Leifsonia xyli subsp. xyli, is widespread in countries and regions where sugarcane is grown and also limited to sugarcane productivity. Although the whole genome sequencing of Leifsonia xyli subsp. xyli was completed, progress in understanding the molecular mechanism of the disease has been slow because it is difficult to grow in culture.Sugarcane is an important sugar and economic crop in the world. Ratoon stunting Disease (RSD) of sugarcane, caused by Leifsonia xyli subsp. xyli membrane protein gene Lxx18460 (anti-sigma K) was cloned from the Lxx-infected sugarcane cultivar GT11 at the mature stage using RT-PCR technique, and the gene structure and expression in infected sugarcane were analyzed. The Lxx18460 gene was transformed into Nicotiana tabacum by Agrobacterium tumefaciens-mediation. The transgenic tobacco plants overexpressing Lxx18460 had lower levels in plant height, leaf area, net photosynthetic rate and endogenous hormones of IAA, ABA and GA3, as well as lower activities of three antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than the wild type (WT) tobacco. With the plant growth, the expression of Lxx18460 gene and protein was increased. To better understand the regulation of Lxx18460 expression, transcriptome analysis of leaves from transgenic and wild type tobacco was performed. A total of 60,222 all-unigenes were obtained through BGISEQ-500 sequencing. Compared the transgenic plants with the WT plants, 11,696 upregulated and 5949 downregulated genes were identified. These differentially expressed genes involved in many metabolic pathways including signal transduction, biosynthesis of other secondary metabolism, carbohydrate metabolism and so on. Though the data presented here are from a heterologous system, Lxx 18460 has an adverse impact on the growth of tobacco; it reduces the photosynthesis of tobacco, destroys the activity of defense enzymes, and affects the levels of endogenous hormones, which indicate that Lxx18460 may act important roles in the course of infection in sugarcane.The Lxx18460 of anti-sigma K (σK) factor in Leifsonia xyli subsp. xyli. Our findings will improve the understanding of the interaction between the RSD pathogen Leifsonia xyli subsp. xyli and sugarcane. The output of this study will also be helpful to explore the pathogenesis of RSD.This is the first study on analyzing the function of the membrane protein gene The online version of this article (10.1186/s12866-018-1378-2) contains supplementary material, which is available to authorized users. Leifsonia xyli subsp. xyli – Lxx), is widespread in worldwide sugarcane production areas [Sugarcane is an important economic crop in Guangxi province which contributes more than 60% of the total sugar production in China , 2. Ratoon areas , 4 and ron areas –8. RSD cLxx colonizes at xylem vessels, mesophyll and bundle sheath cells surrounding the vascular system [Lxx is genetically uniform, and it has no genetic variation [Lxx (CTCB07) was completed by Monteiro-Vitorello et al. [r system , 10. Lxxariation , 11. Theo et al. . The seqo et al. , 14. Otho et al. . No any Lxx is a bacterium, σ factor is one of protein subunits (α2ββ’σ) of the bacterial RNA polymerase holoenzyme and also a major regulatory factor of prokaryotic gene expression regulation. It can be used to identify the target gene promoter regions, promote the combination of the target region and RNA polymerase holoenzyme [ECF and σE in Escherichia coli, which tightly binds to σE, then restricts its identification of promoter and its binding with the core enzyme of RNA polymerase [Mycobacterium tuberculosis encodes anti-Sigma K factor (RskA), which could inhibit the Sigma K regulating action and affect the antigen protein expression of MPB70 and MPB83 ultimately [Streptomyces [loenzyme , and antloenzyme , and sinloenzyme , 18. A tlymerase . Rv0444ctimately . The σR ptomyces . Previouptomyces .Lxx in vitro, and the pathogenesis research on RSD is still at a preliminary stage. The anti σ factors of Lxx and their corresponding features and functionalities have not been sufficiently analyzed. In this study, we isolated and cloned the membrane protein gene Lxx18640  of Lxx, analyzed its biological characteristics, detected its expression in infected sugarcane, and verified its expression in a heterologous system, to further discern its functional role.Transcription is the first step of gene expression, and σ factor plays a key role in starting transcription, so studies on σ factor and anti σ factor are considered the key for regulating the gene expression in recent years. However, it is hard to culture Lxx18460 cDNA was isolated from the infected sugarcane. It was 720 bp in size and the predicted molecular weight of Lxx18460 protein was 24.8 kDa with an isoelectric point (pI) of 5.01  from GenBank was up to 100%, and has been registered with the accession No. JQ740153.A full-length Lxx18460 using software of SMART and Motif Scan showed that the Lxx18460 protein domain located at the 43–238 position is RskA family (anti ơ K factor). The prediction of the transmembrane region of Lxx18460 shows that there is a transmembrane region, which is located between 92 and 114 bp. The amino acid sequence homology of Lxx18460 is low as compared to other strains  , and fifteen of them were found having Lxx18460 gene, NPTII by PCR detection  were compared. As shown in Fig. The SOD, POD, CAT and T-AOC activities in tobacco leaves were measured at different time. The results showed that the CAT, POD and SOD activities were lower in TT than WT plants Fig. a-c. As c3 contents in leaves were significantly lower in the transgenic tobacco plants , respectively. It was noticed that there were downregulated genes only in the terms “signal transduction”, “positive regulation of biological process”, “membrane enclosed lumen”, “molecular transducer activity”, “electron carrier activity”, and “structural molecule activity”. There were upregulated genes only in the terms “cell Killing”, “transcription factor activity”, and “protein binding” , and transformed it into tobacco; the transgenic tobacco showed significant resistance against Tobacco mosaic virus and rot pathogen Pectobactrium carotovorum subsp. carotovorum. Tang et al. [Sugarcane mosaic virus (ScMV) coat protein (CP) gene into sugarcane variety Badila, and the transgenic sugarcane of ScMV-CP gene significantly enhances the ScMV resistance of sugarcane and improve the cane yield and quality greatly. In this study, we analyzed the function of Lxx membrane protein gene Lxx18460 (anti-sigma K) by transforming it into tobacco, and then investigated the phenotypic, physiological, biochemical and molecular changes in the transgenic tobacco plants. We found that the net photosynthetic rate was lower in the transgenic tobacco than that in WT plants. The reason may be that Lxx18460 gene inhibited the expression of photosynthetic enzyme genes, and this speculation was confirmed by quantifying the relative expression of photosynthesis related genes using RT-PCR. We observed that the expression of Rubisco and PEPC genes were inhibited in the transgenic tobacco. Yu et al. [brownblotch strains [pseudomonas solanacearum [Lxx18460 transgenic tobacco declined as compared to the WT plants, which suggested that the defense capability had been decreased