Background: Strawberries have been identified to have antioxidant and anti-inflammatory properties that improve neuronal function and cognition, mostly in animal studies. It is unknown if the consumption of strawberries or related bioactives may reduce the risk of Alzheimer’s dementia risk. Material and Methods: The study was conducted in 925 participants, aged 58–98 years of the Rush Memory and Aging Project. Participants were dementia-free at baseline, completed a food frequency questionnaire, and had at least two annual neurological evaluations. The diagnosis of Alzheimer’s dementia was based on structured clinical neurological examination and standardized diagnostic criteria. The association of strawberry intake and incident Alzheimer’s dementia was analyzed using proportional hazard models adjusted for age, sex, education, physical activity, participation in cognitive activities, APOE-ɛ4 genotype, dietary intake of other fruits, and total calorie intake. Results: A total of 245 participants developed Alzheimer’s dementia over the mean follow-up of 6.7 (±3.6) years. Higher strawberry intake was associated with reduced risk of Alzheimer’s dementia . In separate adjusted models, highest vs. lowest quartile intakes of Vitamin C , Pelargonidin , total anthocyanidins , and total flavonoids  were each associated with lower Alzheimer’s dementia risk. These associations remained after further adjustment for cardiovascular conditions. Conclusion: Consumption of strawberries and foods rich in vitamin C, pelargonidin, anthocyanidins, and total flavonoids may reduce the risk of Alzheimer’s dementia. Alzheimer’s dementia in older adults is associated with loss of well-being, other disabilities, and motor decline ,2,3. Then = 1061). We further excluded those with dementia at baseline (n = 36), less than two cognitive assessments (n = 92), or missing data on model covariates (n = 8). Thus, our analytical sample consisted of 925 participants with a follow-up of 6.7 (±3.6) years. The baseline characteristics of the analytical sample were similar to the overall MAP participants enrolled in the cohort , and there was not much change in reported mean intake (0.64 servings/day at baseline and 0.70 servings/day at the last follow-up). We also assessed the variation in the reporting of strawberry intake by season and found no significant differences in intake by the month of FFQ administration. Total calories and nutrient intakes were based on United States Department of Agricultural (USDA) National Nutrient Database [Diet was assessed annually using a 144-item food frequency questionnaire (FFQ) that had been previously validated in older Chicago residents ,22. StraDatabase . Food leDatabase , which dBased on previous literature various dietary and non-dietary factors were selected as covariates to assess the association between strawberry/bioactive association with Alzheimer’s dementia risk. Dietary intake levels of total energy , other fruits intake (servings /day), leafy green vegetables (servings /day), and seafood (servings/day) were based on the baseline FFQ responses. Other fruits included all fruits other than strawberries in the questionnaire . Leafy green vegetable consumption included three food items  and seafood intake included tuna sandwich, fish sandwich, fish as a main dish, and shrimp/lobster/crab. Non-dietary factors, including age (in years), were computed based on the self-reported date of birth and date of baseline FFQ and education was self-reported years of education. APOE-ɛ4 genotyping was performed using the sequencing as described earlier . Particip ≤ 0.05). Baseline characteristics of the study population were defined using mean (±SD) for continuous variables and percentages for dichotomous variables. Correlations between strawberry intake and various bioactive of interest were assessed using Spearman’s correlation coefficient. To determine the association between strawberry consumption and related nutrients and bioactive with incident Alzheimer’s dementia, we used cox-proportional hazard models programmed in SAS version 9.4  that generated hazards ratio as the estimates. To assess the effect of one serving change in strawberry intake, the frequency of strawberry consumption was modeled as a continuous variable with values of 0 (never or less than once a month); 0.5 (1–3 times/month); 1.0 (once per week), and 2 (2–4 times/ week). Nutrients and bioactives were energy-adjusted using the residual regression method  and modep < 0.0001). Strawberry consumption was highly correlated with dietary intake of pelargonidin (ρ = 0.94), one of the primary anthocyanidins found in strawberries; and in this population, strawberries contributed 75% of the total dietary pelargonidin intake. Intake of proanthocyanidins (ρ = 0.39), cyanidins (ρ = 0.42), total flavonoids (ρ = 0.26), and vitamin C (ρ = 0.25) were moderately associated with strawberry intake  years of age, 75% female, 98% White, had a mean educational level of 15 (±3) years, and 21.5% had an APOE-e4 allele. A total of 245 participants developed Alzheimer’s dementia over the mean follow-up of 6.7 (±3.6) years. Strawberry intake ranged from 0 to 2 servings/week (mean intake of the population: 0.64 servings/week). The baseline characteristics of participants consuming strawberries more than once a week were similar to those consuming strawberries rarely or a few times/month . Strawbey intake .We found that one serving more in strawberry intake was associated with a 24% reduced risk of Alzheimer’s dementia , when cop for trend = 0.34). Participants in the highest quartile intake of pelargonidin (the primary anthocyanidin contained in strawberries) had a 44% reduced risk of Alzheimer’s dementia compared to those in the lowest quartile of intake (p trend = 0.01), total anthocyanidins  pelargonidin , and total flavonoids  from food sources. Additionally, we investigated if the associations of vitamin C and flavonoids intake from foods were confounded by intake of other important antioxidant nutrients associated with cognition, i.e., vitamin E. The effect estimates for all the nutrients analyzed remained similar except that for total flavonoids (p trend = 0.07). Similarly, the effect estimates of total flavonoids were modified when controlled for vitamin C and vitamin E together in a model . However, the effect estimates for pelargonidin  did not change when further adjusted for these nutrients.Strawberries are rich sources of vitamin C and other bioactives, including anthocyanidins , and proanthocyanidins. Intakes of vitamin C (from food sources), total anthocyanidins, pelargonidin, and total flavonoids from food sources were each significantly associated with reduced incidence of Alzheimer’s dementia when controlled for age, sex, education, physical activity, participation in cognitive activities, and Apo-ɛ4 status . Howeverf intake . Furtheravonoids . HoweverWe investigated the potential modification of the observed associations by age (>80 years/≤80 years), sex, education (>15 years/≤15 years) and APOE-ɛ4 status in basic-adjusted models. The only significant interaction effects were for total anthocyanidins and total flavonoids. The association of anthocyanidins with Alzheimer’s dementia risk occurred only among those who were APOE-ɛ4 negative  and not in those without the allele . The protective association of total flavonoid intake with Alzheimer’s dementia was present only in men  and not in women . In this prospective study of older community adults, strawberry intake was associated with a 34% reduced risk of Alzheimer’s dementia compared to no or rare intake. Strawberries are rich in a number of flavonoid bioactives  that we found were each associated with lower Alzheimer’s dementia risk. Pelargonidin intake, which is a primary bioactive present in strawberries, appears to account for the protective effect of strawberries on Alzheimer’s dementia even when controlled for various confounding factors including demographics, genetic predisposition to dementia, lifestyle and other dietary factors including other antioxidant nutrients intake. To our knowledge, this is the first longitudinal cohort study of community older adults to report on the association of strawberries with Alzheimer’s dementia risk. Our results are supported by findings from the Nurses’ Health study in which strawberry consumption, anthocyanidins, and total flavonoid intake were each associated with slower cognitive decline . AdditioThe potential mechanistic links for the beneficial effect of strawberries may be due in part to the neuroprotective properties of the bioactive in strawberries. In animal models, strawberry consumption improved cognitive function, increased neurogenesis, and insulin-like growth factor-1 signaling, reversed neuronal aging by reducing oxidative stress and ameliorated metal-induced neurotoxicity ,14,34. BThe current study has a number of important strengths including the longitudinal study design and 6.7 years of mean follow-up, the community-based sample of older adults, dietary assessment using a comprehensive and validated tool for older adults, structured clinical assessment and standardized diagnostic criteria for Alzheimer’s dementia, and assessment of many health and lifestyle factors associated with Alzheimer’s dementia. However, there are some limitations, as well. This is an observational study design so we cannot establish the temporal relationship and there can be some unaddressed confounding biases too. Our dietary assessment tool had one berry question just for the intake of strawberries. With no other berry question, there is a possibility of misreporting by those consuming other berries  also. Additionally, the study results may not be generalized to younger adults and nonwhite or Hispanic population.Consumption of strawberries may reduce the risk of Alzheimer’s dementia in older adults, probably due to neuroprotective action of pelargonidin, anthocyanidins, and total flavonoids. Once replicated in other cohorts and confirmed by a clinical trial, these findings may have important public health implications, as the addition of strawberries to one’s diet can be an easy adaptation for older adults to reduce Alzheimer’s dementia risk.


