Although preterm birth is associated with respiratory morbidity in childhood, the role of family history of atopy and whether appropriate treatment has been instituted is unclear. Thus we assessed (i) the prevalence of respiratory symptoms, particularly wheezing, in childhood; (ii) evaluated the role of family history of atopy and mode of delivery, and (iii) documented the drug usage, all in preterm-born children compared to term-born control children.We conducted a cross-sectional population-based questionnaire study of 1–10 year-old preterm-born children  and matched term-born controls . Data  was analysed by gestational groups  and by age, <5 years old or ≥ 5 years.Preterm born children aged <5 years  had higher rates of wheeze-ever . Similarly for the ≥5 year age group  wheezing increased with increasing prematurity . At both age groups, inhaler usage was greater in the lowest preterm group but prematurity-associated wheeze was independent of a family history of atopy.Increasing prematurity was associated with increased respiratory symptoms, which were independent of a family history of atopy. Use of bronchodilators was also increased in the preterm groups but its efficacy needs careful evaluation. Preterm birth, defined as birth before 37 weeks’ of gestation, has a major impact on health services across the world, with preterm birth rates varying from 7% in England and Wales to 11.4% in the USA . Childre1) and have increased admissions to hospital for respiratory related illnesses when compared to term-born children [Preterm-born children are at risk of developing deficits in percentage forced expiratory volume in one second  children. Specifically we have quantified the association between gestational age at birth and subsequent respiratory-associated symptoms in preschool (<5 years of age) and school-aged (≥5 years of age) children. We also evaluated the role of a family history of atopy and mode of delivery in the reporting of prematurity-associated wheeze and documented drug usage in preterm-born children classified by gestational groups comparing all the results from the preterm group to the term group.st January 2003 and 31st December 2011. During this period there were 305,894 live births in Wales of which 22,383 were born preterm (<37 weeks’ gestation). We studied all preterm-survivors born in 2003, 2005 and 2007 thus were aged between 5 and 10 years of age in 2013 , and those born between 2009 to 2011 thus were aged less than five years of age in 2013 . In total 13,361 preterm-born children together with an equivalent number of term-born control subjects born on the same day, gender and locality  were invited to take part. All the subjects were identified from the NHS Welsh Informatics Service (NWIS), which collates data from health databases in Wales including Patient Episode Database for Wales , National Community Child Health Database  and Welsh Demographics Service . The South East Wales Research Ethics Committee approved the study. The parents/guardians of the children, by virtue of completing and returning the questionnaires, consented to participate.We conducted a cross-sectional population study of children born preterm between 1Questionnaires were mailed in April 2013 to the parents of the 26,722 subjects with a reminder mailed to non-responders in June 2013. Children aged five years or less were sent the validated Liverpool Respiratory Symptom Questionnaire (LRSQ)  and a moth centile for birth weight (after correcting for gender and gestation) and was compared to children with birth weights between 20-80th centile. Participants with birth weights outside ±3.5 SDs or with unknown gestational age were excluded. Of the 7,149 responders, 97 children were excluded due to implausible birth weight or gestational age at birth.The main exposure of interest, gestational age at birth, was based on antenatal ultrasound scans and maternal reporting and recorded in completed weeks of gestation. It was categorized into four groups: 24 to 32 weeks (very preterm), 33 to 34 weeks (moderate preterm), 35 to 36 weeks (late preterm) and 37 to 43 weeks (term controls) as in our previous report . Birth wRespiratory outcomes were based on questionnaire responses and included wheeze-ever, recent wheezing , other respiratory symptoms , use of inhalers (bronchodilators and corticosteroids), hospital admissions for chest-related problems in the last twelve months, doctor diagnosed asthma and family history of atopy, which was based on parental reporting of first degree relatives having a history of asthma, eczema and hay fever. Diagnosis of CLD was based on parental reporting and checked with the data on the hospital discharge notification. CLD, which was confined to the children born at ≤32 weeks of gestation as need for supplemental oxygen until at least 36 weeks of corrected gestational age , 22. ParDescriptive analyses were used to summarize the characteristics of all responders, matched case-control responders and of all non-responders. Characteristics included age, gender, gestational age, birth weight, WIMD score, maternal age, maternal smoking during pregnancy, mode of delivery and IUGR. For the characteristics of the responders, we compared results between the gestational groups using appropriate statistical tests  after testing for normality by assessing distribution of frequencies and by performing normal probability plots. Since the response rates were greater amongst the preterm group than the term group, we performed sensitivity analyses by comparing wheezing rates between matched preterm- and term-born children. Because of ethical arrangements, we only had access to identifiable data for responders thus the term-borns were matched with a date of birth within 5 days of the preterm-subjects and with same locality and gender.The prevalence rates were calculated as a percentage of the study population for each gestational group. The prevalence rates of respiratory symptoms, inhaler treatment, and hospital admissions are reported according to gestational groups with odds ratio to measure the effect size between groups. Separate univariate analyses by ANOVA were constructed for children less than five years-of-age and those five years-of-age and older. Further univariate analyses using ANOVA were conducted to identify risk factors that were associated with wheezing; those statistically significant (p<0.05) were included in multivariable logistic regression analysis for wheezing. Two generalized linear models of multivariable analyses were performed: the first included all significant risk factors and the second included only family history of atopy. Confounding and significant risk factors included in the generalized linear modeling were gender, family history (FH) of atopy, maternal smoking during pregnancy, mode of delivery, maternal history of asthma, breastfeeding at birth, current maternal smoking, social status, ethnicity, and other family member smoking. Adjusted odds ratios were calculated.A sub-group analysis of wheezers only was performed, by comparing preterm- and term-born wheezers. Univariate analyses using ANOVA were conducted to identify risk factors associated with prematurity-associated wheezing. A separate univariate analysis by ANOVA was performed to assess the influence of family history of atopy on wheezing preterm-born children receiving inhaler treatment compared to wheezing term-born children on inhaler treatment. Analyses were also performed to assess the differences in prevalence of wheezing and inhaler treatment between preterm-born children with and without a family history of atopy and term-born children with and without a family history of atopy. Finally a sub-group analysis of the very preterm group was performed to assess the differences in prevalence of wheezing and inhaler treatment between children with CLD and those without CLD. The statistical analyses were performed using PAWS . p<0.05 was considered significant.There were 7,149 26.7%) responses with 4,284 (60%) from preterm-born and 2,865 (40%) from term-born children . The cha6.7% respLower gestational age at birth, male gender, maternal smoking during pregnancy, current maternal smoking, other family member current smoking, maternal history of asthma, delivery by caesarean section (CS), family history of atopy, lower WIMD rank, and ethnicity were all, individually, significantly associated with increased rates of wheezing for both age groups (p <0.05). These risk factors were all included as in the multivariable analysis of wheezing symptoms Tables  and 4. IThe prevalence of wheeze-ever in children less than five years-of-age was 64.7%, 55.5%, 51.4% and 40.7% in the very preterm, moderate, late preterm and term groups respectively with odds ratios of 2.67 , 1.82  and 1.54  respectively . SlightlThe prevalence of wheeze-ever in children aged five years or older was 55.8%, 41.3%, 37.8% and 27.7% respectively for the very preterm, moderate, late preterm and term groups with odds ratios of 3.29 , 1.84  and 1.58  respectively . SlightlThe results of the sub-group analysis on only wheezing children identified the following significant risk factors for preterm-born wheezy children aged less than five years: being delivered by CS, IUGR, and current maternal smoking  suggesting that atopy does not play a part in the prematurity-associated wheeze . However, preterm-born children d wheeze . Similard wheeze .Children with CLD, who were confined to the very preterm group, had lower birth weight and gestational age than very preterm without CLD  born preterm have greater respiratory symptoms, increased hospital admissions for respiratory related morbidity and greater use of inhaler medication compared to term-born children. A gradient across the gestational age groups was observed with those born very preterm having the highest prevalence rates. Importantly, we show that prematurity-associated wheeze appears to be independent of a family history of atopy. Our results confirm that children born very preterm have significantly higher prevalence of respiratory symptoms including wheezing during pre-school aged years as previously reported , 24 but Our study also noted that preterm-born children were being treated with inhaler medication (bronchodilators and corticosteroids) with greater frequency than term-born children. This too showed a gradient effect with increasing drug usage with decreasing gestational age at birth. Some studies have previously reported under treatment especially in those born very preterm with CLD , 9. WhetOur recent systematic review of studies evaluating efficacy of inhaled bronchodilator identified 21 studies but only one study from 1997, assessed longer-term response to terbutaline showing an improvement . The remA meta-analysis of >147,000 children from 31 European birth cohorts showed that children born a lower gestational age or with accelerated infant weight gain have increased risk of childhood asthma . The autIn our study, the risk factors most strongly associated with wheeze-ever in preterm-born children were the degree of prematurity with decreasing gestation associated with increasing symptoms; mode of delivery and IUGR for both age groups; and current maternal smoking for the less than 5-years of age group. Mode of delivery is likely to be associated with the degree of prematurity but IUGR has been reported to be associated with increased respiratory disease in preterm-born infants in the neonatal period  and in tThe mechanisms underlying wheezing disorders following preterm birth are unclear. Children with atopic asthma have obstructive airflow secondary to airway inflammation and bronchial hyper-responsiveness, which is reversible with bronchodilators. However, preterm-born children, or even those born early-term, with reIn survivors of CLD, the underlying lung pathology may resemble that of pulmonary emphysema rather than asthma . Our datOur study has a few limitations. It is a cross-sectional study relying on parental reporting of symptoms. Thus there is potential for recall bias. This may have affected the wheezing rates for children over 5 years of age, which reports a lower rate of ever wheezing than children less than 5 years of age. However previous studies suggest that parental recall of acute illness correlates well with medical records , 45. We This large population-based study has confirmed and further quantified that preterm-born children have increased prevalence of respiratory symptoms. Children born preterm with or without a family history of atopy had a higher prevalence of wheezing and were more frequently on inhaler treatment when compared to term-born children. Prematurity-associated wheeze is likely to be a separate entity to the wheezing observed in term-born children especially as delivery occurs at an earlier stage of lung growth, which may affect subsequent lung development. It is vital that we identify optimal treatment for this significant group of children by identifying the underlying mechanisms responsible for the airway symptoms and obstruction together with adequately powered randomised controlled trials.S1 FileTable A: Characteristics of non-responders <5 years of age according to gestational groups. Table B: Characteristics of non-responders ≥5 years of age according to gestational groups. Table C: Univariate analysis of risk factors for preterm wheezing children less than 5 years-of-age (unadjusted OR). Table D: Univariate analysis of risk factors for preterm wheezing children 5 years-of-age and older (unadjusted OR). Table E: Characteristics of very preterm born children with and without CLD compared to full term born controls. Table F: Characteristics of matched case-control responders <5 years of age according to gestational groups. Table G: Characteristics of matched case-control responders’ ≥5 years of age according to gestational groups. Table H: Childhood wheezing for all children less than 5 years-of-age compared by gestational age for matched case-controls only (unadjusted OR). Table I: Childhood wheezing for all children 5 years-of-age and older compared by gestational age for matched case-controls only (unadjusted OR).(DOCX)Click here for additional data file.