by transformation of Lxx18460 gene. The transcriptome analysis results also showed that the expression of catalase was decreased. The transgenic plants were more susceptible to membrane damage compared to the WT plants. The pathogen Lxx showed extremely strong infection effect though it had been diluted for hundreds of times [Lxx18460 gene in pathogenic bacteria inhibits the gene transcription of the defense enzymes and damage the defense regulation of host plants, which would help the pathogen to infect the host and to multiply inside the plant cell. Growth of plant is not only affected by the change of various enzyme activities, but also regulated by various endogenous hormones [3, IAA and ABA were inhibited in the Lxx18460 transgenic plants, which suggested that the expression of Lxx18460 gene suppresses the biosynthesis and signaling pathways of the endogenous hormones. The physiological and phenotypic changes in the transgenic tobacco proved that Lxx18460 plays a role in inhibiting the signal transduction and hormone synthesis gene expression.Li et al.  construcg et al.  transforu et al.  reportedu et al.  found thu et al. –30. Exceu et al. . POD act strains , while Cnacearum . In thisof times . It mighhormones . AccordiLxx [Lxx18460 gene expression in tobacco are similar to those caused by Lxx in sugarcane. After transforming the gene Lxx18460 into tobacco, the net photosynthetic rate was decreased and the endogenous hormone synthesis inhibited, and the growth of transgenic plants hindered. As the result the plant height was lower and the leaf area smaller, subsequently, the plants become dwarf and smaller, and the biomass decreased. Furthermore, this study demonstrated that the expression level of Lxx18460 gene and Lxx18460 protein was stably increased with time course during plant growth. The results strongly supported the view that the Lxx18460 gene had been transformed into tobacco, and its expression had slowed down the growth of the transgenic tobacco plants. The higher expression showed the Lxx18460 gene, the more obvious was the effect on transgenic tobacco.It has been reported that the photosynthesis and cane yield were decreased, and defense enzyme activities and endogenous hormone levels were adversely affected by the infection of Lxx –39. InteLxx18460 of anti-sigma K (ơK) factor in Leifsonia xyli subsp. xyli in sugarcane was cloned, and its structure and expression in sugarcane infected by RSD were analyzed. The function of Lxx18460 gene was analyzed by transforming it into tobacco. The results showed that the expression of Lxx18460 gene could inhibit the growth of the transgenic tobacco. Lxx18460 gene plays an important role in regulating signal transduction, gene transcription and plant growth, and in the course of infection in sugarcane. It is important to further investigate the function of Lxx18460 gene and its role in Lxx pathogen. This is the first report of analyzing the function of the membrane protein gene Lxx18460 of anti-sigma K (σK) factor in Leifsonia xyli subsp. xyli. It is necessary to further investigate the interaction between sugarcane and Lxx using Lxx18460 gene to illustrate the molecular mechanism of Lxx infection in sugarcane.The membrane protein gene Lxx-infected sugarcane (Saccharum spp.) variety GT11 grown in the Sugarcane Institute, Guangxi University  was used for cloning Lxx18460. The wild type tobacco (Nicotiana tabacum L. variety 346) was used for transgenic analysis.The Lxx-infected sugarcane stalk was extracted using Trizol Reagent  [Lxx18460 from the GenBank (NC-006087), we design the specific primers as R1: 5’-ATGACCGACGACGAGCGGGG-3′ and R2: 5’ TCAGGAGGCGATGCCTACGA-3′ using Primer Premier 5.0 software. The putative full length Lxx18460 gene was obtained using RT-PCR technique. The reaction procedure was as follows: 10 min at 95 °C for degeneration, 40 cycles for 40 s at 94 °C, for 40 s at 62 °C, for 90 s at 72 °C, and a final extension for 10 min at 72 °C. The amplified target bands were recovered and purified with a Gel Extraction Kit , and then attached to pMD18-T vector , transformed into DH5a competent cells ; and the positive clones were identified by sequencing.Total RNA from the , China) , and thehttps://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to analyze the sequence in the NCBI database. The physical properties of the Lxx18460 protein were predicted using Expasy (http://web.expasy.org/protparam/). Conservative structure and function of the gene encoded protein domains were analyzed using NCBI CDD (Conserved Domain Databases), SMART (http://smart.embl-heidelberg.de/). Transmembrane structures of the protein were analyzed using TMPRED Server  and TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM/).BLAST  and GAPDH tobacco gene (accession number M14419) were used as the internal controls to quantify the relative transcript levels in Lxx-infected sugarcane and transgenic tobacco, respectively [Quantitative real-time PCR technique was applied to detect the expression of u et al. . We calc formula . For traectively , 44.Lxx18460 expression vector primers were designed according to the Lxx18460 gene ORF. EcoR I endonuclease site, Xho I endonuclease site and protective base pair were added to the 5′ terminal of the primer for gene expression detection. The prokaryotic expression vector pET-30a was transformed into E. coli BL21(DE3)pLysS. After the IPTG induction process, the protein was isolated, purified and analyzed by SDS-PAGE with Image Lab 5.0 software. The protein was identified and confirmed by mass spectrometry (MALDI-TOF). The protein induced and over-expressed in E. coli was used as antigen to produce monoclonal antibody. ELISA test was applied to analyze the titer. The specificity was analyzed using Western blot in bacteria and in the healthy and Lxx-infected sugarcane plant samples.The Lxx18460 was cloned using PCR technique, and then inserted into a pBI121 vector (conserved by our lab). This vector was under control of cauliflower mosaic virus 35S (CaMV35S) promoter and nopaline synthase (NOS) terminator [Lxx18460 construction was introduced into the Agrobacterium tumefaciens strain EHA 105, and then transformed into the WT tobacco callus as described by Horsch et al. [The full length open reading frame (ORF) of rminator . The rec2O two times, followed by disinfection for 8–10 min in 4% sodium hypochlorite, rinsing with sterile ddH2O five times before they were sown in plate culture medium (M1) based on MS medium [− 1 sucrose and 0.7% agar (pH 5.8). The infected explants were co-cultured on M1 medium complemented with 1 mg L− 1 6-benzyladenin (BA) and 0.5 mg L− 1 indoleacetic acid (IAA), which is defined as M2 medium, at 25 °C in dark for 4 days. Then the co-cultured plants were rinsed with ddH2O containing 300 mg L− 1 cephalosporin. Positive transgenic plants were selected on M2 medium containing 100 mg L− 1 of kanamycin. The kanamycin-resistant shoots were transferred to M3 selection medium  for rooting. A few days later, the non-transgenic and transgenic status of the T1 plantlets was confirmed by PCR and Western blot analysis.The tobacco seeds were sterilized with 70% ethanol for 30 s and then rinsed with sterile ddHS medium  supplemeLxx18460 in the independent T2 generation was also determined by PCR and Western blot. For