The aim of this study is to report the long-term efficacy and safety of thoracoscopic epicardial left atrial ablation (TELA) in patients with paroxysmal atrial fibrillation (AF).n = 55) patients received an implantable loop recorder (ILR), 30 days post-operatively. Antiarrhythmic and anticoagulation therapy were discontinued at 90 and 180 days postoperatively, respectively, if patients were free of AF recurrence. Failure was defined as ≥two minutes of continuous AF, or atrial tachycardia.This was a retrospective review of medical records. We included all patients diagnosed with paroxysmal AF who underwent TELA at our institution between 04/2011 and 06/2017. TELA included pulmonary vein isolation, LA dome lesions and LA appendage exclusion. All (n = 49/55), 85.5% (n = 47/55) and 76.9% (n = 40/52) at 6, 12 and 24 months, respectively. In the multivariate cox-proportional hazard model, survival at the mean of covariates was 86 and 74% at 12 and 24 months, respectively.Fifty-five patients  qualified for the study. The average duration in AF was 3.64 +/− 3.4 years, mean CHA2DS2-VASc Score was 2.0 +/− 1.6. The procedure was attempted in 57 patients and completed successfully in 55 (96.5%). Two patients experienced a minor pulmonary vein bleed that was managed conservatively. Post procedure, one patient experienced pulmonary edema, another experienced a pneumothorax requiring a chest tube and another experienced acute respiratory distress syndrome resulting in longer hospitalization. Otherwise, there were no major procedural complications. Success rates were 89.1% (In this single center experience, TELA was a safe and efficacious procedure for patients with paroxysmal AF. Atrial fibrillation (AF) is the most common cardiac arrhythmia with several adverse consequences related to a reduction in cardiac output, and to ischemic cerebrovascular accidents or peripheral embolization due to atrial and atrial appendage thrombus formation . Though This study was a retrospective review of medical records. We included all patients diagnosed with paroxysmal AF who underwent TELA at our institution between 04/2011 and 06/2017. The patients did not tolerate or have failed antiarrhythmic drugs. At that time interval, there was no dedicated electrophysiology service at the hospital. The surgeon (JJ), explained the TELA procedure to patients. Patients were also informed that catheter-based PVI was the gold standard for ablation procedures, and they were offered a referral to a center where catheter-based ablation can be performed. Patients who denied a referral and opted to undergo TELA were included in this study.2. Patients’ anticoagulation drugs were held prior to the procedure and resumed the night following the procedure. The procedure included PVI, ganglia mapping and ablation, left atrial dome lesions, superior vena cava lesion, and left atrial appendage exclusion. Under general anesthesia, three 10 mm ports sites were created. The right side was performed first. The pericardium was opened and retracted posteriorly. Using the AtriCure® Transpolar Pen, the autonomic ganglia were mapped and ablated, and then the AtriCure® Lumitip Dissector was used to encircle the pulmonary venous hilum and to guide the AtriCure® Synergy Bipolar RF Clamp around the veins. Three separate lines of ablations were performed with each being repeated twice. Bidirectional block was then confirmed with the pen at each location. An opening was created between the superior vena cava and venous hilum. Working through the transverse sinus, the right-sided portion of the LA dome lesion was created with the AtriCure® Coolrail linear pen. This lesion was repeated twice. Working through the oblique sinus, the right-sided portion of the LA floor lesion was created, and this lesion was repeated twice as well. A superior vena cava lesion is created well above the area of the sinus node with a single application of the radiofrequency clamp. On the left side, the autonomic ganglia and veins were treated identically, and the dome and floor lesions were completed. Bidirectional block was confirmed for the both veins and the box. Autonomic ganglia were localized in the anterior and posterior fat pads, by their response to high-frequency stimulation. Successful ganglion ablation was defined by abolition of the heart rate response  to high-frequency stimulation at the same regions that had elicited clear responses before ablation. Coolrail® device was then used to perform the LA appendage connecting lesion, which consisted of a line on the posterior surface of the LA appendage and several stamping lesions over the base of the LA appendage in the coumadin ridge area. Finally, the LA appendage was excluded with AtriClip® with the aid of transesophageal imaging to confirm a residual of less than 1 cm at the base. Bidirectional block was then confirmed on the LA appendage. The pericardium was then reapproximated, and a chest tube placed. Chest tubes were typically pulled later the day of the procedure. Patients were discharged from the hospital, the day following the procedure, unless a complication occurred. All patients received an implantable loop recorder (ILR), 30 days post-operatively. Antiarrhythmic and anticoagulation therapy were discontinued at 90 and 180 days postoperatively, respectively, if patients were free of AF recurrence. Failure was defined as 2 min or more, of continuous AF or flutter on any ILR download. Procedural complications were defined as major when prolonging or causing a hospital admission within 30 days.Exclusion criteria for the procedure were previous lung lobectomy, or pericardial surgery and a body mass index above 35 kg/mp-value was ≤0.2. Statistical analysis was conducted using MedCalc Statistical Software version 16.4.3 .Continuous data are reported as mean ± standard deviation, and categoric data are reported as number of subjects and ratios. The differences between the groups were compared using the independent-samples Student’s t-test. Chi-square statistics were used to compare categorical variables between groups. Cox-proportional hazard survival analysis was also conducted. Variables were entered in the model if their univariate test 2. The mean follow-up period was 36.8 +/− 21 months. Figure n = 49/55), 85.5% (n = 47/55) and 76.9% (n = 40/52) at 6, 12 and 24 months, respectively. In the multivariate cox-proportional hazard model, survival at the mean of covariates was 88, 86 and 74% at 6, 12 and 24 months, respectively  had previous catheter ablation procedures before the index procedure. Baseline patient demographics, comorbidities, and echocardiographic characteristics are included in Table 2.5% had The procedure was attempted in 57 patients and completed successfully in 55 (96.5%) patients. One patient experienced tension pneumothorax and the other experienced a major bleed during TELA and those two patients were converted to a full sternotomy for AF ablation. Two patients experienced a minor pulmonary vein bleed that was managed conservatively. Post procedure, one patient experienced pulmonary edema, another experienced a pneumothorax requiring a chest tube and another experienced acute respiratory distress syndrome resulting in longer hospitalization. Otherwise, there were no procedural complications, including phrenic nerve injury, esophageal fistula, major bleeding, hemothorax, cardiovascular or cerebrovascular events. At the end of the follow up period, none of the patients experienced any cerebrovascular accidents.This study investigated the long-term efficacy and safety of TELA as a stand-alone procedure in paroxysmal AF patients, using ILR for follow up. The major finding of our study is that the efficacy of TELA was similar to previously published studies using catheter-based PVI.n a metanalysis of 19 studies, the pooled 12-month success rate for the 11 studies reporting outcomes for paroxysmal AF patients was 66.6% (95% CI 58.2 to 74.2%). Single-procedure freedom from atrial arrhythmia at long-term follow-up was 54.1% (95% CI 44.4 to 63.4%) in paroxysmal AF patients. The multiple-procedure long-term success in paroxysmal AF was 79.0% in 8 studies (95% CI 67.6 to 87.1%) [The efficacy results of our study are similar to previous TELA studies. There is a limited number of publications which reported the long-term efficacy of TELA as a stand-alone procedure for restoration of sinus rhythm in AF patients, likely because it was first described -only- a little more than a decade ago. Edgerton et al. reported the results of a prospective, nonrandomized study of 52 consecutive patients with symptomatic paroxysmal AF undergoing TELA . Unlike None of the patients in our study experienced cerebrovascular accidents during follow up. With an average CHA2DS2VASc score of 2, the estimated stroke risk for our population would be 2.2% per year and 2.9% combined risk of stroke, transient ischemic attacks or systemic embolization , 14. TheLimitations of this study include all the inherent limitations of a retrospective study, and the absence of a control group. The Heart Rhythm Society defines failure following an ablation procedure as more than 30 s of AF and this study used a cutoff of 2 min which is the lowest reported by an ILR. Also, there were no postprocedural studies conducted to assess the restoration of atrial mechanical function, in this cohort.In this retrospective single center experience, TELA was a safe and efficacious procedure for patients with paroxysmal AF. Long-term outcomes are similar to the gold standard of catheter-based PVI. Results need to be validated in a randomized clinical trial fashion to compare the safety, efficacy and quality of life metrics between patients with atrial fibrillation undergoing thoracoscopic epicardial versus catheter-based ablation procedures.