Non infectious uveitis in childhood is a relatively uncommon severe disease, with potential significant long-term complications such as cataract, glaucoma and eventually blindness. For these reasons refractory uveitis usually requires early and aggressive immunosuppressive treatment in order to preserve visual acuity and to prevent the significant morbidity of chronic steroid administration. However, the lack of evidence from head-to-head randomized controlled trials (RCT) limits our understanding about the best treatment choices, as well as time of instituting therapy and its duration. Once a child with active uveitis has achieved remission on treatment, there are no evidence-based guidelines with respect to the duration of continued treatment in autoimmune child chronic uveitis. Information regarding the natural clinical history of a child on systemic treatment due to auto-immune chronic uveitis would be helpful in driving therapy: if it would be possible to identify early children at high risk to flare once systemic therapy is stopped, they could have benefits in visual acuity prognosis and quality of life, as well as time free from disease.Aim of our study was to assess the time on remission after discontinuing systemic therapy in a retrospective, comparative, multi-centre, cohort study of childhood non-infectious chronic uveitis.inactive uveitis, which discontinued all related treatments for at least 3 months were enrolled. 23 children previously received Methotrexate, 16 TNF inhibitors. Primary outcome was to assess, once remission was achieved, the time on remission up to the first relapse after discontinuing treatment. Time to remission once systemic not-steroid treatment was started, time to steroid discontinuation, number of relapses before achieving remission and time on remission on therapy before discontinuing all treatments were also considered.39 patients  from 4 different paediatric rheumatology centres, with previously refractory, vision threatening, non-infectious Median follow-up time on remission without treatment was 9 months (range 1–59 months). At last available follow-up after 1 year from discontinuation of treatment, 11/39 (28.2%) children maintained a complete remission over a median period of 18 months. At 49 months of follow-up, 6/8 children with IdCU (75%) compared to 5/31 children with JIA (16.1%) were still on remission without treatment (p<0.003). A higher probability of maintaining uveitis remission after discontinuing treatment was shown in IdCU compared to JIA group . ANA positivity was associated with a higher probability of flare in overall population , in ICU, but not in sub-analysis limited to JIA  and IdCU . None clinical variable, including time on remission on therapy, total length of treatment, and type of treatment, resulted significant predictors of long-lasting remission without therapy.Even if limited to a relatively small group in retrospectively study design, our results suggest that type of disease, rather than the type or the length of treatment, can predict different duration of uveitis remission without systemic therapy.None declared.


Arabidopsis are largely determined by root meristematic activity. Legume roots show strong developmental plasticity in response to both abiotic and biotic stimuli, including symbiotic rhizobia. However, a global analysis of gene regulation in the root meristem of soybean plants is lacking. In this study, we performed a global analysis of the small RNA transcriptome of root tips from soybean seedlings grown under normal and salt stress conditions. In total, 71 miRNA candidates, including known and novel variants of 59 miRNA families, were identified. We found 66 salt-responsive miRNAs in the soybean root meristem; among them, 22 are novel miRNAs. Interestingly, we found auxin-responsive cis-elements in the promoters of many salt-responsive miRNAs, implying that these miRNAs may be regulated by auxin and auxin signaling plays a key role in regulating the plasticity of the miRNAome and root development in soybean. A functional analysis of miR399, a salt-responsive miRNA in the root meristem, indicates the crucial role of this miRNA in modulating soybean root developmental plasticity. Our data provide novel insight into the miRNAome-mediated regulatory mechanism in soybean root growth under salt stress.Root growth and the architecture of the root system in  The root is an important organ that not only supports the aerial parts of plants, but also absorbs water and nutrients for plant growth and survival. The growth rate and architecture of roots are dynamically regulated by internal and external cues, and they are largely determined by the activity of the root apical meristem (RAM)  are small non-coding RNAs that modulate various biological processes at the post-transcriptional level by mediating mRNA degradation or translational repression Bartel, . Recent Arabidopsis, including the maintenance of ion homeostasis, adjustment of cellular responses to osmotic stress, and metabolic responses  were surface-sterilized with 75% ethanol for 1 min, followed by chlorine for 8 h. The sterilized seeds were germinated in plastic pots filled with 400 ml of solid B5 medium containing 0 or 75 mM NaCl. Seedlings were grown in a greenhouse under 16 h of light at 28°C for 5 days. Root tips (0.5 cm) were collected from the plants for high-throughput sequencing, histological sectioning, and indoleacetic acid (IAA) measurement. For 2,4-D treatment, five-day-old seedlings were transferred to liquid B5 medium supplemented with or without 1 μM 2,4-D. After 3 days, root tips were collected.Seeds  were collected from the medium containing 0 or 75 mM NaCl. IAA was extracted and measured as described by Fu et al.  with modTo prepare longitudinal sections of the roots, root tips were collected from the medium with or without 75 mM NaCl and then fixed in formalin/acetic acid/alcohol. The root segments were dehydrated at room temperature in a gradient series of ethanol solutions, followed by two changes of Clear-Rite 3  for 1 h each, and finally embedded in Paraplast  at 62°C. Sectioning was performed using a microtome. Sections were cut every 20 μm and then stained with 0.1% (w/v) Eosin Y. The sections were observed under an Olympus CX31 biological microscope .2. The samples collected from the two libraries were sent to Biomarker Technologies Co. (Beijing) for analysis. Small RNAs were isolated from stressed (SR) and non-stressed (CR) root tips using a TruSeq Small RNA Sample Prep Kit  to construct the libraries. The sequence data were analyzed using miRDeep2 by Biomarker Technologies Co. according to the manufacturer's protocols. The read abundances of miRNAs in the two libraries were normalized to the reads per kilobase of exon model per million mapped reads value . The P-value threshold was determined by the false discovery rate (FDR) to account for multiple tests of significance. To determine the functional annotation of the genes targeted by the differentially expressed miRNAs, a BLAST alignment was performed by searching the NR, SwissProt, GO, COG, and KEGG protein databases. This experiment consisted of three independent biological replicates.For small RNA library construction, root tips (0.5 cm) were harvested from B5 medium supplemented with (SR) or without (CR) 75 mM NaCl, and immediately frozen in liquid NTotal RNA was extracted from root tips using Trizol reagent , and then treated with DNase I to remove contaminating genomic DNA. Stem-loop-specific reverse transcription for miRNAs was performed as described previously  of gma-miR399a was amplified and inserted into the Gateway® binary vector pGWB2. The primers used for plasmid construction are listed in Table cis-elements of the miRNAs, 2000 bp of sequence located upstream of each salt stress-responsive miRNA was chosen as a putative promoter sequence for analysis using PLACE (http://www.dna.affrc.go.jp/PLACE).To analyze the t-test was performed using GraphPad Prism 5.All data were analyzed using SigmaPlot 10.0  and GraphPad Prism 5  software. Student's To investigate the responses of roots to salt stress, we performed a systematic study of soybean root development. As shown in Figure It is well known that the activity of the RAM determines root growth  and four miRNAs were dramatically down-regulated (log2FC < -0.5) under salt stress Figure . This recis regulatory elements in the 2-kb promoter regions of 17 salt stress-responsive miRNAs. As shown in Table cis-element, which is important for the auxin response, was present in the promoters of 13 miRNAs. Another two auxin-responsive cis-elements (NTBBF1ARROLB and CATATGGMSAUR) were found in the promoters of 16 miRNAs , which is an ubiquitin E2-conjugating enzyme  and Glyma.10G036800 (GmPT5), which have been validated as the targets of gma-miR399  in soybean. Furthermore, we found that several novel miRNAs target genes that are involved in various biological processes. For example, Gly13 was predicted to target seven genes, including those encoding cysteine synthase A, ubiquitin-like protease protein 2, and a class I glutamine amidotransferase. Our results suggest that these soybean miRNAs modulate RAM activity by coordinating complicated regulatory networks.Notably, we found that several known soybean miRNAs may target a greater number of genes than their homologs in /b Table . We have35S::miR399a, including primary and lateral roots, were comparable to those from plants carrying the empty vector control when grown under normal conditions (Figures 35S::miR399a exhibited increased sensitivity to salt stress when treated with 75 mM NaCl (Figures 35S::miR399a-overexpressing roots was reduced by 40%, and the lateral root number per hairy root was decreased by up to 70% under salt stress conditions (Figures It has been shown that miR399 family miRNAs mediate phosphate uptake in various plant species (Bari et al.,  Figures ; in cont Figures . Primary Figures . These rArabidopsis as a model plant, several regulators of RAM activity, including PLT1/2, SHR, and SCR, have been identified (Helariutta et al., Plant roots grow post-embryonically, and root growth and the root system architecture in plants are prominently controlled by environmental cues. Therefore, one of the mysteries in plant biology is the regulation of root development with a focus on RAM maintenance and remodeling of the RAM in response to various stresses. Using Arabidopsis and a global analysis of the miRNAs in M. truncatula root tips under salt stress (Dinneny et al., There have been a couple of previous attempts to explore the regulatory networks that modulate root responses to salt stress, including a microarray analysis of different cell layers in Medicago root tips (Lelandais-Briere et al., Populus tremula (Jia et al., Medicago and soybean. A functional analysis of glyma-miR399a further confirmed that miR399 is required for RAM and root development under salt stress conditions (Figure Medicago root tips. We found that gma-miR1512b was significantly down-regulated upon salt stress treatment in the root tip of soybean, suggesting that gma-miR1512 plays an important and specific role in plastic root development in soybean under conditions of salt stress. Additional studies of gma-miR1512 will further our understanding of how the soybean RAM responds to salt stress.Plants constantly encounter various adverse soil stresses, and they have evolved dedicated regulatory networks that are responsible for adaptations at the cell, tissue, organ, and species levels. Our current results indicate that a given miRNA family or members of a family may be involved in diverse biological processes in different plant species. For example, miR2111 is a well-known miRNA that mediates plant responses to phosphate deprivation in Arabidopsis (Hsieh et al., s Figure . These rArabidopsis TAS homolog. One target gene encodes a protein with strong similarity to AtBAM3, a receptor kinase-like protein that regulates shoot and floral meristem development (Depuydt et al., AtBAM3 