physiological and biochemical analysis, the seedlings were grown in the glasshouse condition. During the T2 generation growth period, Lxx18460 expression was observed and analyzed in the transgenic and WT plants using the same method as described above.The T1 plants were just used for phenotypic observation. Seeds of wild type and the transgenic T1 plants were harvested and used for further experiments. The expression of Lxx18460 and marker gene of NPTII. The PCR reaction and PCR procedure was described in Additional file The genomic DNA was extracted from the leaves of transgenic and WT tobacco plants using a modified SDS extraction method , and tesTotal protein was extracted from the leaves of transgenic and WT tobacco plants using a modified phenol extraction method , and quaPlant height was measured from the transgenic and WT tobacco stalk at 50, 80, and 110 days after emergence, respectively. Leaf area was measured using CI-203 Handheld Leaf Area Meter . Net photosynthetic rate was measured using Li-6400 photosynthesis system .Activities of SOD, POD, CAT and T-AOC were measured from leaves of the transgenic and WT tobacco at 50, 80 and 110 days after emergence, respectively. The enzymes were extracted from 0.5 g leaf and grounded in 2 mL extraction buffer containing 0.1 M phosphate buffer (pH 7.4) and 1% polyvinylpyrolidone using a mortar and pestle kept in ice bath. The homogenate was centrifuged at 10,000×g for 10 min at 4 °C and the supernatant was used for enzyme assay. The SOD activity was measured using the nitro-blue tetrazolium method . The actThe fresh leaves of transgenic and WT tobacco were sampled at 50, 80 and 110 days after emergence, respectively. The plant hormone was extracted from 1 g leaf with 2 mL extract buffer (80% methanol containing 1 mM butylated hydroxytoluene) and grounded into homogenate in ice bath. Then solution containing 0.1% Tween-20 and 0.1% gelatin phosphate buffer, pH 7.5) was used to dissolve the hormone extract. The extracts were analyzed using ELISA method with five repeats .Total RNA of each sample was extracted using the Trizol Reagent  according to the manufacturer’s protocol from three biological replicates of each treatment (WT and Transgenic) resulting in 6 samples. The total RNA concentration of each sample was estimated using a Agilent 2100 Bioanalyzer (Agilent Technologies Inc.). The integrity of the RNA samples was assessed with 1.2% agarose gel electrophoresis. The six samples were sequenced using BGISEQ-500 Sequencing performed by Beijing Genomics Institute (BGI)-ShenZhen, China according to the manufacturer’s instructions. Detailed BGISEQ-500 sequencing experimental process and transcriptome data analysis method refer to Additional file P values set at 0.05 and 0.01 levels, respectively, and means were compared by standard error (SE).All the data were analyzed using Microsoft Excel 2003 (Microsoft) and IBM SPSS 21.0. All data points represent means from 15 plants of transgenic or WT tobacco. There were three technical replicates for each experiment, and statistical differences were compared based on the Duncan’s test with Additional file 1:Nucleotide, predicted amino acid sequences (A) and conserved domain (B) of Lxx18460. (DOCX 102 kb)Additional file 2:Identification of transgenic tobacco plants detected with PCR. (DOCX 198 kb)Additional file 3:Lxx18460 transgenic tobacco. (DOCX 541 kb)KEGG pathway analysis of DEGs in Additional file 4:Lxx18460 transgenic tobacco. (DOCX 38 kb)Quantitative real-time PCR analysis of selected DEGs in Additional file 5:PCR identification of transgenic tobacco. (DOCX 16 kb)Additional file 6:BGISEQ-500 sequencing and transcriptome analysis, the primers of DEGs validated by qRTPCR analysis. (DOCX 21 kb)


The Captopril challenge test (CCT) is an easy-conduct confirmatory test for diagnosing primary aldosteronism (PA). Guidelines show that plasma aldosterone is normally suppressed by captopril (> 30%) in primary hypertension (PH) and in healthy people. It is unclear whether this standard is applicable in Chinese subjects. The aim of the present study was to investigate the post-CCT efficacy of plasma aldosterone concentration (PAC) suppression and determine the post-CCT aldosterone renin activity ratio (ARR) and PAC for PA diagnosis.We recruited 110 consecutive patients with PA, 163 with primary hypertension (PH), and 40 healthy volunteers (NC). The CCT was conducted in all patients. Total sodium intake was estimated from 24-h urinary excretions. ROC curves were used to analyze the efficiency of different CCT diagnostic criteria for diagnosing PA.P < 0.05). The mean degree of PAC decline after CCT was approximately 9.3%, and only 11.7% of the patients with PH showed a greater than 30% suppression of PAC after CCT. In patients with PA, the post-CCT change in PRA and PRC was slight. The post-CCT degree of PAC decline was unrelated to dietary salt intake. The areas under the ROC for the post-CCT ARR, PAC and PAC suppression % were 0.994, 0.754 and 0.606, respectively. The optimal post-CCT cutoff value for ARR for diagnosing PA was 20, which yielded a sensitivity and specificity of 94.0 and 99.4%, respectively.In NC and PH patients, PRA was increased and PAC was decreased post-CCT  to the CCT. Primary aldosteronism (PA) is the most common cause of secondary hypertension and affects approximately 10% of patients with essential hypertension . In addiA previous study demonstrated that in China, the overall prevalence rate of PA in adults with resistant hypertension was 7.1% , which iConfirmation tests are a crucial step in the process of obtaining a functional diagnosis of PA. The Endocrine Society’s guidelines for PA recommend 4 confirmatory tests, including the fludrocortisone suppression test (FST), saline infusion test (SIT), captopril challenge test (CCT), and oral sodium loading. Among these confirmatory tests , the CCTThe present study aimed to investigate the usefulness of, optimal interpretation approach to, and cutoff value for the CCT in diagnosing PA in Chinese patients. Furthermore, the correlation between estimated sodium intake and CCT performance was also assessed.The present study was conducted in the Nanjing Drum Tower Hospital. All subjects provided informed consent. We retrieved data from hypertensive patients who were referred to our unit from January 2011 to December 2016 with a suspicion of PA because of resistant hypertension, spontaneous or diuretic-induced hypokalemia, or the finding of an adrenal mass. Potassium-wasting or -sparing diuretics were discontinued at least 4 weeks prior to testing. Other drugs, including β-blockers, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, were withdrawn for at least 2 weeks. Patients with severe hypertension were prescribed α-blockers, and long-acting calcium channel blockers were prescribed if necessary. Dietary sodium intake was unrestricted. Patients with hypokalemia were given adequate oral or intravenous potassium supplements to maintain serum potassium levels higher than 3.0 mmol/L before the study was begun.Data representing normal controls were retrieved from healthy volunteers who participated in the study and had a BP below 140/90 mmHg and no history of hypertension and were not receiving any medication. Finally, we included 110 PA patients, 163 primary