Social and communication (SC) difficulties predict increased social anxiety (SA) symptoms in childhood. Peer victimisation and friendship quality are commonly associated with both SC difficulties and SA. Based on this, we tested for a cascade effect of early SC difficulties, peer victimisation and friendship quality on SA in late childhood, using a population-based sample of 8028 children from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. Parent-reported data were collected on SC difficulties at age 7 and SA at age 7, 10 and 13. Child-reported data on peer victimisation and friendship quality were collected at age 8. Our results revealed that SC difficulties predict increased negative friendship qualities and peer victimisation. Relational victimisation predicted increased SA symptoms at 13 years old. Neither overt nor relational victimisation mediated the developmental relationship between SC difficulties and SA. Furthermore, friendship quality did not moderate the developmental relationship between SC difficulties and SA. In addition, no sex differences were observed. The evidence demonstrates that peer victimisation and friendship quality do not explain why some children with SC difficulties go on to develop SA. Future research clarifying the complex etiological pathways contributing towards the development of SA in childhood and adolescence is warranted. Severe Etiological models of SA in childhood and adolescence have implicated the role of several risk factors, including social skill deficits and negative peer relationships, among others . One risCross-sectional research shows that children diagnosed with SAD exhibit more SC difficulties, as reported by observer, parent and self-ratings, compared to non-anxious children  and chilPeer victimisation and poor friendship quality, are proposed to play a role in the development of SA. Peer victimisation is a multifaceted phenomenon, which encompasses both overt  and relational victimisation (e.g. spreading rumours). Children who experience both subthreshold and clinical SC difficulties, including young people with ASD, often experience high rates of victimisation and peers problems . In lineFriendships are important experiences for developing social and cognitive skills across childhood and adolescence . ExperieEtiological models of SA in normative development and neurodevelopmental disorders propose that, when experienced together, SC difficulties and negative peer relationships will exacerbate a youth’s risk of experiencing SA . For exaIn the present longitudinal study, we aim to examine whether negative friendship qualities, and overt and relational victimisation influence the developmental relationship between SC difficulties and SA symptoms using a population-based sample of children see . In ligh22.1http://www.bris.ac.uk/alspac/researchers/data-access/data-dictionary/. Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. The present study used children who participated in the age 7 data collection phase and had available SC difficulties data (N = 8028). Of those individuals, 6430 (80%) and 5784 (72%) participants had available SA data at age 10 and 13, respectively. Following ALSPACs exclusion criteria for prorated scores, only children with 50% or more complete data on SC difficulties at age 7 and SA at age 10 and 13 years were included in the present study. The final sample of 8028  individuals was used in all final analyses. In the final sample, 42% of mothers were in higher education, 82% owned/mortgaged their own home and 96% were from a white ethnic background. Compared to those in the ALSPAC cohort who were not included in the following analyses, the current sample were more likely to have a mother who had completed higher education , was a homeowner  and was from a white ethnic background .The Avon Longitudinal Study of Parents and Children (ALSPAC) is a population-based cohort of children born in Bristol, UK, between 1991 and 1992. At one years old 13,988 children were alive and formed the original cohort . Beginni2.22.2.1Maternal education at 32 weeks of gestation was used as a measure of SES. Previous research has shown that this is a valid indicator of SES in the ALSPAC cohort . Mothers2.2.2Wechsler Intelligence Scale for Children-Third Edition .The Cambridge Hormones and Moods Project Friendship questionnaire  was used2.2.6r = .41; girls r = .42; boys r = .42). A widely used ordinal victimisation variable was derived from the total victimisation severity score to illustrate the frequency of child victimisation in our sample, using the following cut-offs  pathway analyses were conducted using Mplus V.8. In the pathway analyses, all relationships were tested between latent factors, which are not directly observed, but constitute a measure that captures the common variance amongst the observed variables. Latent factors were used to capture more robust constructs that are free from measurement error. Each latent factor was specified within the model, for example the 12 items from the SCDC loading onto the SC difficulties latent construct. All latent factors were free to covary in each pathway model. All SA latent factor item residuals were specified to covary between time points . All pathway analyses controlled for confounding variables, including IQ, SES and SA symptoms at age 7. All paths are reported as partial regression coefficients. In all pathway analyses, a robust maximum likelihood (MLR) estimator was used to account for the variables that deviate from normality. In addition, full information maximum likelihood (FIML) was used to estimate model parameters from raw data. Using FIML reduces the impact of bias from selective attrition by using all available data .Previous research using ALSPAC data supports the construct validity of the DAWBA-SF and SCDC at 7, 10 and 13 years old . Using MA saturated measurement model was conducted to examine the associations between all latent factors see . To testModel fit was tested using the Satorra-Bentler Scaled chi-square statistic , which i3n = 5784) to those who only had available SA data at 7 years old (n = 2244). No significant difference was observed between individuals who had available SA data at all ages and those who only had data at 7 years old .All questionnaire data were cleaned according to ALSPAC guidelines for data preparation (see Supplement 1). To test for selective attrition in our sample, we compared individuals who contributed SA data at age 7 and 13 years old (Mean scores are presented in n = 1463) had experienced no victimisation, 40% of children (n = 2215) had experienced moderate victimisation (at least one type of victimisation experienced at an infrequent/frequent rate) and 34% of children (n = 1891) had experienced severe victimisation (at least two types of victimisation experienced at an infrequent/frequent rate).In the present sample, 26% of children  were indicative of good/acceptable model fit. The results imply that the Cambridge Hormones and Moods Project Friendship questionnaire measures a coherent friendship construct. All fit indices for the two-factor model with peer victimisation at 8 years old  were indicative of good model fit. The results imply that the Bullying and Friendship Interview Schedule measures two distinct constructs, including overt victimisation and relational victimisation.3.2A saturated model was conducted to examine the associations between all latent factors used in subsequent mediation and moderation pathway analyses see .Table 2C3.3p < .001; SES: β = -.01, p =.45; SA7; β = .49, p <  .001) and 13 years .In all specified models, the effects of IQ, SES and SA symptoms at 7 years old were regressed out of the SA latent factor at age 10 , increased overt victimisation (β(se) = .21(.02), [95% CI] = , p < .001) and relational victimisation (β(se) = .09(.02), [95% CI] = , p < .001), indicating a small to moderate effect of SC difficulties on later peer victimisation and friendship quality.Results from the pathway model analyses revealed that earlier SC difficulties significantly predicted negative friendship quality (β(se) = .15(.02), [95% CI] = , 3.53.63.72[df] = 0.62[6], p = .996). This suggests that no sex differences emerged in the indirect effects of overt and relational victimisation on later SA symptoms. In the moderation model, friendship quality significantly predicted later SA symptoms at 13 years old for boys (β(se) = .07 (.02), [95% CI] = , p < .01), but not girls (β(se) = .02 (.03), [95% CI] = , p > .05), however, this difference was not significant . Our results revealed that SC difficulties significantly predicted SA symptoms at 13 years old for girls (β(se) = .10 (.04), [95% CI] = , p < .01), but not boys (β(se) = .06 (.03), [95% CI] = , p > .01), however, this difference was not significant . Furthermore, in the moderation model, no significant decrease in model fit was observed when comparing a baseline model to a nested model which constrained all interaction paths to be equal across boys and girls . This suggests that no sex differences are observed in the interaction between SC difficulties and friendship quality predicting later SA symptoms.All pathway analyses are reported separately by sex (see Supplement 2). No sex differences were observed in our mediation model pathway analysis. In the mediation model, no significant decrease in model fit was observed when comparing a baseline model to a nested model where the indirect paths from SC difficulties to SA symptoms, via overt and relational victimisation, were constrained to be equal across boys and girls  and the University of Bristol provide core support for ALSPAC. Miss H. Pickard is funded by the Medical Research Council (MRC) and Autistica. This publication is the work of the authors, who will serve as guarantors for the contents of this paper.The