homolog in soybean. Gma-miR399 also has additional putative target genes with different biochemical features beyond the well-known target gene PHO2, which is involved in plant responses to phosphate starvation. Based on our results, GmPHO2 has no significantly change in response to salt stress but another homologous gene Glyma.15G074200 was obviously induced under salt stress. In Arabidopsis, the homologous gene of Glyma.03G021900 function as GROWTH-REGULATING FACTOR 9, regulates the plant growth and development process (Horiguchi et al., Glyma.08G359400 was annotated as a multicopper oxidase which might play roles in plants development to response abiotic stress and phosphate sensing in the root tip (Svistoonoff et al., One efficient way to elucidate miRNA-mediated biological processes and plant stress responses is to find their target genes. Based on computational predictions, we identified putative target genes for all salt-responsive miRNAs in the soybean RAM. As expected, some salt-responsive miRNAs identified in this study, including gma-miR156 and gma-miR172, target the same family of genes encoding various proteins (e.g., AP2 transcription factors; Table cis-element (Table Arabidopsis (Aloni et al., Arabidopsis (Aida et al., Despite the great importance of miRNA-mediated regulatory networks in plant growth and adaptation, the upstream regulators of these key miRNAs remain largely unknown. In the present study, we found that salt stress induced auxin accumulation in the soybean RAM Figure . Intrigunt Table . Second,nt Table . It is wnt Table . To dateThe maintenance of RAM activity is essential for indeterminate root growth in plants, and the reprogramming of RAM activity is crucial for ensuring the correct root system architecture and survival of plants in a constantly changing environment. The current report reveals the critical role of miRNAome plasticity in the soybean RAM in response to salt stress. We propose that the salt-induced activation of auxin signaling is an important upstream event that mediates RAM remodeling through targeting by salt-responsive miRNAs in soybean.YW and XL conceived the study and designed the experiments. ZS, YW, FM, YT, LC, SZ, and QJ performed the experiments. YW, FM, YT, LC, SZ, and QJ collected the samples for high-throughput sequencing. ZS, YW, and LC performed the expression validation of the miRNAs and phenotypic analyses of the transgenic roots. ZS, YW, and FM analyzed the experimental data. ZS, YW, and XL wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neurodegenerative Langerhans Cell Histiocytosis (ND-LCH) is a rare, unpredictable consequence that may devastate the quality of life of patients cured from LCH. We prospectively applied a multidisciplinary diagnostic work-up to early identify and follow-up patients with ND-LCH, with the ultimate goal of better determining the appropriate time for starting therapy.We studied 27 children and young adults with either ND-LCH verified by structural magnetic resonance imaging (MRI) (group 1) or specific risk factors for , but no evidence of, neurodegenerative MRI changes (group 2). All patients underwent clinical, neurophysiological and MRI studies.Seventeen patients had MRI alterations typical for ND-LCH. Nine showed neurological impairment but only three were symptomatic; 11 had abnormal somatosensory evoked potentials (SEPs), and five had abnormal brainstem auditory evoked potentials (BAEPs). MR spectroscopy (MRS) showed reduced cerebellar NAA/Cr ratio in nine patients. SEPs showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for predicting ND-LCH of 70.6% , 100% (69.2%-100%), 100% (73.5%-100%), and 66.7% (38.4%-88.2%), respectively. Repeated investigations in group 1 revealed increasingly abnormal EP parameters, or neurological examination, or both, in nine of fifteen patients while MRI remained unchanged in all but one patient.A targeted MRI study should be performed in all patients with risk factors for ND-LCH for early identification of demyelination. The combined use of SEPs and careful neurological evaluation may represent a valuable, low-cost, well-tolerated and easily available methodology to monitor patients from pre-symptomatic to symptomatic stages. We suggest a multidisciplinary protocol including clinical, MRS, and neurophysiological investigations to identify a population target for future therapeutic trials. Langerhans cell histiocytosis (LCH) is a rare disease of the dendritic cell system that may occur at any age, with a peak incidence between 5 and 10 years and in JDifferent MRI patterns of CNS involvement have been described, 9, 10. cranio-facial” lesions involving the orbital, temporal, sphenoid, ethmoid or mastoid bones, the paranasal sinuses, the anterior or middle cranial fossa and/or with DI, carry a higher risk of developing this serious complication[ND-LCH is a potentially devastating, permanent consequence of LCH. Its real prevalence is still unclear, ranging between 1% and 25% in different cohorts –17. Patiplication.The pathplication.Despite a relatively homogeneous MRI pattern, the clinical manifestations of ND-LCH are extremely heterogeneous, ranging from minimal or no neurological impairment, to a severe clinical picture including ataxia, spastic quadriparesis, intellectual disability and psychiatric symptoms, 12–16. Another largely unmet need concerns effective ND-LCH treatment. Various approaches, including chemotherapy, anti-inflammatory, anti-angiogenic and immune-modulatory medications, have been used in a few symptomatic patients yielding deceptive results. Based on these data from sporadic reports, the current prospective therapeutic trial LCH-IV of the Histiocyte Society (EudraCT number: 2011-001699-20) suggest either intravenous immune globulin or cytarabine in patients with clinical signs of ND-LCH.In this study we describe clinical features, MRI and neurophysiological alterations in ND-LCH and propose a diagnostic work-up for early identification and follow-up of ND-LCH, with the ultimate goal of selecting patients for a future therapeutic intervention in pre- or pauci-symptomatic stages.Our primary endpoints were: a) to analyze clinical features, MRI and neurophysiological alterations in ND-LCH and b) propose a diagnostic work-up for early identification and follow-up of ND-LCH. Secondary endpoints were: a) to follow-up a group of patients with ND-LCH and b) to define selection criteria for a future therapeutic intervention in pre- or pauci-symptomatic stages.As the national referral center for LCH, we launched a survey among all the Italian pediatric hematology-oncology centers to evaluate patients with LCH, either newly diagnosed, on therapy or in follow-up, using a new integrated diagnostic protocol for ND-LCH. In our study we defined as affected by ND-LCH all patients showing typical MRI alterations and we considered structural MRI evaluation as the reference standard, 11, 16.The diagnostic protocol included the following steps: neurological examination (NE), neuropsychological and neurophysiological study , MRspectroscopy (MRS)  by median and range, and made comparisons between groups using Mann-Whitney test. When appropriate, confidence intervals (CI) were calculated using exact likelihood. We set level of significance at 5% two sided.We analyzed diagnostic accuracy by calculating sensitivity, specificity, Receiver operating characteristic (ROC) curves, positive and negative predictive values for all the diagnostic tests included in the protocol.Considering as outcome the ordinal MRI grading, we evaluated the discrimination performance of the diagnostic tests using Harrell’s c, a generalization of the area under the ROC curve, 26.To evaluate the improvement in discrimination of adding to the best performing diagnostics a further test, we calculated the Integrated Discrimination Improvement (IDI) statistics. We alsoBetween April 2010 and February 2012, we enrolled 30 consecutive patients. We subsequentely excluded three patients because they either did not complete the MRI protocol (n = 2) or showed brainstem abnormalities of uncertain nature at MRI (n = 1).Finally, 27 patients  were evaluable for the analysis. Their median age at the study entry was 8.7 years . LCH had been diagnosed at a median age of 2.9 years . Seventeen patients had DI, which was already present at the time of the diagnosis of LCH (n = 9) or developed during the course (n = 8). Nineteen of the 27 patients had multisystem, and eight single-system disease. Recurrent or chronic active disease was present in 21 patients (78%), 24 had received chemotherapy, and 11 of them were being treated during the study.Median age at the study entry was similar in groups 1 and 2 . The duration of the disease , did not differ in the two groups (p = 0.688). Patients in group 1 had a significantly younger age at the onset of LCH than those in group 2 . Multisystem and reactivating disease appear to be more frequent in patients in group 1, although failing to reach the statistical significance .We identified seventeen patients with ND-LCH, ten of whom had no previous evidence of neurodegenerative alterations. All patients exhibited atypical MRI pattern with prevalent involvement of the dentate nucleus and the medium cerebellar peduncles . In six Neurological examination was abnormal in 10 patients, nine with (group 1) and one without (group 2) ND-LCH, of which seven were subjectively healthy. The Scale for the Assessment and Rating of Ataxia (S.A.R.A) revealed an abnormal score  in nine, while only pyramidal signs  were present in the remaining. Barthel index reached the maximum score (100) in all patients except two , who scored 3 and 80, respectively.Evoked potentials were performed in all 27 patients. In one patient VEPs were unreliable due to technical reasons. SEPs were abnormal in 11 patients of group 1, bilaterally in six. The N20 response was delayed in four , of decreased amplitude in four , and absent in three  patients. The P14 response was absent in two patients  and of decreased amplitude in one (#16). The N13 component was absent in one patient (#15). BAEPs were abnormal in five patients of group 1  and in one of group 2 (#23). VEPs were normal in all patients. EEG was normal in all patients but one (#5), who had fronto-central spikes during sleep but never experienced seizures.Neuropsychological evaluation was normal in all the 18 patients tested, with two exhibiting a discrepancy between verbal and non-verbal performances . Two patients had a specific learning disorder .The best performance for prediction of ND-LCH verified using MRI was observed for SEPs  with high specificity and lower sensitivity (70%), followed by NE . When using ordinal grading MRI abnormalities, again SEPs had the best discrimination performance . Defining a cut-off for the ordinal grading MRI abnormalities of 1, SEPs showed 82% specificity and 90% sensitivity.Adding NE to the diagnostic protocol did not improve significantly the discrimination performance, but there was an improvement in average sensitivity on ordinal grading MRI abnormalities over SEPs alone, and overall the integrated discrimination improved by 7.6% . Based on previous findings, we defined a diagnostic protocol including SEPs and NE to assess whether patients with ND-LCH can be predicted as such prior to MRI. Both tests can be negative or positive or only one of the two can be positive. On the basis of these three thresholds, we calculated sensitivity and specificity area under the ROC curve.We monitored clinical, MRI and neurophysiological changes in patients with ND-LCH. All patients but two (#5 and 7) of group 1 underwent repeated evaluations including MRI, MRS, NE, BAEPs and SEPs. We excluded VEPs and EEG due to their low sensitivity. In patient #7 EPs were not re-evaluated as already severely impaired at the first evaluation; patient #5 was lost from the follow-up.The median follow-up duration was 24 months . In five patients neurophysiological and neurological findings remained unchanged. A worsening