hypertension (PH) patients and 40 normal controls (NC).Sodium and potassium levels in blood and urine were measured using fully automated instrumentation. Plasma renin activity (PRA) was measured by radioimmunoassay using a commercial kit . The intra- and inter-assay coefficients of variation (CVs) for PRA were 10 and 15%, respectively. PAC was measured by radioimmunoassay with commercial kits . The intra-assay and inter-assay CVs for this assay were 5.6 and 6.2%, respectively.Blood samples for PRA and PAC were obtained in all patients between 0800 and 0900 h after overnight fasting and at least 2 h lying in a supine position and then again after 2 h of keeping in an upright position. The CCT was performed in the morning. Patients received 50 mg of captopril orally after they were upright  for at least 2 h and seated for 5–15 min. Blood samples were drawn to measure PRA and PA at time 0 and at 2 h after challenge. The patient remained seated during this period.All hypertensive patients underwent an enhanced adrenal computed tomography (CT) scan with fine cuts (3 mm). Adrenal vein sampling (AVS) procedures were performed between 0800 and 1200 h by one radiologist using the bilateral simultaneous technique without cosyntropin stimulation. According to previous recommendations, successful cannulation was considered a selectivity index (SI) ≥ 2, and lateralization of aldosterone secretion was defined as a lateralization index (LI) ≥ 2.PH was defined as a systolic and diastolic BP greater than 140/90 mmHg according to the established criteria and excluding secondary hypertension, such as renal parenchymal hypertension, renovascular hypertension, endocrine hypertension, aortic coarctation, sleep apnea syndrome, and drug-induced hypertension.Identification of PA required all of the following criteria: (1) ARR ≥ 25 and PAC > 12 ng/dL in addition to (2) at least one of the following additional endocrine alterations: a. upright PRA < 1.0 ng/ml/h, b. ARR post-CCT ≥ 20 or post-CCT PAC suppressed less than 30%.Identification of aldosterone-producing adenoma (APA) was based on the following criteria: (1) evidence of PA as defined above, (2) lateralization of aldosterone secretion at CT and/or AVS, (3) evidence of adenoma at CT and/or surgery and/or pathology, and (4) demonstration of normokalemia and hypertension cure or improvement or normalization of PAC and PRA at follow-up after adrenalectomy.Identification of idiopathic hyperaldosteronism (IHA) was based on the following criteria: (1) evidence of PA as defined above, (2) bilateral adrenal lesions on CT scan and/or aldosterone secretion at AVS, (3) demonstration of normokalemia and improvement of hypertension after treatment with mineralocorticoid receptor antagonists.P < 0.05 was considered to be statistically significant. Most statistical analyses were performed using the software package SPSS 21.0 for Microsoft Windows (SPSS Inc.). The ROC curve comparison was performed with MedCalc software 15.2.2 .Normally distributed data are expressed as the means ± standard deviation, and abnormally distributed data are expressed as a median . Values between groups were compared by one-way analysis of variance for parametric data and the Kruskall-Wallis test for nonparametric data. The diagnostic accuracy of post-CCT PAC suppression (as a percentage), PAC and ARR for identifying PA was assessed with a receiver-operated characteristic (ROC) curve and the area under the ROC curve (AUC). The best cutoff and both sensitivity and specificity are provided. A value of P < 0.01). BP was higher in the APA group than in the PH group (P < 0.01). Compared to the NC and PH groups, patients in the PA group had lower serum potassium and higher serum sodium and urine potassium levels (P < 0.01). PAC was markedly higher and PRA was significantly lower in the PA group than in the PH and NC groups (P < 0.01). The ARR was consequently much higher in the PA group than in the PH and NC groups (P < 0.05). The prevalence of hypokalemia was higher in the APA group than in the IHA group (92.7% vs 75%). Compared to patients with IHA, those with APA had higher PAC (P < 0.01). No other significant differences were detected between the APA and IHA groups.We recruited 110 consecutive patients with PA. Of these patients, 82 had an APA, and 28 had IHA. The clinical characteristics of each group are summarized in Table P < 0.01). However, the mean post-CCT suppression percentage of PAC was only approximately 9.3%, and only 11.7% of the patients with PH showed a post-CCT decrease in PAC greater than 30%. In PA patients, the CCT increased PRA and lowered PAC slightly. After CCT, PAC remained higher and PRA was lower in the PA group than in the PH and NC groups (P < 0.01). As a result, ARR remained higher post-CCT higher in the APA and IHA groups than in the PH and NC groups (P < 0.01). The post-CCT PAC was still higher in the APA group than in the IHA group (P < 0.05). No other differences were detected in the indexes between the APA and IHA groups after CCT . As a result, ARR was lower post-CCT than pre-CCT  and 0.754 , respectively. The AUC of the post-CCT ARR was larger than that of the post-CCT PAC (P < 0.01) .According to the Endocrine Society Clinical Practice Guidelines, a post-CCT PAC suppression percentage less than 30% is recommended to confirm PA.osing PA , 11. UsiFour testing procedures  are recommended by the Endocrine Society’s 2008 and 2016 clinical guidelines for PA confirmation diagnosis . Until rThe diagnostic efficiency of the CCT varies substantially among previous studies , 19–21. In addition to our study, other data collected in China have demonstrated that PAC suppression in the CCT or SIT is lower in Chinese than in Western patients , 22, 23.The principle the captopril test is based is the suppression of the serum concentration of aldosterone and an increase in the level of PRA when angiotensin-converting enzyme is inhibited in patients without autonomous aldosterone secretion . PreviouThe optimal post-CCT ARR cut-off value is variable among studies that support the use of post-captopril ARR as a PA diagnostic tool. The divergence in cutoff values may be associated with the assays and techniques used to measure aldosterone and PRA. Consistent with the guidelines of the Japan Endocrine Society, we found that the best cutoff for the post-CCT ARR was 20, which yielded relatively high sensitivity (94.0%) and specificity (99.4%) in this study. Therefore, we found that the post-CCT ARR is a reliable marker for diagnosing PA in a Chinese population.In summary, in this study, we demonstrate that CCT is a reliable functional test for confirming a PA diagnosis. Compared to Western populations, Chinese populations exhibit lower degrees of post-CCT PAC suppression. As a result, the post-CCT PAC decline below 30% recommended as a threshold by the Endocrine Society is ineffective for diagnosing PA in Chinese hypertension subjects. Compared to the post-CCT PAC, the post-CCT ARR is a better approach when used with an optimal cutoff of 20 for interpreting the results of the CCT in Chinese patients. Furthermore, there was no relationship between high salt intake and lower RAS responses in this study. Further study is needed to identify the factors underlying this variation between Chinese and Western populations.