Understanding the DNA methylome and its relationship with non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), is essential for elucidating the molecular mechanisms underlying key biological processes in plants. Few studies have examined the functional roles of the DNA methylome in grass species with highly heterozygous polyploid genomes.Panicum virgatum L.) cultivar ‘Alamo’ using bisulfite sequencing. Single-base-resolution methylation patterns were observed in switchgrass leaf and root tissues, which allowed for characterization of the relationship between DNA methylation and mRNA, miRNA, and lncRNA populations. The results of this study revealed that siRNAs positively regulate DNA methylation of the mCHH sites surrounding genes, and that DNA methylation interferes with gene and lncRNA expression in switchgrass. Ninety-six genes covered by differentially methylated regions (DMRs) were annotated by GO analysis as being involved in stimulus-related processes. Functionally, 82% (79/96) of these genes were found to be hypomethylated in switchgrass root tissue. Sequencing analysis of lncRNAs identified two lncRNAs that are potential precursors of miRNAs, which are predicted to target genes that function in cellulose biosynthesis, stress regulation, and stem and root development.We performed genome-wide DNA methylation profiling in the tetraploid switchgrass  contains supplementary material, which is available to authorized users. Panicum virgatum L.) is a warm season, perennial, C4 grass native to the North American prairie [Switchgrass  and long non-coding RNAs (lncRNAs). MiRNAs are small sequences between 20 and 24 nt in length. These molecules often regulate gene expression post-transcriptionally by either mRNA cleavage or translation repression , 15. LncZea mays), great millet (sorghum bicolor), sugarcane (Saccharum officinarum), and switchgrass. Primary production of these crops annually accounts for roughly 25% of arable land [Oryza sativa) [Brachypodium distachyon [Arabidopsis thaliana, rice, and B. distachyon, have generated several conserved rules: (1) mCG is dominating among the three methylated contexts; (2) methylation may act as a mechanism for controlling gene expression, but the methylation variation depends on types of contexts and gene region; and (3) RdDM pathways are highly associated with mCHH [C4 grasses are the most prominent in the grass family and include some of the most economically important crop species such as maize ( sativa) , Brachypstachyon , and whestachyon ; howeverith mCHH , 29, 30.In this study, we performed global DNA methylation sequencing on leaf and root tissues of tetraploid switchgrass (cv. Alamo) and profiled expression of mRNAs, miRNAs, and lncRNAs in the leaf tissue. Our results revealed that siRNAs positively regulated DNA methylation at the mCHH sites surrounding genes and that DNA methylation may interfere with both gene and lncRNA expression in the polyploid switchgrass genome. In addition, we identified a small subset of genes hypomethylated in the root tissue that were characterized by differentially methylated regions (DMRs), which could potentially be involved in stimulus-related GO processes. Finally, we predicted two precursors (lncRNAs) of miRNAs that might function in cellulose biosynthesis, stress regulation, and stem and root development. Overall, this study described the DNA methylome of switchgrass and its relation to gene and non-coding RNAs. These results provide a platform for future epigenetic studies in biofuel crops.Whole-genome bisulfite sequencing was applied to genomic DNA of switchgrass extracted from leaves and roots. In total, 411 million reads were generated for switchgrass leaf tissue and 392 million reads were generated for switchgrass root tissue. Of the reads generated in this study, 334 (81%) million reads from the leaf sample and 304 (77%) million reads from the root sample could be aligned to the reference genome of switchgrass (V4.1)  were identified in the genomic DNA isolated from switchgrass leaves and roots, respectively. For the leaf tissue, the cytosine methylation levels were higher at the mCG sites (60%) than those at mCHG (40%) and mCHH (4%) sites Table . The datA chromosome-scale view of the methylation sites and their levels was generated from the sequencing data  and mCHG (10%), respectively. For the four Poaceae plants examined in this study, the levels of mCG and mCHG were higher in switchgrass than B. distachyon and rice, but lower than similar sites in maize , and class II  Fig. a–d. WhilArabidopsis, Norway spruce (Picea abies), and castor bean (Ricinus communis) [To identify global differentially methylated DNA between leaf and root tissues, the methylation levels of both genic and TE regions were compared. The analysis of these two genomic components was further divided into upstream, body, and downstream regions  , 20, 34.Differential methylation of the two TE classes was also observed between the leaf and root tissues in the TE body and flanking regions Fig. c; howeveDifferential methylation regions (DMRs) play an important role for epigenetic methylation modifications across plant species . In thisp < 0.05; Additional file Plant roots are essential for responding to abiotic stress stimuli and must quickly react to environmental changes by controlling key stress-related genes , 37. PreTo further determine whether DNA methylation influences gene expression in switchgrass, total RNAs were extracted from the same sets of leaf samples and subjected to RNA-sequencing. A total of 150,545 transcripts were assembled from the RNA-seq data and these transcripts were grouped into four categories according to the criteria of Lu et al. . These cIn the 2-kb regions upstream and downstream of genes, the category of non-expressed gene group contained the highest levels of mCG. Alternatively, the methylation levels at this site were the lowest for the high-expressed gene group Fig. a. This irho = 0.279, p = 2.25E −267), whereas mCG in the upstream  and downstream  regions exhibited a significantly different negative correlation  and body regions  were lower than that of the downstream regions . This suggests a weaker correlation between CHG methylation and gene expression in the upstream and body regions than in the downstream regions , whereas the gene body and downstream regions displayed negative and significant correlations  . Other methylated regions, which had zero model coefficients less than 0, had negative associations with gene expression. The predictivity of the regression model was tested using a tenfold cross-validation method [Q2 values, suggesting a limited ability of the model to predict gene expression  in the Spearman analysis. Regardlessly, these weak correlations supported all of the relationships predicted through RNA-seq analysis with the exception that no association was found in the downstream region for mCHH sites  higher than in the regions not targeted by any siRNAs , but negative correlations in the upstream  and downstream regions . Similarly, the mCHG sites were found to have positive and significant correlations between siRNA and DNA methylation in the gene body region , and a negative correlation in the downstream flanking region . No correlation between the two elements was determined for this site for the upstream gene region (p > 0.05). In contrast, the gene body , as well as the flanking regions , were found to have positive and significant correlations at the mCHH sites. Regression analysis was conducted based on a negative binomial model [p = 1.84E−69) and upstream regions , and a negative and significant correlation in the downstream region . The mCHG sites displayed positive correlations in the upstream and body regions ; however, there was no significant correlation in the downstream region (p > 0.05). In contrast, mCHH sites in all regions exhibited positive and significant correlations . Through the tenfold cross-validation analysis, the model showed a limited power  to predict the siRNA expression  [http://rfam.xfam.org/) [LncRNAs have been shown to be closely associated with DNA methylation . A totall#about) . Surprisam.org/) . Seven lam.org/) , Xist_exam.org/) , SOX2OT_am.org/) , H19_3 [am.org/) , ZEB2_ASam.org/) , and RFPam.org/)  non-codiam.org/) . The funDNA methylation allows for an organism to quickly respond to environmental stimuli by altering levels of mRNA expression . LncRNAsp > 0.05) in the regression analysis. This predictivity of regression model was also tested via the tenfold cross-validation method. The yielded small positive Q2 values from the model exhibited a restrictive ability to predict the lncRNA expression.  