of EP results was observed in eight patients , five of which  also showed neurological worsening.In patient #14 neurological symptoms worsened without EP impairment. In patient #15 SEPs slightly improved.MRI remained unchanged in all patients but one (#14) in whom a progression of white matter and globus pallidus abnormalities was observed. MRS findings appeared modified in two  patients.We applied a multidisciplinary diagnostic protocol for assessing and following-up patients with early-stage ND-LCH. In this study,an extended structural MRI protocol allowed to identify neurodegenerative abnormalities in patients at risk for ND-LCH before neurological impairment and neurophysiological abnormalities became obvious. This result is of relevance as brain MRI is not routinely performed in patients at risk for ND-LCH and targeted MRI protocols to detect neurodegenerative abnormalities have not been developed yet. MRI abnormalities in patients with ND-LCH have been described since 2000 , 9. ThisWhile structural MRI appears to be the reference standard, SEPs is the test with the highest capability to predict ND-LCH and discriminate its grading. Remarkably, SEPs were abnormal also in patients with noneurological signs or who only exhibited subtle deficits. In two previous studies on patients with ND-LCH, which included VEPs, EEG and BAEPs, abnormal EPs were found in up to 40% of patients, 19. In We noticed that subtle abnormalities may be difficult to identify in a single patient in the early pre-symptomatic stages. Regarding this issue, the combination of neurological examination, targeted MRI protocols, and SEPs had the highest diagnostic value. MRS might provide interesting correlations between morphology and function. A decreased peak of N-acetyl-aspartate in the cerebellum of patients with ND-LCH was described in single case reports, 29. In During follow-up we also identified neurological and neurophysiological changes in most patients with ND-LCH, despite structural brain MRI did not show signs of progression. An integrated neurophysiological and neurological protocol appears necessary to monitor the progression of the disease, also in view of possible new therapeutic approaches. Yet, limited knowledge of the pathogenic mechanism of demyelination, without any available animal model of ND-LCH, has impaired our ability to interpret MRI alteration and their changing over time .Recent studies of large cohorts of patients in the prospective trials LCH I to III have shown that a younger age at diagnosis does not represent an independent, adverse prognostic factor for treatment response and survival–32. In oThe main unmet medical need in ND-LCH remains treatment. Since reversal of clinical manifestations in patients with overt impairment is unrealistic, hopes should point to an early start of a potentially effective therapy. Our multidisciplinary approach can allow to identify asymptomatic or pauci-symptomatic patients with ND-LCH in order to start treatment early and possibly improve prognosis. A longitudinal therapeutic study is currently ongoing in our center and will hopefully help solve this issue. The possible therapeutic use of target therapy at least in the subset of patients with V600EBRAF mutation should be carefully explored in a scientific setting.Our study has limitations. The number of patients is relatively small; yet, given the rarity of the disease, and especially of ND-LCH, recruiting a larger number of patients would have needed a very long time or a multicenter setting, thus introducing additional potential biases. We also acknowledge that a study limited to prospective observation of newly diagnosed patients with LCH could have helped in describing the natural history of the disease, but this might require an extremely long time interval, which would impair feasibility. A correlation between time of follow-up and the probability to observe ND-LCH has been proposed . To thisWe propose a novel protocol of evaluation for patients with LCH, with the main purpose of identifying and monitoring patients with ND-LCH while still in a pre- or pauci-symptomatic stage. Patients with ND-LCH deserve a targeted structural MRI study for early identification of demyelination. Spectroscopy may then usefully complement structural MRI. SEPs are promising in detecting initial brain dysfunction. The combined use of SEPs and careful neurological evaluation may represent a valuable, low-cost, well-tolerated and easily available methodology to monitor patients from pre-symptomatic to symptomatic stages of ND-LCH. The wider use of our multidisciplinary protocol might allow the selection of patients for early therapeutic intervention, hopefully providing the basis for future therapeutic trials.S1 Fig(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S1 References(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Tablep-values of testing the null hypothesis of no contribution of adding NE to SEPs in the diagnostic protocol.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


PHAS) in soybean with a focus on those involved in lipid metabolism. The results showed that a large number of eTMs and PHAS genes could be found in soybean. Additionally, we found that lipid metabolism related genes were potentially regulated by 28 miRNAs, and nine of them were potentially further regulated by a number of eTMs with expression evidence. Thirty-three miRNAs were found to trigger production of phasiRNAs from 49 PHAS genes, which were able to target lipid metabolism related genes. Degradome data supported miRNA- and/or phasiRNA-mediated cleavage of genes involved in lipid metabolism. Most eTMs for miRNAs involved in lipid metabolism and phasiRNAs targeting lipid metabolism related genes showed a tissue-specific expression pattern. Our bioinformatical evidences suggested that lipid metabolism in soybean is potentially regulated by a complex non-coding network, including miRNAs, eTMs, and phasiRNAs, and the results extended our knowledge on functions of non-coding RNAs.A wide range of RNA species interacting with microRNAs (miRNAs) form a complex gene regulation network and play vital roles in diverse biological processes. In this study, we performed a genome-wide identification of endogenous target mimics (eTMs) for miRNAs and phased-siRNA-producing loci ( Glycine max) is one of the most important crops in the world. One of its main characteristics is its capacity to fix atmospheric nitrogen through symbioses with microorganisms , and natural antisense siRNA (nat-siRNA), play vital roles in plant development as well as in adaption to biotic and abiotic stresses  to trigger production of phased tasiRNAs. On the other hand, the activity of miRNAs can be attenuated or abolished by endogenous target mimics (eTMs), which are usually non-coding transcripts and are able to sequester miRNAs in an uncleavable manner , INDUCED BY PHOSPHATE STARVATION1 (IPS1) from Arabidopsis thaliana , while TAS1, TAS2, and TAS4 are found only in Arabidopsis and its close relatives  family, including ARF2, ARF3, and ARF4 , nucleotide-binding and leucine-rich-repeat-containing proteins (NB-LRRs), and MYB transcription factors in Arabidopsis, Medicago, Malus, Prunus, and Nicotiana  and other four oil crops (or their progenitor species), Ricinus communis (v0.1), Linum usitatissimum (v1.0), Brassica rapa (v1.2), and Gossypium raimondii (v2.1), were obtained from Phytozome  , and those with =4 and >4 mismatches compared to miRNAs of other plants in miRBase were considered as conserved miRNAs and soybean-specific miRNAs, respectively  bulges composed of three nucleotides are only permitted at the positions corresponding to the 9th to 12th nucleotides counting from the 5′ end of a miRNA sequence; (2) perfect pairing is required from the 2nd to 8th positions at the 5′ end of a miRNA sequence; (3) the total number of mismatches and G/U pairs within the eTM and miRNA pairing region  should be no more than three; and (4) the distance between an eTM and its upstream/downstream genes is longer than 200 nucleotides  with default settings. The abundance of RNA-seq reads aligned (at least three reads) to an eTM sequence indicates the expression level of the eTM. To investigate the conservation of the soybean eTMs in other oil crops or their progenitor species, including R. communis, L. usitatissimum, B. rapa, and G. raimondii, miRNA pairing sites of the G. max eTMs were used to Blast against the genomes of the four species . Alignment of eTMs from soybean and other species was achieved using sequences including the miRNA pairing sites along with their flanking regions of the predicted eTMs .To investigate the expression profiles of the predicted eTMs, publicly available RNA-Seq data generated from various tissues and organs of soybean Table  were mapPHAS) loci were identified in the intergenic regions of the G. max genome using the following algorithm previously described by Howell et al. . In this study, all phasiRNA-producing loci were considered as PHAS loci, although they were located in the non-coding regions of the G. max genome. For a miRNA to be considered as a putative phasiRNA trigger, (1) it has to be able to bind to a PHAS locus within the regions generating phased siRNAs or their flanking 200-nt regions; (2) the putative miRNA cleavage site (corresponding to the position between the 10th and 11th nucleotides counting from the 5′ end of a miRNA) should be at a position corresponding to a phase register.  aligned to all phases of the PHAS locus.PhasiRNA-producing  with the default settings   examined in this study. Similarly, only the target mimic sites but not their flanking regions were conserved in these predicted eTMs  were selected to predict putative eTMs for all 554 soybean miRNAs. Of the 554 soybean miRNAs, 334 (60.3%) had eTMs predicted. In total, we predicted 10,410 eTMs for 144 conserved miRNAs and 32,555 for 190 soybean-specific miRNAs Table . As an es Figure , consists Figure . PredictExpression of the predicted eTMs is a prerequisite for them to be functional. To examine whether the predicted eTMs are expressed in soybean, we analyzed 20 published RNA-Seq datasets generated from a wide range of organs or tissues, including vegetative and reproductive tissues as well as developing seeds Table . In totaTo investigate a role of miRNAs and their eTMs in lipid metabolism in soybean, we first identified soybean homologs of Arabidopsis genes involved in lipid metabolism. This analysis found 1507 genes with a potential role in lipid metabolism in soybean. We then predicted potential targets of all soybean miRNAs  downloaded from miRBase (Release 20). Based on these two analyses, we found that 89 soybean homologs of Arabidopsis genes involved in lipid biosynthesis were predicted targets of 97 miRNAs (45 conserved and 52 soybean-specific miRNAs) Table . AccordiOf the 457 expressed eTMs, 119 were eTMs for 33 miRNAs predicted to target genes involved in lipid metabolism, including 37 eTMs for 14 conserved miRNAs and 82 eTMs for 19 soybean-specific miRNAs Tables , S6. SomPHAS) loci of the G. max (Williams 82) genome were identified based on small RNA reads from eight small RNA libraries (Table PHAS loci generating 21-nt or 24-nt phasiRNAs were detected in the G. max genome (phase score >1.4). Even when using a stringent phase score (phase score >20), 1573 of 21-nt and 4681 of 24-nt PHAS loci could still be identified  had at least a miRNA binding site predicted within the regions generating phased siRNAs and/or their flanking 200-nt regions  located in soybean chromosome 17, and a phasiRNA from this locus was predicted to target Glyma03g31570, which encodes an acylhydrolase involved in oxylipin metabolism  and MYB protein family and disease resistance by nucleotide binding leucine-rich repeat NB-LRR . This phenomenon may suggest that eTMs and phasiRNAs are two types of important regulators co-evolved with soybean-specific miRNAs to regulate soybean-specific biological processes, and that the co-evolution of the non-coding RNA