This study determined the effects of a novel combination of vitamin D and probiotic on metabolic and clinical symptoms in chronic schizophrenia.9 CFU/day probiotic (n = 30) or placebo (n = 30) for 12 weeks.This trial was conducted among 60 patients with chronic schizophrenia to receive either 50,000 IU vitamin D3 every 2 weeks plus 8 × 10P = 0.004) and total PANSS scores . Vitamin D and probiotic co-supplementation also significantly increased total antioxidant capacity , and significantly decreased malondialdehyde  and high sensitivity C-reactive protein levels  compared with the placebo. Moreover, taking vitamin D plus probiotic significantly reduced fasting plasma glucose , insulin concentrations , homeostasis model of assessment-estimated insulin resistance , triglycerides  and total cholesterol levels  and total−/HDL-cholesterol ratio .Vitamin D and probiotic co-supplementation was associated with a significant improvement in the general  for clinical trials registration (http://www.irct.ir: IRCT2017072333551N2). 07-31-2017 2This study was retrospectively registered in the Iranian website ( Over 50 million people around the world suffer from schizophrenia . It is tIt has been shown that crosstalk between nutrition and mental health is associated with neurotransmitter and hormonal pathways in the gut which regulate the function of brain . Low levGiven the anti-inflammatory, antioxidant and immune-modulatory impacts of vitamin D and probiotics, we hypothesized that their combination might be beneficial in chronic schizophrenia. Therefore, conducted to evaluate the impacts of vitamin D and probiotic on clinical symptoms, biomarkers of oxidative stress and cardiometabolic risk in chronic schizophrenia.http://www.irct.ir: # IRCT2017072333551N2). It was conducted at a psychiatry clinic in Kashan, Iran from July to October 2017. Any participant who diagnosed with schizophrenia using DSM-IV-TR criteria with disease duration of at least two years, had PANSS score of 55 or greater, treated with chlorpromazine  and agents anticholinergic  during the last 6 months and aged 25–65 years old were included in the study. Any subject with mental retardation (Intelligent Quotient of< 70), substance or alcohol addiction (except caffeine or nicotine) within the last 6 months of screening, a score of≥14 on a 17-item Hamilton Depression Rating Scale or a score of ≥4 on PANSS (depression item), anyone under treatment with lithium, carbamazepine, sodium valproic acid, with existing chronic and acute medical illness, with lactation or pregnancy, the use of antidepressants including MAO, TCA, SSRI in the last 6 months were excluded from this trials. Study protocol followed the principals of the Declaration of Helsinki and was approved by the ethics committee at Kashan University of Medical Sciences (KAUMS) (No. IR.KAUMS.REC.1396.10). Informed consent was signed by all participants prior to the intervention.This randomized, double-blind, placebo-controlled trial was registered by the Iranian registry of clinical trials (2) and type and dosage of psychiatric medications were taken into consideration. Then, participants were equally allocated into either taking 50,000 IU of vitamin D3 every 2 weeks plus 8 × 109 CFU/day of probiotic supplements containing Lactobacillus acidophilus, Bifidobacterium bifidum, Lactobacillus reuteri, and Lactobacillus fermentum (each 2 × 109) for 12 weeks  or placebo which were capsules in the similar shape and packaging as vitamin D and probiotic . Participants were instructed not taking other vitamin D and probiotic supplements, while maintaining their regular diet and physical activity during the trial. Placebos, vitamin D, and probiotic were produced by Zahravi Pharmaceutical Company , LactoCare®, Zisttakhmir Company  and Barij Essence Pharmaceutical Company , respectively. Study compliance was determined by counting the remaining tablets in the returned containers as well as measuring serum 25-OH-vitamin D concentration. All participants were asked to record their food intake for 3 days (one and two weekdays) at week 1, 5, 9 and 12 of the trial. These recorded data were used to calculate participants’ nutrient intake for the Iranian food pattern. Physical activity was assessed through participants’ physical activity record at the first day of each month [Computer-generated random numbers were used by an instructed staff to randomize study participants at the psychiatry clinic. While randomizing, age (< 65 vs. ≥65 y), gender , BMI (< 25 vs. ≥25 kg/mch month .The Height and weight of subjects were obtained without shoes and in light clothing by a trained staff in the psychiatry clinic at baseline and the end of the intervention. Body Mass Index (BMI) was reached as weight in kg divided by height in square meter.Severity of psychiatric symptoms was assessed using PANSS  and diffFasting blood (10 mL) were collected from study participants at the beginning and after the 12-week intervention at Kashan reference laboratory . Serum 25-hydroxyvitamin D concentrations were measured using an ELISA kit  with inter- and intra-assay coefficient of variations (CVs) below 7%. Total antioxidant capacity (TAC) concentrations were measured using the method of ferric reduction antioxidant power developed by Benzie and Strain . Total gSuggested formula for clinical trial sample size calculation was used to calculate sample size in this study. Considering type one (α) and type two errors (β) of 0.05, and 0.20, of the power of study was 80%. According to Sheikhmoonesi et al. , we usedt-test was performed to compare the differences in baseline characteristics and daily dietary macro- and micro-nutrient intakes between the two treatment groups. Pearson Chi-square test was conducted for the comparison of categorical variables. To assess the impacts of vitamin D and probiotic on clinical symptoms, biomarkers of inflammation and oxidative stress, lipid profiles, insulin metabolism and repeated measures analysis of variance (RM ANOVA) was used. To control confounding variables, such as baseline values of each biochemical variable, age and baseline BMI, we were done analysis of covariance (ANCOVA). Significance level was the p-value of less than 0.05. Statistical Package for Social Science version 18  was applied to run all statistical analyses in this study.The Kolmogrov-Smirnov test was employed to check the normal distribution of data. Intention-to-treat (ITT) analysis was applied for all randomly allocated cases. Independent samples Four participants in the placebo groups and supplemented dropped out Fig. . However(Table ).There was no significant difference between the two groups in terms of height, age, weight, BMI and METs at baseline as well as mean changes in weight and BMI throughout the trial Table .Table 1G(Table ).Based on 3-days dietary records, we observed no significant difference in dietary energy, protein, carbohydrate, saturated fatty acids, fat, polyunsaturated fatty acids, monounsaturated fatty acids, total dietary fiber, cholesterol and micronutrients Table .Table 2MP < 0.001), the general  and total PANSS score  (Table 3). In addition, probiotic and vitamin D significantly increased plasma TAC , and decreased MDA  and serum hs-CRP levels . Moreover, taking vitamin D plus probiotic, significantly reduced FPG , serum insulin concentrations , HOMA-IR , triglycerides , total- , and total−/HDL-cholesterol ratio . Vitamin D and probiotic co-supplementation also significantly increased QUICKI . Taking vitamin D and probiotic had no significant effect on BPRS score and other metabolic profiles. Linear regression analysis revealed no association between serum 25-OH-vitamin D status and PANSS score at study baseline .After the 