to verify the predicted relationships of lncRNAs within the 2-kb flanking regions  were negatively correlated with gene expression Fig. a, b. TheOverall, the mCG and mCHH were found to be positively related to gene expression in the gene body and upstream flanking regions, respectively. These results differed from other regions that displayed a negative correlation Fig. . Taken tRdDM is a process by which 24-nt siRNAs direct the de novo methylation of cytosine of complementary DNA sequences. The de novo methylation of DNA at all mCG, mCHG, and mCHH contexts is usually performed by domains rearranged methylase 2 (DRM2) through the RdDM pathway . In thisLaSCR1 was shown to decrease root numbers in transformed roots of white lupin  and Medicago truncatula [Previous studies suggest that lncRNAs could be precursors of miRNAs , 86. By uncatula . TherefoThis study utilized a combination of omics techniques to conduct genome-wide single-base-resolution methylome, lncRNA, miRNA, and mRNA sequencing in switchgrass. The results presented here support that siRNAs positively regulate DNA methylation at mCHH sites and that DNA methylation may interfere with gene and lncRNA expression in the polyploid switchgrass genome. In addition, a total of 96 genes were identified that are covered by DMRs in leaf and root tissues. These genes are believed to be involved in stimulus-related GO processes and 79 of them were hypomethylated in the root tissue. We also identified 9244 novel lncRNAs in switchgrass and predicted two lncRNA precursors of miRNAs that may function in cellulose biosynthesis, stress regulation, and stem and root development. Overall, the successful DNA methylome and transcriptome sequencing of switchgrass presented in this study provides a reference for other highly heterozygous Poaceae grasses with similar characteristics. These results could also serve as genomic resources for identifying further methylation patterns and additional non-coding RNAs analysis in switchgrass.The switchgrass cv. Alamo was propagated through tiller-splitting and planted in pots  containing 1500 g soil . The plants were maintained in a greenhouse  at 28 °C/20 °C (day/night) with a photoperiod of 16 h/8 h (day/night). Four months after transplanting and approximately 1 week before the emergence of flower primordium (E5 stage), the flag leaves and roots of six individual plants were collected . The leaThe flag leaves and roots were pooled from six random individuals with equal masses for each one and were ground to a fine powder in liquid nitrogen. Genomic DNA was extracted using a plant genomic DNA kit  following the manufacturer’s instructions. The DNA integrity and concentration were measured by agarose gel electrophoresis and NanoDrop spectrophotometer, respectively.Bisulfite sequencing libraries were prepared using the TruSeq Nano DNA LT kit  as described in Du’s study . The genQ-value ≤ 20) bases were removed.The raw reads generated from the Illumina HiSeq™ 4000 were filtered to get high-quality reads using fastq-mcf (v1.04) tool  accordinhttps://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvirgatum_er;V4.1) using BSMAP software (v 2.90) [http://miyoviqo.tha.im/) according to the average methylation levels for each 100-bp interval. ANOVA analysis was applied to test the significance of the average methylation levels of both genic and TE regions between leaf and root tissues [We mapped the clean reads to the switchgrass reference genome ((v 2.90)  with the tissues .Differentially methylated regions (DMRs) between leaf and root tissues of switchgrass for CG, CHG, and CHH were identified based on the following criteria:χ2) value was p ≤ 0.05.(1) The length of each DMR region was between 40 bp and 10 kb; (2) the distance between adjacent methylated sites was < 200-bp; (3) more than ten reads needed to be present for each cytosine, and more than four reads were need for coverage for each methylated cytosine; (4) more than five methylated cytosines must be present in at least one sample; (5) the fold change of the average methylation level was > 2; (6) Pearson’s chi-square test , and reads containing more than 10% of unknown nucleotides (N) using fastq-mcf (v1.04) tool [https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Pvirgatum_er; V4.1) using TopHat2 (v 2.0.3.12) with default parameters [The six individual plants were pooled into two independent biological replicates, and each replicate contained three individuals. Total RNAs were isolated from the flag leaves of the two replicates using TRIzol reagent  according to the manufacturer’s instructions. rRNAs were removed from the samples, retaining only mRNAs and non-coding RNAs. The sequencing libraries were built following Illumina’s standard protocol for RNA-seq library construction as previously described , and the04) tool . Bowtie204) tool  was usedrameters . Reconstrameters  and TopHrameters . The aburameters  and tranhttp://cpc.cbi.pku.edu.cn/) were used to evaluate the protein-coding potential of new transcripts with default parameters. Transcripts that had both CPC and CNCI scores less than 0 were chosen as long non-coding RNAs.Two programs: CNCI (v2)  and CPC http://eddylab.org/infernal/) was used in the sequence alignment and lncRNAs were classified by sequence consensus and secondary structures.Rfam divides non-coding RNAs into families based on their evolution from a common ancestor. Producing multiple sequence alignments of these families can provide insight into their structure and function, similar to the case of protein families. To better annotate lncRNAs at the evolution level, the software Infernal   using BLAST (v 2.2.25), and those with greater than 90% identity were selected. Additionally, the software miRPara (v 6.2)  was emplhttp://www.genome.jp/kegg/) using KOBAS [p value  cut-off was set to 0.05.GO analysis and KEGG analysis were applied to determine the biological roles of the targets genes of identified lncRNAs in switchgrass. GO terms were assigned based on the Gene Ontology (GO) database  using GOng KOBAS . The p vRNAs extracted from the flag leaves of the two biological replicates were used for small RNA-sequencing. Small RNA molecules with a size range of 18–30 nt were enriched by polyacrylamide gel electrophoresis (PAGE) and libraries were built using the NEBNext multiplex small RNA library prep set  based on the manufacturer’s protocol. The small RNAs were then sequenced using an Illumina HiSeq 2500 by Gene Denovo Biotechnology Co. . Raw reads were filtered by removing the following: reads without 3′ adapters, reads containing 5′ adapters, reads containing 3′ and 5′ adapters but no small RNA fragment between them, reads containing poly A sequences in the small RNA fragment, and reads shorter than 18-nt (not including adapters). The high-quality sequences were then BLAST (v 2.2.25)  against www.statstutor.ac.uk/resources/uploaded/spearmans.pdf): (1) rho > 0 equated to a positive correlation, (2) rho < 0 signified a negative correlation, (3) very weak was indicated by the absolute value of rho < 0.2, (4) weak was indicated by 0.2 ≤ the absolute value of rho < 0.4, (5) moderate was determined by 0.4 ≤ the absolute value of rho < 0.6, (6) strong was determined by 0.6 ≤ the absolute value of rho < 0.79, and (7) very strong was indicated by 0.8 ≤ the absolute value of rho < 1.0. The relationships of mRNA, siRNA and lncRNA with methylation were evaluated by modeling gene expression as a function of the number of methylated cytosines in the body of the gene and both flanking regions [R2  values were calculated by a formula: Q2 = 1 − PRESS (predictive error sum of squares)/TSS .The relationships between methylation and mRNAs, siRNAs, and lncRNAs were all analyzed using Spearman and regression analysis. For Spearman analysis, the evaluation of correlation was based on the following criteria  and root (b) tissues of switchgrass. The x-axis represents the sequencing depth. The y axis represents the percentage for cytosine with a sequencing depth over a specific depth of the whole genome cytosine.Additional file 4: Figure S2 Chromosome distribution for root (a) and leaf (b). Methylation level in 80-kb windows throughout chromosomes in the leaf tissue of switchgrass. The red line means ‘−’ strand, and the blue line means ‘+’ strand.Additional file 5: Table S2. The levels of mCG, mCHG, and mCHH and genome size in genomes of different species.Additional file 6: Figure S3. Correlation between genome sizes and methylation levels for different species as Arabidopsis, B. rapa, G. max, M. truncatula, P. trichocarpa, Z. mays, O. sativa, B. distachyum, and P. virgatum (leaf tissue) used in Figure 1e.Additional file 7: Figure S4. Methylation levels of Class I and Class II TEs in the leaf (a) and root (b) of switchgrass.Additional file 8: Table S3. Comparison of methylation levels between class I and class II transposons in leaf and root tissues.Additional file 9: Table S4. Comparison of methylation levels in genic and TE regions between switchgrass leaf and root tissues.Additional file 10: Table S5. Comparison of methylation levels in different TE types between switchgrass leaf and root tissues.Additional file 11: Data S2. GO annotation of genes covered by the DMRs between switchgrass leaf and root tissues.Additional file 12: Data S3 . The methylation levels of 96 genes covered by DMRs and involved in stimulus related GO processes in both leaf and root tissues of switchgrass.Additional file 13: Figure S5. Molecular function for genes covered by the CHH DMRs in the GO annotation in gene upstream (a), body (b), and downstream (c). The colored boxes mean the adjust p < 0.05, and the more deeper color, the less adjust p values for the boxes.Additional file 14: Data S4. The CHH methylation of DMR covered genes annotated in the oxidation-reduction process and oxidoreductase in both leaf and root tissues.Additional file 15: Table S6. The number of mRNA and lncRNA in the four expression levels.Additional file 16: Table S7. Correlation analysis between methylation levels and gene expression in different genic regions.Additional file 17: Table S8. Regression analysis and 10-fold cross-validation between DNA methylation and gene expression based on zero hurdle model.Additional file 18: Table S8. Length distribution of siRNAs (from 20 to 25 nucleotides) in switchgrass for two biological repeats including Alamo_1 (a) and Alamo_2 (b).Additional file 19: Table S9. Comparison of methylation levels between siRNA uniquely mapping region and without that region.Additional file 20: Table S10. The correlation analysis between gene methylation and siRNA expression.Additional file 21: Table S11. Regression analysis and 10-fold cross-validation between DNA methylation and siRNA expression based on negative binomial model.Additional file 22: Figure S7. Proportion of six different types of lncRNA in switchgrass.Additional file 23: Table S12. Prediction of lncRNA families in switchgrass.Additional file 24: Table S13. Annotation of targets of miR169 and miR171.Additional file 25: Table S14. The correlation analysis between methylation levels and lncRNA expression.Additional file 26: Table S15. Regression analysis and 10-fold cross-validation between DNA methylation and lncRNA expression based on zero hurdle model.Additional file 27: Figure S8. siRNA levels (per bp per million reads) in TEs relative to the distance from the nearest gene.