network in soybean may be a result of species-specific adaptations. In addition, we found that some biological processes were significantly enriched in the predicted target genes of miRNAs that have eTMs with expression evidence, suggesting a specified role of the interaction between miRNAs and their eTMs in these biological processes  were found for the 190 soybean-specific miRNAs than for the 144 conserved miRNAs. Similarly, we also found that soybean es Table . For exahttp://aralip.plantbiology.msu.edu/) (Li-Beisson et al., PHAS genes, in lipid metabolism in soybean. Understanding the functions of the miRNA network in regulating lipid metabolism in soybean will be of great value for the cultivation of soybean cultivars with increased oil content. Overall, our results revealed that lipid metabolism related genes in soybean are potentially directly regulated by 28 miRNAs with degradome data support, and that nine of these 28 miRNAs are potentially further regulated by a number of eTMs with 40 of them supported by expression data. The lipid metabolism pathways regulated by the network involving these nine miRNAs and 40 eTMs includes fatty acid synthesis, elongation, degradation, oxylipin metabolism, and phospholipid signaling (Table PHAS genes, which were able to target lipid biosynthesis related genes (Figure At least 120 enzymatic reactions and more than 600 genes are involved in the 24 pathways related to Acyl-lipid metabolism in Arabidopsis (ng Table . In addis Figure . We founs Figure . These rPHAS genes generating phasiRNAs that were predicted to target lipid biosynthesis related genes also showed a tissue-specific expression pattern (Figure PHAS genes based on the publicly available degradome data (Table PHAS genes to produce phasiRNAs that in turn to target genes involved in lipid metabolism (Tables This hypothesis was supported by several pieces of evidence. Firstly, of the 97 miRNAs predicted to target lipid biosynthesis related genes, 28 were confirmed to cleave their targets based on the data from seven publicly available degradome libraries Table . Secondln Figure , and miRta Table . Fourthlm Tables , S9. Furm Tables , suggestIn this study, our focus was on non-coding RNAs interacting with miRNAs in soybean; however, in addition to non-coding RNAs, our analyses also found that 120 protein-coding genes were potentially targeted by miRNAs to generate phasiRNAs Table . AdditioLongjiang Fan conceived and designed the study. Chu-Yu Ye, Hao Xu, Enhui Shen, Yang Liu, Yu Wang, Yifei Shen, and Jie Qiu analyzed the data. Chu-Yu Ye, Qian-Hao Zhu, and Longjiang Fan wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed.Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus—plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of  However, the production of potato is hindered by various environmental and biological factors, with Potato virus Y (PVY) being economically the most important potato viral pathogen. PVY belongs to the family Potyviridae and was first described over 80 years ago as a causative agent of potato degeneration [In 2012, potato showed the fifth highest production among all of the crops produced worldwide  [The mock-inoculated potato leaves of cv. ‘Igor’ did not show any morphological changes. PVYnd 7 dpi . By 13 dt shown) .N-inoculated, and PVYNTN-inoculated leaves were harvested at three time points: 1 dpi to observe any early responses to inoculation; 3 dpi to observe responses before symptom development and 6 dpi when the symptoms were developed. At 3 dpi and 6 dpi, upper non-inoculated leaves were also harvested. These upper leaves from 1 dpi were not included in the study as previous data have shown that the virus does not spread to upper leaves before 3 dpi [Mock-inoculated, PVYre 3 dpi .m/z values. The metabolites identified were linked with their metabolic pathway ontologies for easier interpretation  in relation to time and treatment. Here, 83 metabolites (32 of known identity) showed significant changes in relation to time . Also, tThe analysis with t-tests showed that in the inoculated leaves, a total of 100 peaks changed significantly for at least one of the times or treatments; however, only 41 of these peaks were identified as known metabolites . The higN-inoculated leaves and in the PVYNTN-inoculated leaves, respectively, relative to mock inoculated leaves.When comparing the levels of the metabolites in the leaves inoculated with one of the virus isolates relative to the mock-inoculated leaves, the metabolite concentrations were generally lower in the virus-inoculated leaves at 1 dpi . For 11%Metabolites involved in primary metabolism processes were examined into more detail, in particular for major carbohydrate metabolism, amino-acid metabolism, and the TCA cycle. The metabolites associated with secondary metabolism also showed interesting patterns of accumulation over the observed times, such as for the phenylpropanoids and the redox-state-related metabolites.NTN-inoculated leaves, relative to the mock-inoculated leaves  in the PVYd leaves . The sucSeveral unidentified metabolites also showed interesting patterns of accumulation. Indeed, these might be important in the potato responses to PVY infection; however, chemical identification would be needed to provide a full biological interpretation of their roles .Gene expression analysis of the selected genes involved in the metabolic pathways indicated in CWINV) decreased in the PVYN-inoculated leaves, relative to the mock-inoculated leaves at 1 dpi (FRK) increased in the PVYNTN-inoculated leaves. The PVYNTN-inoculated leaves also showed an increase in the expression of CWINV, hexokinase (HKS) and β-amylase (BAM3) at 6 dpi, relative to the mock-inoculated leaves.Expression of cell-wall invertase (at 1 dpi . At the GAD) gene that codes for one of the three most important GABA-shunt enzymes increased through disease development in the leaves inoculated with both of the PVY isolates, in comparison to the mock-inoculated leaves (SSADH) had a unique pattern of expression through disease development.Expression of the glutamate decarboxylase  and cinnamate 4-hydroxylase (C4H) had similar expression patterns through disease development  showed statistically significant changes for at least one of the times or treatments . As was NTN-inoculated plants  to inoculation [NTN-inoculated leaves, while on PVYN-inoculated plants, only mild or no symptoms were observed at 7 dpi.Responses of potato plants to infection with PVY have until now been studied at different levels, from morphological to biochemical, and from proteomic to transcriptomic ,9,26,27.culation . In the Three different approaches were employed to get a general overview of the response of individual potato leaves and changes in metabolic pathways after inoculation with PVY isolates. The distance matrix and hierarchical clustering show high variability between plants in the response at 3 dpi, which is most probably the consequence of different disease response rates in the individual potato plants . At 1 anGBSS gene in the PVYNTN-inoculated leaves, relative to the mock-inoculated leaves [Tomato mosaic virus , the concentrations of sucrose and glucose were in opposite proportions, with glucose being in abundance in the inoculated asymptomatic leaves [GBSS [CWINV in PVY-inoculated leaves. However, no direct functional confirmation is available to support this hypothesis.Soluble carbohydrates and starch have been shown to have important roles in plant responses to virus infections, where different biological systems have their specific modes of response . In PVY-inoculated potato leaves, a two-stage response was observed for the sugar concentrations, with an initial decrease followed by a strong increase. Decreased concentrations of sugars at 1 dpi might be the consequence of soluble sugar uptake for starch synthesis  which isd leaves . In tomac leaves . Similarc leaves . Accumules [GBSS  and CWINN-inoculated and symptomatic PVYNTN-inoculated leaves. However, differences in the dynamics of the responses between the PVYN-inoculated and PVYNTN-inoculated potato leaves were detected at the gene expression level in this study and previous [NTN-inoculated leaves than in the asymptomatic PVYN-inoculated leaves .There were no clear differences at 6 dpi in the levels of the soluble sugars in the asymptomatic PVYprevious  study. OGBSSI in upper leaves of the PVY-infected potato cv. ‘Igor’ [In these studied potato—PVY interactions, no changes were seen, however, for the sugar balance in the upper non-inoculated leaves over the time period of this study . Albeit,. ‘Igor’ . Soluble. ‘Igor’ ,10,14,16Arabidopsis plants with various viruses at the late stages of infection [Amaranthus hypochondriacus L. infected with Ageratum enation virus (AEV) [An often-observed characteristic of plant responses to virus infections is a decrease in photosynthesis and its associated mechanisms ,26,27,30nfection . In thisnfection  and in Aus (AEV) .GAD) and GABA catabolism and the levels of GABA have been shown [GAD was in agreement with the GABA precursor (glutamate) and GABA concentrations. Expression of the SSADH transcript was shown not to be significantly affected by the inoculation with these PVY isolates in potato plants. The SSADH products are succinate and NADH, and these are substrates for the mitochondrial respiratory chain, and they have roles in the prevention of accumulation of ROS, and the consequent cell death. Therefore, other levels of regulation are expected here [The Krebs cycle is directly affected by oxidative stress, and plants have the possibility to avoid the Krebs cycle via the GABA shunt, which uses less sensitive enzymes and to maintain the required NADH levels . In tranen shown ,33. The ted here . One of ted here .Arabidopsis leaves, some amino acids were reported to decrease and some to increase [GABA is involved in different processes that are connected to abiotic and biotic stress, presumably through different pathways ,35–37. Tincrease ,41. In tincrease ,42. The increase ,42 or inincrease , needs tArabidopsis plants [Glutamate also has an important role in ROS scavenging mechanisms, via glutathione and the production of NO and polyamines , e.g., ps plants .NTN-inoculated potato leaves than in PVYN-inoculated ones. Moreover, at 3 dpi, there was greater accumulation of dehydroascorbate in the PVYN-inoculated leaves than in the PVYNTN-inoculated leaves. On the other hand, a strong early increase in the expression of the genes related to antioxidant metabolism was previously described in the PVYNTN-inoculated potato leaves of cultivars Igor [O inoculation [One of the first and most important protection and signalling mechanisms that is induced in infected plants is the production of ROS ,20, and ars Igor  and Premars Igor . Interesculation .In the upper non-inoculated potato leaves, the responses were not so uniform, with only dehydroascorbate showing accumulation at 3 dpi, and lyxonate at 6 dpi. Similarly, the accumulation of intermediates in glutathione and ascorbate metabolism was observed in systemic leaves of SuCMoV-infected sunflowers , and decSynthesis of phenylpropanoids starts with the shikimate pathway, which has been shown to be responsive to biotic stress . The fintrans-caffeoylquinic acid and 4-trans-caffeoylquinic acid showed significant accumulation -infected tomato, expression of the genes associated with the phenylpropanoid pathway decreased [N-inoculated leaves, which might be at least partially accountable for the milder symptoms in these leaves, compared to those with PVYNTN inoculation. Similarly, differences in early expression (up to 10 hpi) of phenylpropanoid related transcripts were shown in potato cv. ‘Premier Russet’ response to two PVY isolates, PVYNTN and PVYO [In the upper non-inoculated potato leaves, shikimate, 3- as well . Accumulecreased . Phenylpecreased ,51, and ecreased . In the and PVYO .N-infected and symptomatic PVYNTN-infected leaves. Similar dynamics were observed also for the abundance of several