12-week intervention, vitamin D and probiotic led to a significant enhancement in 25-OH-vitamin D levels , BPRS (P < 0.001), GSH (P = 0.002) and plasma NO (P < 0.001). Hence, we adjusted the analysis for baseline values of biochemical parameters, age and baseline BMI. After controlling for these potential confounders, the difference in changes in total−/HDL-cholesterol ratio (P = 0.06) between the two groups became non-significant, while difference in changes in negative PANSS score (P = 0.03), BPRS (P = 0.03) and plasma GSH (P = 0.004) became statistically significant (Table ). Other metabolic profiles did not change statically after this adjustment.There was observed a statically difference in the baseline levels of positive PANSS score ( P = 0.00 became s2) by using computer random numbers. Therefore, the difference in positive PANSS score and BPRS existing in both groups occurred by chance. In the current study, there was no significant difference between two intervention groups in terms of weight and BMI after 12-week intervention. Further studies are required to examine the effects of probiotic and vitamin D on weight, BMI and metabolic outcomes in schizophrenia, considering longer duration, which may affect clinical symptoms.We found that probiotic and vitamin D for 12 weeks to patients with chronic schizophrenia had ameliorated effects on the general and total PANSS scores, as well as their metabolic profiles. This clinical trial reporting the effect of vitamin D and probiotic on clinical symptoms, biomarkers of oxidative stress and inflammation, lipid profiles and glycemic control in chronic schizophrenia. In current study, all patients were being hospitalized during the intervention; otherwise there was no difference in treatment protocol between outpatient and inpatient. The only difference might be the compliance rate which was closely monitored for inpatients. Moreover, to our best knowledge, the overall treatment protocol for schizophrenic patients is similar around globe. So, this study can be generalized to other countries as well. It should be taken into account that there was a significant difference in positive PANSS score and BPRS between placebo and vitamin D plus probiotic groups at study baseline. There are several reasons to explain this difference. In the current study, we did not randomize subjects based on their PANSS score and BPRS due to being diagnosed with chronic schizophrenia. Random allocation into two groups was done after stratification for age (< 65 vs. ≥65 y), gender  and pre-intervention BMI  criteria, 41% of US in schizophrenia have metabolic syndrome . Anti-inThis study is related to some limitations. In the current report, we did not characterize the microbiota and thus cannot establish whether probiotic administration over 12 weeks altered microbiota composition which leads to funding limitations. In addition, we could not evaluate an analysis of 16 s RNA of fecal matter. Therefore, analysis of 16 s RNA of fecal matter is suggested in future studies. Moreover, one cannot explain if the treatment impacts found in the present report was due to the impact of which component of the combined supplementation. Hence, future research is needed with single supplement in the present study in order to assess the ameliorate impacts of each supplement on clinical symptoms and metabolic profiles in chronic schizophrenic patients with longer duration of supplementation.Overall, probiotic and vitamin D for 12 weeks to chronic schizophrenia had impacts effects on the general and total PANSS scores, as well as their metabolic profiles. In addition, when we adjusted the analyses for baseline values, baseline BMI and age, the difference in changes of negative PANSS score and BPRS became statistically significant.


Long non-coding RNAs (lncRNAs) have recently emerged as one of the critical epigenetic controllers, which participate in several biological functions by regulating gene transcription, mRNA splicing, protein interaction, etc. In a previous study, we reported that lncRNAs may play a role in developing depression pathophysiology. In the present study, we have examined how lncRNAs are co-expressed with gene transcripts and whether specific lncRNA/mRNA modules are associated with stress vulnerability or resiliency to develop depression. Differential regulation of lncRNAs and coding RNAs were determined in hippocampi of three group of rats comprising learned helplessness , non-learned helplessness , and tested controls (TC) using a single-microarray-based platform. Weighted gene co-expression network analysis (WGCNA) was conducted to correlate the expression status of protein-coding transcripts with lncRNAs. The associated co-expression modules, hub genes, and biological functions were analyzed. We found signature co-expression networks as well as modules that underlie normal as well as aberrant response to stress. We also identified specific hub and driver genes associated with vulnerability and resilience to develop depression. Altogether, our study provides evidence that lncRNA associated complex trait-specific networks may play a crucial role in developing depression. The onset of depression is primarily associated with stressful life events, which act as precipitating factors in individuals with increased risk of depression vulnerability . FactorsSince long-term exposure to stress develops a feeling of helplessness, pervasive sadness, and despair in individuals with a high degree of susceptibility , an animTo further examine the role of lncRNAs in stress resiliency and in the vulnerability to develop depression, in the present study we constructed a co-expression network based on differentially expressed mRNAs and lncRNAs in the hippocampus of LH and NLH rats. We also used another group of rats that were not given stress but tested for depressive behavior  to examine the non-specific effect of behavioral testing per se. To construct such networks, weighted gene correlation network analysis (WGCNA) algorithm  was usedDetailed methods are provided in the Supplementary methods section.All animal experiments were performed in male Sprague–Dawley rats (Holtzman strain) between the age of 6 and 8 weeks under the guidelines of Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham. The experimenter was blinded to all the behavioral measurements.n = 7) and NLH . Another group of rats , which was tested for ET without giving any shock, was also included to rule out the non-specific effects of stress caused by restraint, tail shock, or testing. We used 6–7 rats per group based on our previous study in which we found a robust change in lncRNA expression in hippocampus of LH and NLH rats [The study was performed in learned helpless model of depression in which rats were divided into two groups based on escape latency (ET): LH . One additional package, used in the analysis, was downloaded from Bioconductor open source platform . Normalip < 0.05 was set for statistical significance.After obtaining modules from each group, module eigengene, summarized as the first principal component of expression dataset, was calculated with the “ModuleEigengenes” function. The module eigengene is a weighted average of module gene expression profile. Association analysis between a module and the trait of each group was performed as the function of “corPvalueStudent” based on the module eigengene. i represents the strength of a correlation between a gene and a phenotypic trait. Ki, which means intramodular connectivity, was calculated from the sum of its connection strengths with all the other genes in the same module.The two important parameters, including gene significance (GS) and intramodular connectivity (Ki) were used