We present a case of a nearly 3-year-old girl who was admitted to hospital due to severe hypernatremia (196 mmol/l). Her medical history included central hypothyreosis and growth hormone deficiency. Rehydration and normalization of sodium was achieved according to guidelines. On the fourth day of hospitalization, the patient developed tremor, ataxia, and rigor. Cranial magnetic resonance imaging (cMRI) was performed and (mis)interpreted for meningoencephalitis, with corresponding diagnostic and therapeutic implications. The patient had extrapontine myelinolysis. The child recovered completely after hospitalization for nearly 2 weeks. Extreme hypernatremia is rare in infants, and rehydration therapy can be challenging, as sodium levels should be reduced very slowly . There aIn this case report, we present the pitfalls of severe hypernatremia, its treatment and complications including (mis)interpretation of radiological imaging, and therapeutic conclusions.A 2-year-and-8-month-old Swiss girl presented to a pediatric office with fever of 38.8°C, vomiting, and refusal to eat for 3 days. Prior to admission, according to the parents, the patient had drunk ca. 500 ml of fluids. This was a response to a reviewers remark concerning signs of thirst. Blood analysis demonstrated severe hypernatremia (196 mmol/l), prompting urgent hospital admission.The patient was born at term (40 + 1 weeks of gestation) and had a birth weight of 3390 g. She was delivered through a C-Section due to pathological cardiotocography (CTG) and green amniotic fluid. The APGAR score was 6/8/10. On the second day of life, she developed bilateral parenchymal and intraventricular grade III brain hemorrhage diagnosed by ultrasound. Additionally, she had recurring seizures, which were successfully treated with phenobarbital (3 mg/kg/d). cMRI at two weeks of age showed hydrocephalus with intraventricular hemorrhage in the caudothalamic groove displacing, but not including the thalamus, as well as a small intraparenchymal hemorrhage of the right parietal side and subarachnoid hemorrhage of the left occipital side, along the tentorium and the cisterna cerebellomedullaris, with signs for slight hypoxia.Postnatally, the patient also had hypernatremia of 180 mmol/l, which was treated with infusion therapy (glucose 5%). We are not aware of any further urine or serum measurements . Neonatal ultrasound showed, slight hyperplasia and no adrenal hemorrhage and tumor. She was discharged with a sodium level of 160 mmol/l. A “central dysregulation” etiology was hypothesized. Sodium levels in the first year were normal (or slightly elevated) and ranged from 136 to 154 mmol/l.After birth, fT3 and fT4 serum levels were decreased; thus, thyroxine substitution was initiated. The therapy was ended after 1 month because of a hyperthyroid metabolic state . Neonatal screening was unremarkable. Two weeks later, at the age of 6 weeks, fT4 was decreased again to 15.6 pmol/l , and thyroxine substitution was restarted.3, normal values 148 ± 37). Growth hormone deficiency was postulated followed by substitution with Norditropin. At the age of 12 months, cMRI was performed again, which showed partial pituitary dysgenesis and hydrocephalus malresorptivus.At the age of 1 year, growth arrest  as well as reduced oral intake were observed. IGF1 was reduced to 1.8 nmol/l , IGFBP3 was normal (1.07 mg/l), with no adrenal insufficiency , sodium was 136 mmol/l, no celiac disease, and bone age was normal. After re-evaluation of cMRI at 2 weeks of age, retrospective diagnosis of pituitary hypoplasia was carried out , length (93 cm), and head circumference (46 cm) were all below the 3rd percentile. Body temperature was 37.6°C, blood pressure was 97/65 mmHg, and heart rate was 102/min. The remainder of the physical examination was normal.The patient was admitted to the pediatric intensive care unit. Blood gas analysis showed excessive hypernatremia (187 mmol/l) and hyperchloremia (148 mmol/l) with normal pH and base excess . OsmolalInfusion with isotonic glucose-electrolyte solution (sodium 140 mmol/l and glucose 5%) was initiated. Potassium chloride (7.46%) was added due to mild hypokalemia of 3.02 mmol/l. Blood gas analysis was performed hourly, revealing a slow decrease of sodium to a minimum of 143 mmol/l , decreasBlood glucose on admission was 11.1 mmol/l and normalized with rehydration, suggesting the high blood glucose on admission was due to stress hyperglycemia. Supplementation of thyroid and growth hormones was continued. On the day of admission, urinary excretion was slightly reduced (ca. 2.4 ml/kg/h), and on the second day, the excretion increased (ca. 4 ml/kg/h). There was no fever or edema, vital parameters were stable, and other laboratory tests showed no abnormalities.µg/dl .The following values were measured on admission: Urine osmolality was 876 mosmol/kg , urine antidiuretic hormone (ADH) level was 23.70 ng/l , plasma aldosterone level was 7.0 ng/dl , and copeptin pro-arginine-vasopressin (AVP) was 4.4 pmol/l  was normal. MRI of the head showed vague, nonischemic diffusion impairment in the basal ganglia, corpus callosum, and subcortical regions on both sides , with onCryptococcus neoformans, Escherichia coli, Enterovirus, Haemophilus influenzae B, human herpesvirus 6 (HHV-6), human parechovirus, herpes simplex virus (HSV) 1/2, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae (group B streptococcus (GBS)), Streptococcus pneumoniae, and varicella zoster virus (VZV). Additional PCR for Mycoplasma and Rotavirus was negative. The antibody specificity index (ASI) for mumps, measles, rubella, varicella, CMV, Epstein–Barr virus (EBV), herpes simplex and Borrelia, as well as tick-borne encephalitis (TBE) antibodies was normal. CSF examination showed an absence of oligoclonal bands, and only a slight increase of immunoglobulin M (IgM) of 2.4 mg/l  and a slight increase in albumin quotient of 8.5  were noted.The results of cell count, protein, and glucose in the cerebrospinal fluid (CSF) were normal. Cefotaxime and aciclovir intravenous were initiated. Multiplex polymerase chain reaction (PCR) of the CSF was negative for cytomegalovirus (CMV), Creatine kinase (CK) was extremely elevated (12794 U/l), with normal values for other muscle enzymes (aspartate transaminase (AST), alanine transaminase (ALT)). Although CK-isoenzyme analysis was not performed, the elevated CK levels were assumed to be mostly brain creatine kinase (CK-BB). Rhabdomyolysis due to excessive hypernatremia remains a possible explanation. No seizures, as a possible explanation, were observed throughout the hospitalization.After 5 days, the child was transferred by plane to Switzerland, where the family resides.At the accepting hospital, the neurological symptoms remained. Rehydration was continued and slowly tapered over time. Brain computed tomography (CT) scan was performed to rule out sinus vein thrombosis. Antibacterial and antiviral therapy were stopped. Central dysregulation was discussed again concerning the origin of the hypernatremia. A retrospective review of the MRI was interpreted as extrapontine myelinolysis following extreme hypernatremia. After hospitalization for nearly 2 weeks, the child recovered completely.In emergency admissions, rapid evaluation of serum electrolytes and acid-base balance is indispensable. Imbalances require rapid medical intervention and application of evidence-based treatment using established guidelines when possible. Excessive hypernatremia is rare in infants and needs to be treated, as in this case, in a pediatric intensive care unit (PICU) due to high mortality.In this case, hypernatremia could have been caused by hypertonic dehydration , although the almost normal urine output of 2.4 ml/kg/h and normal urine osmolality are not typical of severe dehydration. Dehydration could have possibly been aggravated by a disturbance of thirst regulation (adipsic hypernatremia), which is often caused by intracranial lesions and injury to osmoreceptors , 7. On tRehydration therapy in hypernatremia is challenging for every pediatrician. Serum sodium levels should be carefully monitored and reduced very slowly (up to 0.5 mmol/l/h or 12 mmol/l/d) over time.hyponatremia is overcorrected. There are some reported cases of children with extrapontine myelinolysis with hypernatremia [The child in our case report was treated according to established guidelines, and sodium levels were decreased slowly as recommended . Still, natremia –4, makinPatients with central pontine myelinolysis have a poor prognosis, with mortality of around 25%. Mortality has decreased in the last few decades, and even children with severe neurological deficiencies can have a complete recovery .Although normalization of sodium levels was achieved according to guidelines, the development of subsequent neurological deficiencies on day 4 was puzzling and led to additional procedures. Impairments of diffusion in the basal ganglia, corpus callosum, and subcortical regions in the cMRI with normal angiography and only subtle signal variations in T2-FLAIR were misinterpreted as meningoencephalitis. The misinterpretation was probably because the radiologists not being aware of the extreme electrolyte imbalance on admission. The T2 “shine-through” effect can effectively be ruled out, as our radiologists intentionally use high B-values to prevent such an effect. Contrast agent administration was not performed.Typical radiological features of ODS in the T2 sequence are hyperintensity in the central pons (CPM) or basal ganglia (EPM). Other possible locations include the hippocampus and lateral geniculate bodies.Impairment is mostly symmetric, and in acute situations, a restricted diffusion (hyperintensity in DWI) with a lower apparent diffusion coefficient (ADC) is possible. These changes can often be reversible in subacute cases . EnhancePatients should be assessed neurologically daily during rehydration therapy for pronounced electrolyte imbalance, aiming to detect neurological status changes promptly and to be able to respond quickly by modifying therapy regimes.Imaging techniques should be performed in a timely manner, and interpretation requires integration of clinical context and laboratory findings to avoid misdiagnoses and treatment.Hypertonic dehydration can be life-threatening and often correlates with neurological complications (EPM and CPM).Quick and potentially repetitive radiological imaging is the diagnosis method of choice.Not every MRI enhancement means inflammation. Interpretation must involve active collaboration between radiologists and clinicians. Loss of information can lead to improper conclusions.The following points can be learnt:


