimental effects on reproductive health . TherefoThird, structural changes in the pelvic organs and pelvic floor organization following pregnancy may induce a decreased risk for BC in parous women. Childbirth is an established risk factor for lower urinary tract dysfunction, such as increased urinary frequency, stress incontinence, and overactive bladder , but theMeta-analyses of observational studies are prone to confounding, selection, and information bias as the included observational studies and can present particular challenges that may distort the results. However, our study has some strengths. First, we searched 2 electronic databases, which ensured a broad scope, and we included seven cohorts and seven case-control studies that involved a total of 6,214 cases and 2,693,350 non-cases. This large sample size should have provided sufficient statistical power to detect this potential relationship. Second, to control misclassification, we conducted a dose-response meta-analysis. This is the most comprehensive meta-analysis evaluating the associations between parity and BC. Third, no publication biases were detected in present study and outcome may be unlikely to remain unreported as studies with complex assessment of exposure.This current dose-response meta-analysis indicates that women with parity have an inversely associated BC risk compared with nulliparous women. The exact mechanism underlying this protective effect requires further investigation.


MicroRNAs have recently been verified as useful diagnostic biomarkers in various diseases. In this study, we investigated whether miR-217 is a useful diagnostic biomarker and the possible pathological mechanism of miR-217 in this disease. Patients with focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and diabetic nephropathy (DN) and control patients were enrolled in this study. The miR-217 inhibitor and mimics were transfected into human podocyte cells to investigate the pathological mechanism of miR-217 in this disease. Relevant indicators were detected and tested. Compared with control patients, miR-217 was significantly downregulated and TNFSF11 was significantly upregulated in MN. Then, miR-217 had obvious separation between patients with MN and control patients, with an AUC of 0.941, a cutoff value of <750.0 copies/ul, and sensitivity and specificity of 88.9% and 75.9%. In addition, the TNFSF11 was confirmed to be the target gene of miR-217. Finally, in in vitro experiments, the upregulation of miR-217 could decrease the expression of TNFSF11 and not induce human podocyte cells apoptosis; however, the downregulation of miR-217 could bring about an opposite change. miR-217 is a useful diagnostic biomarker and is involved in human podocyte cells apoptosis via targeting TNFSF11 in membranous nephropathy. Membranous nephropathy (MN) is a renal disease, is a common factor causing nephrotic syndrome in adults, and eventually causes end-stage renal failure for many patients –4. The pHowever, the diagnosis of MN based on clinical characteristics alone is incomplete and usually needs to be confirmed by the histological and immunohistochemical analyses under renal biopsy . In addiMicroRNAs are a kind of small noncoding RNAs, which are involved in the posttranscriptional regulation of gene expression by binding the translation section and lead to either mRNA degradation or translational inhibition and have been found to regulate crucial biological processes, including proliferation, differentiation, and apoptosis –12. SomeA previous study showed that there were 20 upregulated and downregulated microRNAs with the highest fold changes in the peripheral blood from patients with MN, and miR-217 was most significantly downregulated, with more than 10-fold reduction in the peripheral blood [84 patients with membranous nephropathy (MN), 67 patients with diabetic nephropathy (DN), 82 patients with focal segmental glomerulosclerosis (FSGS), and 72 age- and sex-matched control patients were enrolled in this study at the Sichuan Cancer Hospital, Chengdu, China, between December 2014 and February 2016. Inclusion criteria were age < 65 years, the excretion of urine albumin > 30 mg/24 h, and serum creatinine level < 3 mg/dL. MN was diagnosed according to the previously described criteria , FSGS wag for 5 minutes and plasma supernatants were also collected in free ribonuclease tubes to be centrifuged at 15,000g for 5 min for eliminating cell debris and stored at −80°C.Pathological renal tissues were collected from patients undergoing renal biopsy and peripheral blood was also collected from these patients. All samples were assigned within 2 hours after collection. The tissue samples were collected in free ribonuclease tubes and stored at −80°C. The blood samples were centrifuged at 3,000The human podocyte cell line AB8/13 was purchased from American Type Culture Collection and cultured. In brief, human podocyte cells were maintained at 33°C for proliferation and cultured at 37°C to induce differentiation for 14–16 days. An inverted microscope was used to observe the cell morphology. When the human podocyte cells grew to about 80% confluence, they were exposed to 100 nmol/L Angiotensin II  for 24 h.Synthetic miR-217 mimics, miR-217 inhibitor, and the relative controls (empty vector) were purchased from BioVectra . Synthetic microRNAs were transfected into human podocyte cells to induce upregulation and downregulation of miR-217 with Lipofectamine 2000™ , according to the manufacturer's protocol. After being infected, the cells were incubated for 8 hours and cultured in a fresh medium for further experiments.n = 2 − ΔΔCt, where ΔCt = Ct related microRNA−Ct U6 and ΔΔCt =  ΔCt experimental−ΔCt control.Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen), according to the manufacturer's protocols. Total RNA concentrations were detected by the NanoVue plus  for RNA concentrations. The cDNA was synthesized with the PrimeScript RT Reagent Kit . The TaqMan miRNA assay  was used for quantification of microRNAs at 95°C for 10 min, followed by 95°C for 15 s (40 cycles) and 60°C for 1 min (40 cycles). The miR-217 was measured with the TaqMan miRNA assays and U6 as an internal control. The expression of miR-217 was evaluated based on the threshold cycle (Ct) as  C. elegans microRNAs  were established and validated to normalize for the microRNAs in the plasma. Synthetic single-stranded RNA oligonucleotide miR-217 (miRBase release v.19.0) was purchased from Shanghai GenePharma, China. Synthetic miRNAs were input into the RT reaction according to the range of copies from reference [ C. elegans miRNAs [Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen), according to the manufacturer's protocols. The syntheticeference , 26. Thes miRNAs , 26.Databases of miRanda, TargetScan, and PicTar were used to predict the potential target genes of miR-217. Among those target genes, the gene of TNFSF11 had a binding site in the 3′UTR of miR-217.Luciferase reporter vector was purchased from Saierbio  and the QuickChange Lightning kit  was used for site-directed mutagenesis. Expression miR-217 plasmid and TNFSF11 wild-type and mutated 3′UTR luciferase reporter were cotransfected into HEK293T cells. Renilla luciferase  served as the control. The luciferase signal was measured with the Dual-Luciferase Reporter Assay System .μg) was boiled and then chilled and separated and finally transferred to the PVDF membrane . The membranes were incubated overnight with primary antibody and incubated with secondary anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibodies according to the manufacturer's protocols .A lysis buffer was used to extract whole cell lysates for western blotting, and a protein assay kit  was used to determine total protein concentration according to the manufacturer's instructions. Total protein  following the instructions strictly. In short, cell suspension was attached to the surface of a glass slide with polylysine and fixed with 4% paraformaldehyde. Paraffin sections were used and cells were incubated with terminal deoxynucleotidyl transferase using FITC-labeled nucleotides. The percentage of TUNEL-positive cells was calculated as follows: TUNEL-positive cells/total number of cells × 100%.t-test and the one-way analysis of variance test were used to analyze continuous data. The chi-square test was used to analyze categorical data. Receiver operating characteristic (ROC) curve analysis was used for the separation level. A p value < 0.05 was considered to represent a statistically significant difference.The data were presented as mean ± SD, median (range), or categorical data. The unpaired Renal tissue was collected from those patients undergoing renal biopsy and plasma was isolated from the blood sample. The relative expression of miR-217 was detected by the qRT-PCR method. The results showed that, compared with control patients, the expression of miR-217 was not statistically different in renal tissue and plasma from FSGS, was significantly upregulated in those from the DN , and was significantly downregulated in those from MN  Figures . (fold change = −2.02 in plasma)  .n = 84) and control patients (n = 72), with an AUC of 0.942 , a cutoff value < 750.0 copies/ul, and sensitivity and specificity of 88.9% and 75.9%  is a common nephrotic syndrome in adults and leads to severe complications . NowadayIt has been reported that microRNAs are small, noncoding RNAs and have the ability to regulate protein-coding genes by binding the translation section and lead to either mRNA degradation or translational inhibition . Recentlα could induce cytosolic phospholipase A2 expression via JNK1/2- and p38 MAPK-dependent AP-1 activation [α could also induce cytosolic phospholipase A2 expression via inhibition of PKCα-dependent NADPH oxidase/ROS and NF-κB [To date, the recognized pathological mechanism of MN is the combination of relative phospholipase A2 antibody and receptor that damages the podocytes and then destroys the glomerular filtration barrier and generates proteinuria –7. Some tivation ; TNF-α cnd NF-κB . Therefond NF-κB . In MN, nd NF-κB , 34. Somnd NF-κB , 36. Thend NF-κB . In humand NF-κB . WhetherThe study also had some limitations. First, the sample size was small. Second, all of the patients only had membranous nephropathy, diabetic nephropathy, or focal segmental glomerulosclerosis, not other relative diseases.In summary, our study identifies that miR-217 is a useful diagnostic biomarker and is involved in human podocyte cells apoptosis via targeting TNFSF11 in membranous nephropathy and provides a new diagnostic strategy for membranous nephropathy.