for identifying hub genes of behavior-associated modules. Intramodular hub genes were selected based on a strong correlation with depression (GSi > 0.9) and higher connectivity (Ki > 0.9). GSTo conduct gene-ontology (GO) function for detected modules of lncRNA datasets, we measured the correlation between lncRNAs and mRNAs by Pearson product-moment correlation. We selected the top tenfold change of each module in the lncRNA dataset for correlation analysis. The absolute correlation values were calculated between each lncRNA and the entire DE mRNA dataset in the same group. We ranked absolute correlation values and selected the top ten mRNAs with higher correlation values. For each module of lncRNA dataset, we identified 100 mRNAs highly correlated with lncRNAs for further functional analysis.p values were calculated for GOs enriched among different modules. Pathway analysis was performed with the function of “Pathview” and “org.Rn.eg.db” (the rat Genome-wide annotation). The p value of the enriched pathway was derived from Database for Annotation, Visualization and Integrated Discovery (DAVID) [GO analysis was explored as the function of each module. We used the “topGo” function in R platform to identify the significant enrichments. The GO category was separated into three groups: the molecular functions, the biological processes (BP), and cellular components. The two-sided Fisher’s test was used to classify the GO category and  (DAVID) .We also constructed gene co-expression networks to identify the interactions among genes or lncRNAs of each module. To make a visual network, only the strongest correlations (≥0.94) were included in these representations. The node in the networks represented a coding transcript or lncRNA in the modules. In addition, the two genes connected by an edge indicated a strong correlation . The co-expression networks were produced using “igraph” package on the R platform.To specifically identify differentially expressed lncRNAs or mRNAs (DEGs) associated with either vulnerability or resiliency to depression, a Venn diagram analysis was performed with the package “draw.triple.venn” on the R platform. For vulnerability (LH), we subtracted the DEGs overlapping for “LH vs. TC and LH vs. NLH” and overlapping for “NLH vs. TC and LH vs. NLH” from the group of LH vs. TC. For resilience behaviors (NLH), we subtracted the DEGs overlapping for “NLH vs. TC and LH vs. NLH” and overlapping for “LH vs. TC and LH vs. NLH” from the group of NLH vs. TC.To explore the relationship between the depression-related hub lncRNAs and hub mRNAs, we performed CCA analysis using the function “CCP” with R from open source code platform GitHub. The underlying principle of CCA is related to multivariate integrative analysis of paired data. Here the CCA can detect the linear components that are directly involved in the development of depressive behavior.st strand cDNA for coding transcripts, conventional oligo dT priming method was used. Primer sequences are provided in the Supplementary Table Relative quantification of coding transcripts was determined following the ∆∆Ct method  by usingp < 0.01) between TC, NLH, and LH groups when tested on three different days . Individual group comparison showed significantly higher EL for LH rats compared with TC and NLH rats on days 2 (p < 0.001), 8 (p < 0.001), and 14 (p < 0.001). NLH rats did not show any significant difference in EL when compared with the TC group at any time point   (19) Fig. .p value was <0.05 and the fold change was >1.3  and LTGturquoise  modules were negatively correlated with phenotype changes within the two groups. This shows that these two modules comprised of genes that have higher expression levels in the LH group. In contrast, the other three modules: LTGyellow , LTGbrown , and LTGgreen  showed positive correlation . We implemented the dynamic branch-cutting algorithm with a robust measure of interconnectedness using DynamicTreeCut and the WGCNA R library. Each module was assigned a unique color label, which is visualized underneath the cluster dendrogram shown in Fig. ion Fig. .Table 1MA total of 395 DEGs were significantly altered in the NLH vs. TC comparison. Four modules were found in this group comparison: NTGbrown, NTGturquoise, NTGblue, and NTGyellow  and LTGgreen (p = 0.023) modules showed significant correlations between intramodular connectivity and gene significance. The LTGturquoise module was by far the largest module containing 290 transcripts. GO-enrichment analysis showed that among others, the biological process related to “G-protein coupled receptor (GPCR) signaling pathway” and “detection of chemical stimulus” involved in sensory perception were most significantly associated with turquoise module (Supplementary Table p = 4.30E−08) associated with 44 DEGs and (2) neuroactive ligand–receptor interaction (p = 3.10E−02)   that were significantly associated with LH behavior. The relatedness of traits with significantly altered hub genes often comprise an intramodular connectivity within the network module and represent a convenient course to pathway-based gene screen procedure depending on the degree of intramodular connectivity. As shown in Fig. n p = 0.03 modules116 Fig. , which wnes Fig. .Fig. 2IdSigmar1 and Lpal2. Both of them were significantly downregulated  in the LH compared with the TC group (Supplementary Table The LTGgreen module consisted of 52 DEGs Table . The netp = 0.9) between intramodular connectivity and gene significance  and neuroactive ligand–receptor interaction (p = 3.70E−03) pathways (Supplementary Tables p = 0.49) between intramodular connectivity and gene significance  and viral myocarditis (p = 6.10E−02) pathways were the most enriched ones (Supplementary Table p = 0.39)  Fig. . Moreovep = 0.0011) , whereas the pathway associated with “olfactory transduction” was represented by both NTGturquoise and NTGblue modules   except LNGturquoise (p = 0.091) , Olr8 (olfactory receptor 8), and Sgpl1 (sphingosine-1-phosphate lyase 1)  was identified as hub mRNA, which seems to play a crucial role in the entire network . Top ten mRNAs having the highest correlations with lncRNAs were selected for functional annotations. Some lncRNAs had the overlapping correlations with the same mRNAs. Thus, the number of mRNAs was less than 100 within each module.p = 7.50E−02) and cardiac muscle contraction in the LTCbrown (p = 3.70E−02) and LTCturquoise (p = 1.80E−02) were associated with LH (Supplementary Table r = 0.16) and LTCbrown (r = 0.32)  Fig. . Therefo32) Fig. .Fig. 4Cop = 0.0039)  of the NLH group  and was comprised of 314 DE lncRNAs  associated with olfactory transduction pathway , Sptlc3 , Sult2a2 , Six1 (SIX homeobox 1) , and Agxt2  were the top five ranked genes , Lmo2 , Tmem163 , B3gnt8 , and Sfrs14  were the top significantly altered coding transcripts  without any change in other variates (variats 2–5).The canonical correlation coefficients (CCA) are shown in Supplementary Table Rnf29, Tas2r116, Myl3, Inexa, and Olr8) across various comparison groups and analyzed their expression by qPCR using the RNA isolated from hippocampus. It was found that the two hub genes (Rnf29 and Tas2r116) from TC vs. LH group were significantly upregulated  similar to that observed in the microarray analysis  was more in the LH vs. TC group than in the other two comparing groups. Interestingly, similar to lncRNAs, mRNA dysregulation in LH vs. TC was the most significant among the three groups. The striking alteration of transcripts in LH vs. TC was found to be associated with different phenotypic changes. Among all 458 upregulated mRNA transcripts in LH vs. TC, the “Olr60” related to olfactory receptor 60, showed the highest expression change. On the