Background: There have been some gradual changes in the distribution of renal biopsy pathological diagnoses during recent years. This study aimed to show changes in renal disease prevalence in China by investigating 10 patients diagnosed at our Kidney Disease Centre during the last 15 years.Methods and results: All patients aged 15-year-old or older who underwent renal biopsy at the First Affiliated Hospital, Zhejiang University, from 2001 to 2015 were enrolled. There were 5 common types of primary glomerulonephritis: IgA nephropathy (IgA N), membranous nephropathy (MN), mesangial progressive glomerulonephritis (MsPGN), minimal change disease (MCD), and focal segmental glomerulosclerosis (FSGS), which represented 50%, 16.8%, 15.9%, 8.1% and 2.5% of total cases, respectively. IgA nephropathy was the most common type of primary glomerulonephritis (PGN).Conclusions: Our results mostly showed a new trend that the diagnosis of IgA nephropathy was not increasing and the prevalence of membranous nephropathy had increased, becoming the second most common type of primary glomerulonephritis.Distinguished with other domestic studies, IgA nephropathy did not show a trend of continuous growth although it still had about the half proportion of PGN, whereas membranous nephropathy kept rising and became the second common PGN.Concerning SGN, LN peaked in the younger-age and middle-age groups with a significant female prevalence, DN, BANS and SV had a male predominance peaking in the middle-age and old-age groups. Moreover, lupus nephritis (LN) and Henoch-Schonlein nephritis (HSN) remain the most common secondary glomerulonephritis (SGN) while diabetic nephropathy (DN) is less prevalent in our study compared to reports from some Western countries. Therefore, we aimed to evaluate changes in the proportion of renal pathologic diagnoses in East China during the last 15 years.Diagnostic information obtained from renal biopsy is critical for accurate diagnosis, selection of the most appropriate treatment, and prediction of patient prognosis. In recent years, retrospective studies of renal biopsies, with statistical analyses of different regions, races and time periods, have provided informative findings. For adults, IgA nephropathy is the most common primary glomerulonephritis (PGN)  in many Overall, 10 877 patients  underwent renal biopsy at the First Affiliated Hospital, Zhejiang University during the 15 years period from January 2001 to December 2015. We retrospectively analyzed records of patients aged ≥15 years old. Patients with incomplete records, inadequate biopsies, and repeated biopsies were excluded. In the end, 10 779 cases were included in our analysis. The date of renal biopsy, age, sex, pathologic type and pathologic diagnosis was collected.Clinical indications for renal biopsy were as follows: (1) nephrotic syndrome or nephrotic range proteinuria, (2) acute nephritic syndrome, (3) rapidly progressive glomerulonephritis, (4) chronic nephritic syndrome, (5) asymptomatic hematuria or proteinuria, and (6) acute or chronic renal failure without definite cause. There was no significant change in the indications for renal biopsy during the observation period. Almost all patients, without obvious contraindications, will have a biopsy as long as they meet the criteria. This study was approved by the local ethics committees. Informed consent was obtained for renal biopsy from each patient.Data were divided into 3 groups according to 5-year intervals: 2001–2005, 2006–2010 and 2011–2015. Biopsies were also divided into 6 groups according to patients’ age for stratified analysis: 15–24 years old, 25–34 years old, 35–44 years old, 45–54 years old, 55–64 years old, and elderly (≥65 years old). All biopsy specimens were prepared and examined by the same group of clinicians, pathologists and technicians. A renal biopsy was processed for light microscopy, immunofluorescence microscopy and electron microscopy in all submitted cases. The final diagnosis made for each patient was based on both clinical and histologic investigations.All biopsies were categorized for the purpose of this analysis as PGN, SGN, hereditary nephropathy (HN), tubulointerstitial nephropathy (TIN), or other miscellaneous or undefined histological diagnoses. In this study, we analyzed only the common categories of PGN and SGN. PGN included IgA N, MN, non-IgA mesangial proliferative glomerulonephritis (MsPGN), minimal change disease (MCD), and focal segmental glomerulosclerosis (FSGS). SGN included LN, HSN, DN, systemic vasculitis (SV), hepatitis B virus-associated glomerular nephropathy (HBV-GN), benign nephrosclerosis (BANS) and amyloidosis nephropathy (AMYN).p < .05 defined as significant. All statistical analyses were performed using SPSS software, version 22.0 .Results are presented as the means and the standard deviations or the medians with the interquartile ranges, and categorical variables are presented as frequencies with percentages. The distribution of patients with various renal biopsy diagnoses compared between the 5-year intervals, age groups and sex was calculated using Pearson’s chi-square analysis. Bonferroni method was accepted when involving multiple comparisons among groups. All statistical tests were two-sided, with a value for In total, 10 779 patients were reviewed. Mean patient age at the time of biopsy was 40 ± 14.86 years old ranging from 15 to 87 years. There was a gradual increase in the mean age upon renal biopsy with subsequent 5-year intervals, including 37.07 ± 13.77 years old in 2001–2005, 39.71 ± 14.37 years old in 2006–2010 and 42.98 ± 15.18 years old in 2011–2015. There was a sharp increase in the number of patients who underwent renal biopsy throughout the period of analysis, from less than 50 cases in 2001 to more than 1000 cases during the most recent 5 years .As shown in p < .001). The diagnosis of TIN and HN were comparable across all three time periods. In total, a significant difference in the sex distribution, the male-to-female ratio was 1.04:1 . Multiple comparisons by Bonferroni method revealed sexy distribution difference of respective pathological pattern. The results showed male predominance among PGN biopsies (1.09:1) and HN biopsies (2.44:1) but a female predominance among SGN biopsies  of all biopsies; this included 1478 biopsies (82.7%) from 2001 to 2005, 2794 biopsies (79.9%) from 2006 to 2010 and 4222 biopsies (76.8%) from 2011 to 2015, with a slight decreasing trend over time. To the contrary, the rate of SGN increased from 14.2% in 2001–2005 to 17.4% in 2006–2010 and 20.1% in 2011–2015. The chi-squared test revealed a significant difference in the number of PGN patients among three 5-year intervals ; IgA N showed a gradually decreasing trend in the most recent 5 year time period. The next most common PGN diagnosis was MN (16.8%), followed by MsPGN (15.9%), MCD (8.1%), and FSGS (2.5%). MsPGN was the second most common primary glomerular disease in the initial year (2001), but the rate of MN dramatically increased to account for an equivalent number of biopsies in the middle time period and surpassed all diagnoses except IgA N to become the second most common PGN after 2009. During the same period, the diagnosis of MsPGN decreased significantly, whereas the proportion of biopsies diagnosed with FSGS remained constant over the entire study period. The rate of MCD diagnosis underwent a rapid rise during the middle time period, and the most recent time period showed approximately the same rate of diagnosis. Except MsPGN, other categories of PGN show a distinguished sexy ratio: more female patients in IgA N and more male persons in MN, FSGS and MCD (The most frequent diagnosis within the PGN group was IgA N ( and MCD .There were 7 common SGN categories diagnosed at our center: LN (29.2%), HSN (19.2%), DN (9.9%), SV (8.5%), HBV-GN (7.8%), BANS (10.3%) and AMYN (4.8%). There was a male predominance among DN, BANS, AN and HBV-GN biopsies with statistically significant difference whereas females were absolutely prevalent among LN biopsies . LN was p < .001) and nearly increased 2 times from 2006–2010 to 2011–2015. The analysis by age category indicated that the frequency of MN in all age categories increased significantly over time (p < 0.001).The distribution of IgA N by age group appeared a ‘triangle’ shape, peaking in the 25–34 years old age group  and 35–44 years old age group , which corresponded with previous reports from Peking University in China [p < .001), DN increased over twice (p < .001).LN, HSN, DN, SV, BANS, AMYN and HBS-GN were the most common SGN diagnoses. As shown in Type IV and type V were the most common histologic diagnoses within LN, both types in total occupying about 60%. The following was IV + V miscellaneous type (11.9%). As for other types, each type respectively was less than 10% with a small share .There was a dramatic increase in the number of renal biopsies at our center during the past 15 years, which correlates with an increased number of ward beds and improvements in renal biopsy technology. In addition, with improvements in economic status and health care reform, more patients could afford payment for renal biopsy. Based on the stratified analysis of age, the proportion of biopsies in patients over 45 years old increased, consistent with the recent report on the prevalence of biopsy-proven renal diseases in elderly Chinese patients from the Nan Jing . The incWe found IgA N to be the most common PGN, comprising about 50% of cases of PGN; this is similar to previous reports from our country ,12,17. HAnother significant difference we found was that MN dramatically increased during the study period, representing a larger proportion (25.2%) of PGN in 2014 than in the other years. We found a similar trend reported in previous studies in China . The resst century, which continued through the most recent 5 years. As we know MsPGN is an early stage of many diseases, we are currently used for a small amount of proteinuria or/and hematuria, that with certain mesangial cells and mesangial matrix hyperplasia but without a dense substance by LM and EM. And as the disease diagnosis progresses, the number of diagnoses of this disease in our center is also gradually decreasing. This is similar to previous domestic reports [There was a noticeable decrease in the diagnosis of MsPGN during the first decade of the 21 reports ,12,17. OMoreover, we found a higher proportion of LN and a lower proportion of FSGS and DM compared with European  and AmerThere are some limitations of our study. Firstly, as a single-center study, the enrolled patients may not have been an adequate representation of the entire Chinese population. Our hospital is the referral medical center for renal disease in the Zhejiang Province in the eastern part of China, we received many referral patients, particularly severely ill patients. Thus a detection bias was unavoidable. It is also worth mentioning that this study only reflects the frequencies of kidney diseases among those who have undergone renal biopsy, and thus these findings may not be applicable to the general population. However, the reporting of renal biopsy data from a single center with stable biopsy and diagnostic criteria is useful for the assessment of changing patterns in renal disease. Secondly, as it is an observational study, we could only observe the changes in the frequencies of kidney diseases and thus could not fully explain the reasons behind these changes. Some studies are limited by a lack of clinical details and variability in renal biopsy practices across the whole country; we could not determine whether the changes in the proportion of renal biopsy diagnoses were due to differences of environment, patient-specific factors, social development, or differences amongst geographic regions. Hence, follow-up studies are required to elucidate the roles of various factors in the frequencies of kidney diseases.In conclusion, the pathologic spectrum of renal disease has changed within the last 15 years, especially concerning PGN. IgA N remains the most common PGN, although there was a steady decline in the most recent 5 years. In the 5-year interval comparison, the relative frequency of MN has continued to increase, while the relative frequency of MsPGN has dramatically decreased over the past 15 years. Concerning SGN, LN peaked in the younger-age and middle-age groups with a significant female prevalence, whereas DN, BANS and SV had a male predominance peaking in the middle-age and old-age groups.