amino acids, for the GABA shunt and TCA cycle intermediates, and for the phenylpropanoids. The accumulation of these metabolites coincided with the expected onset of virus multiplication. The upper non-inoculated leaves were not yet fully infected at 6 dpi. As expected, the differences in the metabolite profiles in these upper non-inoculated leaves were less pronounced compared to the differences in the PVY-inoculated leaves. However, by 6 dpi, the phenylpropanoids and the amino acids valine and isoleucine accumulated to high levels also in these upper non-inoculated leaves. The comparisons between the responses to each of these PVY isolates in inoculated leaves reveal a more rapid onset of accumulation of ROS scavengers in the leaves inoculated with PVYN than in those inoculated with PVYNTN . This could be connected with the lower damage seen for the leaves of the potato plants infected with this milder PVYN strain, and at least partially explain the differences in the phenotypes observed.The process of disease development was studied at the gene expression and metabolome levels in the potato—PVY interaction. In PVY-infected potato leaves, the levels of carbohydrates and ROS scavengers initially decreased (at 1 dpi) and then later increased (at 6 dpi), and the responses were similar in both the asymptomatic PVYSolanum tuberosum) cv. ‘Igor’ were grown from node tissue cultures and planted in soil in growth chambers that were kept at 21 ±2°C, with a photoperiod of 16 h and relative humidity of 70%. After 4 weeks, three basal leaves were mechanically inoculated with the sap of healthy plants (mock inoculated), PVYN-infected plants , or PVYNTN-infected plants . The inoculums were left on leaves for 10 min and afterwards washed off with tap water. At 1, 3 and 6 dpi, the inoculated leaves from four plants per time point per treatment were harvested. At 3 and 6 dpi, three upper non-inoculated leaves were also harvested from the same plants. The harvested leaves were stored in tubes and immediately frozen in liquid nitrogen. This plant material was stored at -80°C prior to further analysis. A separate group of six inoculated plants was left for symptom observation.Potato plants , using up to 300 mg frozen homogenised leaf material. MagMAX-96 Total RNA Isolation kits (Ambion) and KingFisher apparatus (Thermo) were used, following the manufacturer protocols. The isolated RNA was treated with DNase, quantified with NanoDrop spectrophotometer, and checked for quality by gel electrophoresis, as previously described .Up to 2 μg total RNA was transcribed into cDNA using High-Capacity cDNA Reverse Transcription kits (ABI), following the manufacturer recommendations. The final volume of the reaction mixture was 25 μl, and random hexamer primers were used.BAM), fructokinase (FRK), hexokinase (HKS), glutamate decarboxylase (GAD), succinic semialdehyde dehydrogenase (SSADH), phenylalanine-ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and cell-wall invertase  (Table ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Solanum_tuberosum/), the Potato Oligo Chip Initiative (POCI) database (http://pgrc-35.ipk-gatersleben.de/pls/htmldb_pgrc/f?p=194:1), and the Potato Genome Sequencing Consortium (PGSC) database (http://www.potatogenome.net/index.php/Main_Page) were aligned in the VectorNTI software (InforMax), and the regions of homology were determined. Primers were designed using the PrimerExpress software (Applied Biosystems)  transcript assembly database (ems) see . The conUniversal Master Mix  together with primers and probes were used for the analysis of 2 μl cDNA. The final concentrations of all of the probes in the reaction mix was 150 nM, while the primer concentration was 300 nM, except for PVY-uni and reference amplicons COX and EF-1, where it was 250 nM and 900 nM for probe and primers, respectively. The probes for all of the newly designed amplicons were modified with the 5’-FAM reporter and with the 3'-Zen Iowa Black FQ quencher.A 10-fold dilution series standard curve (10-times to 10000-times diluted) was prepared from a pool of all cDNA samples and tested with all amplicons. Ten-fold and 100-fold diluted cDNA was tested for each sample in two parallel reactions to check for the inhibition of amplification. All RT-qPCRs were carried out in 384-well reaction plates on a Lightcycler LC480 apparatus (Roche). For the amplifications, standard cycling conditions were used with an added melting curve stage for the SybrGreen reactions.The data were first analysed using the Lightcycler 480 software (Roche). For quantification of the relative gene expression the standard curve method was used, as previously proposed by Pfaffl . The effg for 4 min at 4°C. The supernatant was transferred to a new tube, and 500 μl MilliQ water was added to create two separate phases. The samples were briefly vortexed and then centrifuged at 14,000× g for 2 min at 4°C. The aqueous upper phase (500 μl) was transferred to a new tube, dried in a speed-vac, and stored at -20°C until derivatisation. Five microlitres of 13C-sorbitol standard (0.1 g/l) was added to the dried samples, which were dried again in the speed-vac. The samples were resuspended in 20 μl 40% methoxyaminohydrochloride in pyridine (w/v). The mixture was incubated and agitated for 90 min at 30°C. After this incubation, 80 μl N-methyl-N-(trimethylsilyl)trifluoroacetamide  was added to the samples. The samples were incubated at 37°C for 30 min, and then centrifuged at 14,000× g for 2 min at room temperature. Fifty microlitres of the supernatant was the transferred to the GC microvials.Plant material (50 mg) from the inoculated third leaf (from the ground) and a non-inoculated leaf were used separately for the extraction of the metabolites. Chilled  methanol: chloroform: water  was added to the plant material that was homogenised in liquid nitrogen, and this was vortexed for 10 s and incubated on ice for 8 min. Samples were centrifuged at 14,000× http://gmd.mpimp-golm.mpg.de/) were used. Representative ions were selected for quantification. The peaks were quantified using the LCquan software, and the data were exported to MS Excel. To test the reliability of the selected ions for some of the metabolites, two ions were selected for quantification . The same data were obtained regardless of the ions selected, and the quantity of one ion was used for further analysis. The identified metabolites were annotated with MapMan ontology (http://mapman.gabipd.org/web/guest/mapman), and new potato metabolites were added to MapMan.The GC-MS analysis was carried out as described by . For theN, PVYNTN) and harvested at three different times , distance matrix analysis and hierarchical clustering  was carried out using MultiExperiment Viewer (http://www.tm4.org/mev.html), where the table of all of the determined peaks and set of identified metabolites only in each individual sample was used, respectively. In addition the PCA was carried out on the set of all determined peaks from 1 and 6 dpi in XLSTAT add-in for MS Excel (http://www.xlstat.com/en/).To determine the metabolite composition in different samples under the different treatments . For determining the statistically significant changes in metabolite concentrations and in transcript copy numbers, the data were also analysed using two-sided Student t-tests implemented in MS Excel. The cut-off p-value for statistical significance was set to <0.05.Two-way ANOVA was applied to determine which metabolites showed statistically significant changes in relation to time or treatment, and their combination. ANOVA was carried out in MultiExperiment Viewer , and more pronounced chlorotic and necrotic ring spot lesions on the PVYNTN-inoculated leaves (right).No symptoms were observed on the mock-inoculated leaves (left), no symptoms or mild clorotic or necrotic ring spot lesions on the PVY(TIF)Click here for additional data file.S2 Figm/z 347 is shown in red. The spectrum of a compound eluted at 27.0 minutes is shown on the middle panel, where raw spectrum (black ions) is overlaid with extracted spectrum after deconvolution (white ions). Hit from the library  overlaid with extracted spectrum (white ions) is on the lower panel.The chromatogram is shown in AMDIS software. The total ion chromatogram (TIC) represents all ions eluting in a scan of the potato sample (upper panel). Chromatogram of an ion (TIF)Click here for additional data file.S3 FigSymbols: white, mock inoculated; grey, PVYN inoculated; black, PVYNTN inoculated; circles, 1 dpi; squares, 6 dpi. Clear separation can be seen for the first component (F1) for the samples collected at the two different times , with separation of the three out of four mock-inoculated samples at 6 dpi from all of the other samples according to the second component (F2).(DOC)Click here for additional data file.S1 TableAmount of individual metabolite, normalised to internal standard is shown.(XLSX)Click here for additional data file.S2 TableThe ratios were calculated between the PVYN-inoculated and mock-inoculated leaves (N:mock), the PVYNTN-inoculated and mock-inoculated leaves (NTN:mock), and between the virus isolates used for the PVY-inoculated plants (NTN:N). S, significance determined by student t-tests. Significance determined by two factor ANOVA is also given, where the time and treatment (treat) were given as factors and the significance of the interactions (inter) between these factors is given as well. *, p <0.05; **, p <0.01; ***, p <0.001.(XLSX)Click here for additional data file.S3 TableThe ratios were calculated between the PVYN-inoculated and mock-inoculated leaves (N:mock), the PVYNTN-inoculated and mock-inoculated leaves (NTN:mock), and between the virus isolates used for the PVY-inoculated plants (NTN:N). The corresponding metabolic pathways are assigned. CHO, carbohydrates; S, significance determined by student t-tests. *, p <0.05; **, p <0.01.(XLS)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Fabry disease. an X-linked deficiency of α-galactosidase A coded by the GLA gene, leads to intracellular globotriaosylceramide (GL-3) accumulation. Although less common than in males, chronic kidney disease, occurs in ∼15% of females. Recent studies highlight the importance of podocyte injury in Fabry nephropathy development and progression. We hypothesized that the greater the % of podocytes with active wild-type GLA gene (due to X-inactivation of the mutant copy) the less is the overall podocyte injury.Kidney biopsies from 12 treatment-naive females with Fabry disease, ages 15 (8–63), median [range], years were studied by electron microscopy and compared with 4 treatment-naive male patients.In females, 51 (13–100)% of podocytes (PC) per glomerulus had no GL-3 inclusions, this consistent with a non-Fabry podocyte phenotype (NFPC). In PC with GL-3 inclusions [Fabry podocyte phenotype (FPC)], GL-3 volume density per podocyte was virtually identical in females and males, consistent with little or no cross-correction between FPC and NFPC. %NFPC per glomerulus (%NFPC/glom) correlated with age in females , suggesting a survival disadvantage for FPC over time. Age-adjusted %NFPC/glom was inversely related to foot process width (FPW) , an indicator of PC injury. GL-3 volume density in FPC in females correlated directly with FPW.These findings support important relationships between podocyte mosaicism and podocyte injury in female Fabry patients. Kidney biopsy, by providing information about podocyte mosaicism, may help to stratify females with Fabry disease for kidney disease risk and to guide treatment decisions. Fabry disease is a storage disease caused by deficiency of the α-galactosidase A  enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins. This leads to the accumulation of its substrates, predominately globotriaosylceramide (GL-3) in various cell types and organs, causing a constellation of complications including skin lesions, strokes, cardiac arrhythmias and cardiomyopathy, neuropathies and renal failure. These studies were performed in accordance with principles of the Declaration of Helsinki and were reviewed and approved by the Institutional Review Board of the University of Minnesota, Comité. de Protection des Personnes “Ile-De-France II.” and the Regional Ethics Committee of Western Norway. Informed consents approved by the institutional board review committees were obtained prior to these studies.Kidney biopsies from 12 ERT-naive females with Fabry disease, age 15 (8–63) years were studied by electron microscopy for distribution of podocyte involvement by the Fabry phenotype. Biopsies were obtained for assessment of the severity of the lesions of Fabry nephropathy in order to aid clinical decision-making regarding ERT initiation and/or as a baseline biopsy prior to ERT initiation. Biopsies from 4 ERT-naive males with Fabry disease, age 14 (7–18), were studied for comparison; 7/16 patients presented here were included in our previous publications. 