A Zika virus disease outbreak occurred in Roatán, Honduras, during September 2015–July 2016. Blood samples and clinical information were obtained from 183 patients given a clinical diagnosis of suspected dengue virus infection. A total of 79 patients were positive for Zika virus, 13 for chikungunya virus, and 6 for dengue virus. Zika virus is a mosquitoborne flavivirus that is clinically nonspecific and associated with severe congenital injury  and laboratory diagnostic findings for cases of presumptive dengue. As of September 2015, Zika virus had not been identified in Roatán. We report emergence of Zika virus on Roatán.We conducted a cross-sectional survey of patients who came to Public Hospital Roatán during September 2015–July 2016 and were given a clinical diagnosis of suspected dengue fever. This hospital, the only public hospital in Roatán, has 58 inpatient beds. Approximately 90% of dengue cases on Roatán are diagnosed at this hospital; most case-patients came to the emergency department, which had 18,578 visits in 2015 .We used a convenience sampling approach. All patients >2 years of age given a diagnosis of clinically suspected DENV infection at the hospital during the enrollment period were eligible. Suspected DENV infection was diagnosed by using criteria of the Honduran Ministry of Health, which are fever plus 2 of the following: nausea/vomiting, rash, headache/retroorbital pain, myalgias/arthralgias, petechiae/positive tourniquet test result, leukopenia, and bleeding.am–4:00 pm) Monday through Friday. Patients underwent a phlebotomy and completed a clinical questionnaire, administered verbally in English or Spanish that addressed demographics, migration history, employment, medical history, and symptoms and signs of arbovirus infection.Patients were enrolled after we obtained informed consent; minors were enrolled only after we obtained parental permission. Ethical approval was granted by institutional review boards of the University of California, San Francisco, and Universidad Nacional Autónoma de Honduras. Enrollment of participants and blood collection were restricted to workday hours (8:00 Samples of whole blood (5–9 mL) were collected in EDTA-treated vacutainers  within 1 h of diagnosis. Samples were centrifuged , which yielded sufficient plasma to prepare 5 equal aliquots . Aliquots were labeled with patient identification numbers and stored frozen at −30°C pending for testing.In July 2016, samples were shipped on dry ice to Blood Systems Research Institute  where initial testing was performed by using the Trioplex Assay  for detection of DENV, chikungunya virus (CHIKV), and Zika virus RNA. RNA was extracted from 140 μL of plasma and eluted in 60 μL of buffer .Multiplex real-time reverse transcription PCR (RT-PCR) was performed using the SuperScript III Platinum One-Step qRT-PCR Kit  with DENV, CHIKV, and Zika virus primers and probes developed at the Centers for Disease Control and Prevention. Each duplicate reaction contained 10 μL of sample RNA in a reaction volume of 25 µL. Samples were tested in a 96-well format in a real-time instrument . Results were considered positive if the cycle threshold was <38.A more sensitive Zika virus–only test based on transcription-mediated amplification , which processed 0.5-mL plasma into each amplification reaction, was performed in parallel for all samples to confirm Zika virus infections detected by the Trioplex Assay and detect low levels of Zika virus RNA , which generated global positioning system coordinates for their homes. The survey, laboratory test data, and global positioning system coordinates were uploaded into ArcGIS version 10.3.1 software . All potential predictors of Zika virus infection were analyzed by using univariate logistic regression models in Stata version 13 . Characteristics that were significant (p = 0.10) by univariate analysis were entered into a multivariable logistic regression model.A total of 183 patients participated and provided blood samples . Most  and headache  were independently associated with Zika virus infection. Fever  and vomiting  were associated with a decreased risk for Zika virus infection .This study had limitations. Our exclusive enrollment of patients who came to Public Hospital Roatán excluded patients who came to other healthcare facilities or outside daytime working hours who were not sufficiently symptomatic to seek care. Alternative body fluids  were not available for testing, and serologic testing for Zika virus was not performed because of cost constraints and cross-reactivity between Zika virus and DENV antibodies. We did not perform confirmatory testing of CHIKV- and DENV-positive samples. This study could be biased for detecting Zika virus and not DENV/CHIKV because of use of a more sensitive assay.We demonstrated the evolution of the Zika virus disease outbreak in Roatán, Honduras, in early 2016. Findings highlight challenges in case ascertainment based only on clinical diagnosis. In the absence of laboratory confirmation, clinical diagnosis has low specificity because of overlap of signs and symptoms between infections with Zika virus, DENV, CHIKV, and a host of other regionally endemic infections. Enrollment was contingent upon a suspected diagnosis of dengue. However, DENV was detected in <5% of case-patients, whereas 43% of case-patients had acute Zika virus infections.After a rapid peak in early February 2016, there was a slow decrease in reported cases through July 2016, when enrollment concluded. Access to laboratory testing remains a barrier to surveillance and clinical management in low-resource countries where Zika virus disease outbreaks are predominantly focused, but laboratory infrastructure is lacking (