Swallowing function and swallowing-related quality of life (QoL) can be adversely affected in patients after partial laryngectomy, but are often neglected by patients and clinical workers. This study aimed to investigate the degree of swallowing function and swallowing-related QoL after partial laryngectomy in patients with laryngeal carcinoma.Sixty-eight hospitalized patients undergoing partial laryngectomy due to laryngeal carcinoma at the Second Affiliated Hospital of Xi’an Jiao Tong University were included in this prospective study. A general information questionnaire was used to collect baseline characteristics. The water swallow test and swallowing quality of life questionnaire (SWAL-QOL) were carried out the day before surgery and at 2, 4, 12, 24 and 48 weeks after surgery.P < 0.05). There was no statistical difference between postoperative 24 and 48 weeks (P = 0.379).Swallowing dysfunction occurred in 1 case (1.5%) the day before surgery and in 49 (72.1%), 44 (64.7%), 33 (49.3%), 19 (28.4%) and 8 (11.9%) cases at 2, 4, 12, 24 and 48 weeks after surgery, respectively. Mean SWAL-QOL total scores were 4266.3 ± 232.0 the day before surgery, and 1992.9 ± 1062.4, 2473.9 ± 962.9, 3169.2 ± 753.6, 3696.7 ± 718.3 and 3910.8 ± 1510.4 at 2, 4, 12, 24 and 48 weeks, respectively. SWAL-QOL total scores increased gradually after operation, and the differences were statistically significant (Partial laryngectomy affects swallowing function and swallowing-related QoL in patients with laryngeal carcinoma. While swallowing function and swallowing-related QoL increase gradually over time, in some patients, nearly a year after surgery they are not fully restored. Therefore, attention should be paid during postoperative nursing to improve swallowing function. Laryngeal cancer is one of the most common malignant tumors encountered in the otorhinolaryngology department . In ChinThe purpose of partial laryngectomy for laryngeal cancer patients is to preserve and reconstruct laryngeal function while ensuring the safety margin. Considering the tumor location, size, shape, range, pattern of growth and differentiation extent, the resection area can be expanded or reduced , using mIt takes a long time for most patients after partial laryngectomy to recover the swallowing function as they rely on self-exercise or natural rehabilitation of nerve and muscle function . In addiConsecutively laryngeal carcinoma patients undergoing partial laryngectomy in the department of otorhinolaryngology surgery, the Second Affiliated Hospital of Xi’an Jiao Tong University, were enrolled between January 2015 and December 2016 in this prospective study. Laryngeal carcinoma was staged according to the seventh edition of TNM staging of the American Joint Committee on Cancer (AJCC). Selection of partial laryngectomy was mainly based on the location, size and local invasion of the tumor. The inclusion criteria were: (1) postoperative histopathological diagnosis as laryngeal squamous cell carcinoma; (2) planned first partial laryngectomy; (3) no operation history for the head or neck within three months; (4) no existing or potential disease causing swallowing dysfunction; (5) no severe surgical complications; (6) consent to participate in this study. The exclusion criteria were: (1) abnormal communication due to consciousness disturbance; (2) recurrent tumor or partial laryngectomy performed several times. The study was carried out in accordance with the declaration of Helsinki and approved by the ethics committee of the Second Affiliated Hospital of Xi’an Jiao Tong University. Informed consent was obtained from all patients.In this study, swallowing function was assessed by the water swallow test. A general information questionnaire along with the swallowing-related QoL questionnaire were used to investigate swallowing-related QoL. The water swallow test  is an exA general information questionnaire was designed by the authors with reference to the relevant literature combined with clinical data of laryngeal carcinoma patients, including age, gender, ethnic, education level, marital status, occupation status, smoking and drinking statuses, condition of laryngeal diseases and operation selection. The Swallow Quality-of-Life Questionnaire  , with goMedical records and preoperative discussions were consulted, and the patients were included according to the above inclusion and exclusion criteria. The general information questionnaire was completed after the patient provided a signed informed consent. Swallowing function was assessed by a trained investigator using the water swallow test. Then, SWAL-QOL was completed during outpatient follow-up the day before surgery and at 2, 4, 12, 24 and 48 weeks after operation. All the patients accepted unified guidance to fill out the questionnaire by themselves. The questionnaire was completed in about 10 min. The investigator checked the questionnaire to make sure that there were no missing items, and answers were not repeated several times in a random way; the collected data were valid.www.EpiData.dk). Continuous variables were expressed as mean ± standard deviation (SD), and repeated measures analysis of variance was used to compare differences in SWAL-QOL at different times. Categorical variables were expressed as frequency and percentage, and differences in swallowing function at different times were compared by the Chi-square test. P < 0.05 was considered statistical significant.SPSS 17.0  was used to analyze the data obtained from the survey with the EPIDATA software  were issued, but one patient died after completing only three questionnaires, so a total of 405 effective questionnaires were collected, for a recovery rate of valid questionnaires of 99.3%.Patient characteristics are shown in Table Cancer stage distribution is shown in Table P = 0.047), 24 (P = 0.022) and 48 (P = 0.021) weeks, but eight (11.9%) patients were still affected after 48 weeks.Swallowing function was adversely affected by surgery in 49 patients (72.1%) 2 weeks later Table . SwallowP < 0.001) except the final one, since total scores were similar at 24 and 48 weeks (P = 0.379).SWAL-QOL total scores were decreased after surgery, but slowly recovered over the 48-week follow-up, as shown in Table The aim of this study was to investigate swallowing function and swallowing-related QoL in patients with laryngeal carcinoma after partial laryngectomy. Swallowing dysfunction occurred in 72.1% of patients immediately after surgery but recovered slowly until 48 weeks, at which time 11.9% of patients were still experiencing abnormal swallowing function. The SWAL-QOL total score was negatively impacted upon by surgery but increased gradually after operation, but there was no statistical difference between SWAL-QOL total score at 48 weeks and 24 weeks postoperatively. These results revealed that in some patients, nearly a year after surgery, swallowing function and swallowing-related QoL were not restored. Therefore, more attention should be paid during postoperative nursing to improve swallowing function. For instance, patients should be trained to overcome the psychological barriers of swallowing. Because of the fear that coughing resulting from swallowing may affect wound healing, many patients do not dare eat or swallow, just keeping food in the mouth. In this case, nurses should be patient in explaining that the wound has already healed and mild coughing would not affect recovery, in order to encourage the patients to relax and swallow boldly. In addition, health care professionals should help the patients identify their suitable eating position.After partial laryngectomy, the degree of swallowing dysfunction is directly related to the type of operation, the resection area and the larynx reconstruction method , 24. In P = 0.862) between postoperative 24 and 48 weeks. This may be explained by the following reasons. (1) The nerves and muscles could recover, and swallowing function could gradually improve after partial laryngectomy. (2) Most laryngeal cancer patients could psychologically confront difficulties such as persistent cough or expectoration with positive attitudes after medical staff education when they are aware of the likely good prognosis with high survival rate.Because the larynx has important functions in swallowing, breathing and speaking , laryngeThe results of this study highlight the need to implement methods for improving swallowing function after laryngectomy. A previous study suggested that a patient support network may help solve swallowing problems using a patient-to-patient communication model . Then, aThis study also has some limitations. First, healthy patients were not included, and the data lack reference values. In addition, data relating to postoperative exercise and nursing intervention for patients were not collected and analyzed. Meanwhile, the quite small sample size may have introduced some biases. Larger studies with longer follow-up are needed to determine comprehensive and long-term impacts and related factors of partial laryngectomy on swallowing function and swallowing-related QoL.Partial laryngectomy is a commonly used therapy in laryngeal carcinoma to possibly maintain laryngeal function. Long-term swallowing function and swallowing-related QoL are both adversely affected in some patients even 48 weeks after surgery. This highlights the need for nursing staff to pay more attention to changes in swallowing function and swallowing-related QoL for laryngeal carcinoma patients after partial laryngectomy, and provide the necessary education and/or training to patients.