9/12 female and 3/4 male patients had results of GLA mutation analysis in their medical records confirming the diagnosis of Fabry disease . The diaBiopsies from 6 healthy living kidney donors, age 37 (16–52) were used to estimate normal control values for podocyte foot process with (FPW), as described below.Electron microscopy specimens were fixed in 2.5% glutaraldehyde, and embedded in PolyBed. Random glomerular sections were prepared as previously described. Observers were masked to any of the patient characteristics. Montages of complete glomerular profiles were prepared from the above images in Adobe Photoshop software . Twice digital magnification was applied to the images. Podocyte nuclei were identified and glomerular profiles with less than 10 podocyte nuclei (n = 3) were excluded from these studies. The cytoplasmic profiles surrounding each podocyte nucleus were carefully examined for presence of GL-3 inclusions. Podocyte nuclear profiles with cytoplasmic GL-3 inclusions, consistent with Fabry phenotype podocytes (FPC) or without cytoplasmic GL-3 inclusions, consistent with non-Fabry phnotype podocytes (NFPC) cytoplasmic GL-3 inclusions were counted in each glomerulus and %NFPC/glom was calculated. The maximum number of immediately adjacent podocytes, including podocytes on the other side of the same capillary loop, which were NFPC was recorded as an estimate of the size of podocyte mosaic patches on the section.vs. NFPC in female patients with Fabry disease, FPW was separately estimated in systematically and uniformly obtained electron micrographs with and without FPC.Based on the best quality of tissue preservation and images, one glomerulus per biopsy was arbitrarily selected for detailed GL-3 volume density measurement in podocytes with visible nuclei on the section. Boundaries of nuclei and cell membranes, excluding the tertiary foot processes were traced using the magnetic lasso tool in separate layers in Adobe Photoshop . SimilarStatistica 8.0  was used for statistical analysis. Comparison between groups was made by student t-test after confirming homogeneity of variances. Relationships between variables were evaluated using Pearson correlation. Partial correlations were performed to control for confounding variables. Random effects model variance component analysis was performed to estimate % contribution of biopsies (inter-subject) and glomeruli (intra-subject) variations to total variance of %NFPC per glomerulus (%NFPC/glom). p<0.05 was considered statistically significant.e.g., mesangial or endothelial cells). In 4 female patient biopsies, no GL-3 inclusions were identified in endothelial and/or mesangial cells, thus, comments about mosaicism could not be made in those cell types. In 7/12 female patient biopsies, parietal epithelial cell profiles with no GL-3 inclusions were identified while occasional parietal epithelial cells with enlarged cytoplasm had abundant GL-3 inclusions. Characteristic lamellar GL-3 inclusions were not easily identified in proximal tubular epithelial cells. Distal tubular epithelial cells showed variable GL-3 inclusions in both males and females. Thus, this variability could not be accounted for X-inactivation mosaicism in those cells.Examination of electron micrographs from all biopsies allowed easy and reliable distinction between male and female patients by the identification in females of podocytes with visible nuclei without GL-3 cytoplasmic inclusions, termed non-Fabry podocytes (NFPC)  in contr54 (27–87) podocytes per biopsy were examined for presence of GL-3 inclusions in female patients. 51 (13–100)% of podocyte profiles per biopsy with visible nuclei and no GL-3 inclusions, were classified as NFPC in these females. NFPC were distributed as single cells among FPC or in patches composed of 2–6 podocytes .Two biopsies from female patients (one with 3 and another with 2 glomeruli) had no GL-3 inclusions in podocytes with visible nuclei , median (range)) available for electron microscopy were used to compare inter- and intra- subject variability of podocytes with the Fabry the phenotype. The %NFPC/glom in 2–4 glomeruli per biopsy in female patients is shown in Values of %NFPC/glom, Vv(Inc/PC), volume fraction of GL-3 inclusions per mesangial cells [Vv(Inc/Mes)], volume fraction of inclusions per endothelial cells [Vv(Inc/Endo)], and foot process width (FPW) are provided in To better visualize the relationships between %NFPC/glom and FPW and UPCR, 10 female patients were grouped into 5 age-matched pairs, 3 pairs with identical ages and the 2 other pairs with ages no more than 2 years apart. Except for one pair, in each pair, the subject with lower %NFPC/glom had greater FPW value, suggestive of an inverse relationship between the extent of podocyte injury and the number of NFPC in age-matched Fabry females . Even an9 the latter a strong predictor of renal disease risk among Fabry disease patients.23 Increased FPW is a common concomitant of injury in podocytes and is seen in a variety of conditions known to be associated with injury in these cells. et al first described the heterogeneous distribution of GL-3 inclusions in podocytes in 3 females with Fabry disease. et al also described variable podocyte GL-3 accumulation in 4 females with Fabry disease. We recently demonstrated that the fraction of the volume of podocytes occupied by GL-3 [Vv(Inc/PC)] increases with age in children with Fabry disease and is directly correlated with FPW and to proteinuria,Importantly, we documented, through unbiased morphometric measurements, that the average fraction of podocyte cytoplasm occupied by GL-3 was virtually identical in male and female patients, suggesting that FPC do not benefit from the enzymatic activity of the adjacent NFPC or from the generally higher residual plasma αGal A in females than on males. Also important, we found that inter-glomerular variation in podocyte phenotype mosaicism in a given female Fabry patient’s renal biopsy is much smaller than inter-patient variation. This suggests that estimation of podocyte phenotypic mosaicism even in a single glomerulus is representative of this phenomenon in the biopsy and validates the study of podocyte phenotype mosaicism in a few glomeruli. It also suggests that podocyte mosaciasm in females is established relatively early in embryogenesis. It should be noted that in these studies we used random profiles of glomeruli. Although we observed NFPC in patches of up to 5 cells in these two-dimensional glomerular cross-sections, these patches are likely larger in three dimensions. Nonetheless we posit that these patches are not very large, otherwise we might have expected greater inter-glomerular variability as a result of random sectioning through glomeruli.et al. reported that the trend line between the age and the Mainz severity score index (MSSI) vs. 28 without unfavorably skewed X-inactivation in leukocytes, urinary and salivary cells. This suggested that random X-inactivation could influence the severity of the Fabry phenotype in female patients. et al. found that in 46% of the 28 Fabry females studied, skewed X-inactivation did not correlate with phenotype severity. This study is the first to show a quantitative relationship in females with Fabry disease between the X-inactivation phenomenon and podocyte injury manifest as increased FPW. Although X-inactivation may play a role in the clinical phenotypic expression of X-linked diseases, a direct link between skewed X-inactivation and severity of the Fabry phenotype has been controversial. Dobrovolny The relationship between podocyte mosaicism and podocyte injury in female Fabry patients suggests that the status of podocyte X-inactivation, after making adjustment for the age of the patient, could help to identify females with greater risk of progression of Fabry nephropathy. Although it would be desirable to use less invasive methods to obtain information about the proportion of cells with active mutant GLA, the status of mosaicism may be cell type and organ specific. Moreover, as outlined above, studies attempting to link X-inactivation in peripheral blood leukocytes and clinical manifestations of Fabry females have produced contradictory results. Importantly, the present study showed that not only podocyte mosaicism for Fabry phenotype and podocyte injury are linked in female patients, but also podocyte injury in these patients is not limited to FPC and extends to NFPC, evidenced by increased FPW in GBM areas covered by NFPC compared with normal control biopsies. This is consistent with experimental studies performed by Matsusaka et al. where following administration of the immunotoxin LMB2 to the mice chimeric for hCD25 (LMB2 receptor), not only hCD25+ podocytes, but also hCD25- podocytes were injured and showed foot process widening, suggesting that specific injury to some podocytes can induce non-specific injury to other podocytes, To our knowledge, this is the first report of the finding of rare cells with GL-3 inclusions in kidney biopsies from female Fabry patients. We encountered two females, ages 34 (case #9) and 39 years (case #11), with known Fabry disease by mutation analysis with very rare kidney cells with GL-3 inclusions. One (case #11), had a cardiac variant GLA mutation (N215S) This study has some limitations. Although, it is the largest kidney biopsy study of females with Fabry disease, given the heterogeneity of Fabry mutations and clinical manifestations, additional studies are needed to confirm our results. We did not examine the status of X-inactivation in podocytes directly, rather we studied their apparent phenotype as to presence/absence of GL-3 inclusions in cell body profiles as a surrogate for X-inactivation. However, the easy discrimination between males and females on biopsies based on podocyte mosaicism supports validity of this surrogate. We cannot exclude the possibility of overestimation of %NFPC/glom in this study due to missing GL-3 inclusions in a single section through podocyte cell bodies. However, among 4 biopsies examined from male patients, only 2 of 98 podocyte nucleated profiles, both of very small size, had no GL-3 inclusions. Thus, we believe the extent of %NFPC/glom overestimation due to random sectioning is trivial.In summary, this is the first study showing that mosaicism of podocytes in females with Fabry disease is related to podocyte injury. The extent of podocyte mosaicism for the Fabry phenotype is quite uniform among the glomeruli. The fraction of the cell body occupied by GL-3 in affected podocytes in females is the same as in males, indicating the absence of significant cross-correction. The relative number of podocytes without the Fabry phenotype increases with age in female patients, suggesting either a disproportionate loss of Fabry-affected podocytes over time and/or selection bias. Information about podocyte mosaicism in kidney biopsies may be useful to identify females with Fabry disease with increased risk of developing progressive podocyte and nephron loss. The methodology we introduced is applicable to future longitudinal biopsy studies to test this hypothesis.


We compared the circulating microRNA profiles of Qi-stagnation (QSB) and Qi-deficiency (QDB) in coronary heart disease (CHD) patients with blood stasis syndrome. Twenty-nine CHD patients were divided into QSB group and QDB group. The analysis was carried out through comparing their circulating microRNA profiles and the following bioinformatics analysis. The number of differential miRNAs in QDB group was much more than that in QSB group. Functional annotations of the differentially expressed miRNAs target genes in the QSB group and QDB group were, respectively, related to regulation of cellular component organization, regulation of glucose metabolic process, and so forth and protein kinase cascade, phosphate metabolic process, and so forth. KEGG pathway analysis showed that the process Qi-deficiency was associated with phagocytosis including endocytosis and mTOR signaling pathway. Specifically, pathway of cell adhesion molecules played the crucial role in the pathological process of Qi-stagnation, with a unique upregulation except for pathways associated with cancer signal. MicroRNA-gene-net analysis indicated that let-7c, miR-4487, miR-619, miR-8075, miR-6735, and miR-32-5p and miR-17-5p, miR-130a, and miR 320 family had the most important and extensive regulatory function for Qi-stagnation syndromes and Qi-deficiency syndromes, respectively. Differentially expressed miRNAs and concerned pathways suggest different molecular mechanisms that may mediate the pathological process of QSB and QDB syndromes. Coronary heart disease (CHD), characterized by myocardial ischemia or necrosis caused by vascular stenosis or occlusion, is one of the leading causes of deaths and hospital admissions worldwide and constitutes the leading cause of disease burden in the world according to the 2010 Global Burden of Disease Study (GBD) , 4. TCM Chinese medicine holds that blood stasis syndrome is a common reason responsible for CHD in clinic of Chinese medicine due to Qi-stagnation (QSB) or Qi-deficiency (QDB). At present, many researchers consider that objective signs of blood stasis are reflected in microcirculation related to vessel and cell function, such as vascular diastolic dysfunction, abnormal platelet function, blood viscosity, and blood cell adhesion. As for QSB and QDB, there exists different biological basis and objective signs, with their respective unique characteristics of pathological changes.MicroRNAs (miRNAs) are endogenous small RNA molecules best known for their function in posttranscriptional gene regulation. More than 60% of protein-coding genes may be targeted by miRNAs . Since tThe characterization and differentiation of QSB and QDB syndromes have played an important role in the clinical practice of TCM for CHD. We speculated that miRNAs of peripheral blood have been a major parameter in discriminating the QSB and QDB syndromes and affected the appearance of the CHD patients with QSB and QDB syndromes. Therefore, we investigated the possible relationship between the syndromes as described in TCM practice and miRNAs profiles to bridge the gap between traditional syndrome diagnosis and molecular systems biology.This study was approved by the Medical Ethical Committee Xiyuan Hospital of China Academy of Chinese Medical Sciences (2011XL008-2). All 19 healthy volunteers reported no CHD and exhibited a normal syndrome as judged by TCM doctors. Delayed, written informed consent was obtained from all enrollees after they were clinically stabilized. 29 patients presenting with CHD were identified in Xiyuan Hospital of China Academy of Chinese Medical Sciences during the time between March 2012 and June 2013. Of the 29 patients screened for inclusion, 7 fulfilled study inclusion criteria for QSB group and 22 fulfilled study inclusion criteria for QDB group.10 milliliters of venous blood was obtained from ulnar vein and transferred in a sterile fashion into vacuum blood collection tubes. Samples were placed immediately on ice and then centrifuged at 4°C for 20 min at 3000 rpm to obtain the serum which was taken to the study laboratory within 10 minutes.A260/A280 = 1.8–2.1, using NanoDrop 2000 .Total RNA including small RNA and miRNAs was isolated from serum samples by TRIzol reagent  and purified using RNeasy Mini Kit , including a DNase digestion treatment. We ensure that the purification method retains slow molecular weight (LMW) RNA. RNA concentrations were determined by the absorbance at 260 nm and quality control standards were t-test analysis. Differentially expressed miRNAs with at least 1.5-fold change in either direction with P < 0.05 were considered to be up- or downregulated.RNA was labeled using FlashTag Biotin HSR labeling kit as the manufacturer's instructions . Labeled RNA was hybridized to GeneChip microRNA 3.0 array  according to the user manuals. Affymetrix Expression Console Software (version 1.3.1) was used for microarray analysis, including data normalization, summarization, and quality control assessment. Median-centric normalization was used for the custom microRNA oligonucleotide chips. Affymetrix chips were normalized using the robust multichip analysis (RMA) procedure. Differentially expressed miRNAs were identified based on RVM −ΔΔCt method.The cDNA was subjected to real time quantitative PCR with defined primers and Power SYBR Green PCR Master Mix . The data were analyzed using the ABI 7000 system SDS software . All the experiments were performed in duplicate and relative expression levels of these microRNAs were determined by the 2Gene ontology analysis has been carried out using DAVID and GSEA. ID of differential expressed genes was uploaded to the DAVID database and the analysis was performed based on their respective molecular function, biological process, or cellular component. Functional categories were enriched within genes that were differentially expressed between the QSB and QDB groups. Top ten GO functional categories that genes mainly involved in were selected for specific analysis.P < 0.05 and fold change > 1.5 were identified for further analysis.Normalised signal intensities from each experimental condition for the differentially expressed genes were uploaded to the KEGG database which is to collect pathway maps that computerize the network information of molecular interaction. KEGG analysis  is used for discovering the relation that is not easily visible from the changes of individual genes. Pathways that had significant changes of http://www.targetscan.org/) in conjunction with the miRanda version August 2010 Release (available at: http://www.microrna.org/) was used to predict the targets of the miRNAs. The circles represented target genes and the squares represented miRNAs. The relationships between miRNAs and target genes were represented by edges. The center of the network was represented by degree which is the contribution of one miRNA to the genes around or the contribution of one gene to the miRNAs around [To build a miRNA-gene-network, the relationship between miRNAs and genes was counted by their differential expression values and according to their interactions in the Sanger (Targetscan&miRanda) miRNA database. TargetScan 6.2 (s around . The keyAs shown in P < 0.05) between QSB and QDB by microarrays. Compared with the healthy patients, we detected 21 differentially expressed miRNAs, with 16 (76.2%) upregulated miRNAs and 5 (23.8%) downregulated miRNAs. 33 differentially expressed miRNAs were found in QDB group, with 21 (63.7%) upregulated miRNAs and 12 (36.3%) downregulated miRNAs. MiRNAs that are differentially expressed between QSB and QDB groups which are expected to contribute to functional differences among the CHD patients. Therefore, we identified miRNAs that are differentially expressed . All randomly selected miRNAs were in agreement with the microarray analyses in terms of the direction of the observed differential expression  were the same positive tendency as obtained with the microarray data. Equally, the observed log2 fold changes with qRT-PCR of the down-regulated miRNAs (miR-320c and miR-619) were the same negative tendency as obtained with the microarray data.To determine that the microarray-based miRNAs detections were reliable for the determination of miRNAs expression patterns in the serum, we performed SYBR green-based real time quantitative PCR on upregulated or downregulated miRNAs in the QSB and QDB groups from microarray data  or efflux  is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and the defined intracellular membrane compartments [According to the KEGG analysis, 6 pathways were related to the QSB, 5 of which were related to the cancer concerned pathways. The only pathway that did not belong to the cancer concerned pathway was cell adhesion molecules (CAMs). Cell-cell and cell-matrix interactions are mediated through several different families of CAMs including the selectins, the integrins, the cadherins, and the immunoglobulins. CAMs play a very significant and critical role in both heart development and different pathophysiological heart disease . Blood sartments . Some reartments . In consartments –22. Consartments , 24. Thehttp://www.microrna.org/). The biologic actions of miR-320 involve carcinogenesis, development, and ischemia reperfusion injury [The construction of microRNA-gene-network in QSB group demonstrates that let-7c, miR-4487, miR-619, miR-8075, miR-6735, and miR-32-5p are located in the center of diagram, suggesting that the abnormal miRNAs may have the most important and extensive regulatory function for Qi-stagnation. The construction of microRNA-gene-network in QDB displays that miR-17-5p, miR-130a, miR-320a, miR-320b, and miR-320c are located in the center of diagram, suggesting that the abnormal miRNAs may have the most important and extensive regulatory function for Qi-deficiency. Further, we found that though miR-4487, miR-619, and miR-8075 were the overlapping miRNAs in both QSB and QDB syndromes, their regulatory function seemed more wide and involved in more regulatory networks in QSB syndromes than that in QDB syndromes. Of note, miR-320 family including miR-320a, miR-320b, and miR-320c regulates QDB syndromes predominantly. MiR-320 has been found to have widespread biological effects as it regulates multiple important molecules. Its potential targets include ET-1, ERK1, VEGF, and FN (n injury , 26. ComIn this paper, we compared molecular networks of two syndromes of CDH patients with blood stasis and analyzed their common and different microRNAs mechanisms. We achieve a better understanding of the biological characteristics of syndrome differentiation of CHD which is beneficial to explore different therapies to enhance the effectiveness and pertinence of treatment.