other hand, out of 298 downregulated transcripts, Olr601 coded by olfactory receptor 601 was found to be strikingly downregulated in this group. Similarly, our analysis in the NLH vs. TC group identified Txndc8 (thioredoxin domain containing 8), which was the top ranking mRNA based on the magnitude of its expression. In the LH vs. NLH comparison, we identified several interesting mRNAs including Slc10a5 , Vom1r42 , and Olr1222 (olfactory receptor 1222), primarily for their potential involvement in the central nervous system (CNS)-related functions [Rnf29 and Tas2r116 driver genes, unlike the other three tested genes  from TC vs. NLH and LH vs. NLH groups, they showed a similar pattern of expression. This validates our findings from microarray expression assay regarding hub gene identification.In our study, we conducted transcriptome-wide expression array using 8977 lncRNA and 14605 mRNA probes and identified differentially expressed transcripts with fold change > 1.3 and unctions –37. We aThe DEG extracted from the gene-expression array experiment provided interesting results considering their potential value as biomarkers for mental illnesses. In the past, compelling evidence has suggested that individual genes only play a partial role in the development and progression of psychiatric phenotype. Increasing number of studies have suggested that complex phenotypic disorders are associated with a correlation between genes and are often supported by intricate co-expressional network, which connects clinical traits, genome and transcriptome ,38,. To Interestingly, pathway analysis demonstrated that several modules from LH vs. TC comparison  and LH vs. NLH comparison  were significantly associated with olfactory transduction. Also, GO analysis suggested that these modules were significantly associated with sensory abilities. Based on these findings, it can be strongly hypothesized that dysfunctionality in olfactory processing might be associated with the development of depression. In fact, removal of olfactory bulb (OB) induces changes in neurochemical, neuroanatomical, physiological, and endocrine functions . PreviouNext, we integrated the data from three comparisons and selected the candidate genes specific to depression vulnerability or resiliency. Interestingly, the enrichment of olfactory transduction pathway appeared again both in the susceptibility and resiliency. Our results also revealed a significant correlation between lncRNAs and mRNAs using the CCA, which elucidated their potential regulatory mechanisms. A similar correlation between the lncRNA- and mRNA-expression profiling has recently been reported in the peripheral blood of depressed patients .Tas2r116, Expi, Rnf29, Oprs1, and LOC690326) and 20 lncRNAs were identified as hub genes. Tas2r116 is a member of vertebrate taste receptor family and represents the GPCR superfamily [Tas1r3) in taste tissue and reduced gustatory nerve excitation by sweet compounds [Expi is located on chromosome 10q26, includes three exons encoding for extracellular proteinase inhibitors, and is involved in the proteinase inhibiting capacity, cell invasive, and cell metastatic potential [Myl3) and four lncRNAs in the LH vs. NLH group, four mRNAs  and six lncRNAs in the NLH vs. TC group were analyzed with the stringent criteria. These hub genes were not only highly correlated, but also had high-intramodular connectivity with the other genes in the same module. At present, it is difficult to illustrate the biological functions of these hub genes; however, they indicate new molecular pathways that can be associated with resiliency or development of depression phenotypes. Further studies will be needed to dissect their phenotypic association.Hub genes play a central role in the co-expression networks. For the LH vs. TC comparison, 5 hub mRNAs  compared to humans. Although it is difficult to translate rodent findings to humans, the association of lncRNA in major depression has been elaborated in two recent studies using human subjects. One study, conducted in peripheral blood, showed the differential regulation of both lncRNAs and mRNAs in MDD patients, which were mostly associated with metabolic process and neurodevelopmental disorders . AnotherSupplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9Primer sequences used for qPCR expression assaySupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8qPCR-based expression changes of select hub genes associated with rat model of depressionIllustration of the workflow analysis followed for data generation


Little data is available on awareness of hemophilia carrier condition or associated bleeding risk and management in Sub-Saharan African countries. This study sought to identify hemophilia carriers in Côte d’Ivoire in order to collect data on demographics, bleeding phenotype, and laboratory results. Another purpose was to provide Ivorian hemophilia carriers with counseling on their risk of bleeding and of having children with hemophilia. A 12-month prospective study was conducted involving Ivorian hemophilia carriers recruited trough pedigree analysis pertaining to 81 hemophilia patients followed-up at the Yopougon Hemophilia Treatment Center in Abidjan. They were assessed using in-depth interviews, pedigree analysis, and laboratory testing.Sixty-one subjects comprising 27 obligate and 34 possible carriers were recruited. None had previously been assessed, with 64% unaware of their carrier status despite a familial history of hemophilia in 69%. The most frequently reported bleeding symptom was menorrhagia (31%). Prolonged bleeding was reported after vaginal delivery in 19.6%, post-surgery in 4.9%, and post-dental extraction in 4.9%. Only one carrier was treated with tranexamic acid, with no other hemostatic therapy recorded. The median (range) clotting FVIII was 0.85 IU/mL (0.24–1.90 IU/mL) and FIX 0.60 IU/mL (0.42–1.76 IU/mL) in hemophilia A and B carriers, respectively. HA carriers had a FVIII < 0.5 IU/mL in 12.5%.This study highlights the need of implementing care for hemophilia carriers in developing countries, and the high value of pedigree analysis for carrier identification, along with the relevance of diagnosis, treatment, and education of carriers, families, and caregivers. Hemophilia is a congenital X-linked recessive bleeding disorder causing low levels of Factor VIII  or Factor IX , occurring in approximately 1 in 5000 for HA and 1 in 20,000–30,000 for HB) live male births .A wide range in clotting FVIII or FIX levels is observed in carriers, independently of hemophilia severity within the family , which iPreviously, pedigree analysis and clotting FVIII/IX levels were applied for diagnosing hemophilia carriership . Since tIn developing countries, carrier detection proves even more crucial for early hemophilia diagnosis in children, because they often die early in life due to the lack of diagnostic opportunities, absence of care facilities, and limited access to clotting factor concentrates (CFC) . As circCentre Hospitalier universitaire (CHU) of Yopougon in Abidjan and the international HTC of the Cliniques universitaires Saint-Luc in Brussels, Belgium. This collaboration has provided the opportunity to identify and assess potential and obligate hemophilia carriers among families of patients with hemophilia (PWH) in this location.In 2015, a twinning partnership was established between the Ivorian hemophilia treatment center (HTC) of the This study aimed to identify obligate and potential carriers of hemophilia in Côte d’Ivoire and collect detailed information on their demographics, pedigree, bleeding phenotypes, and laboratory results. Yet, its ultimate goal was to provide Ivorian hemophilia carriers with appropriate counseling on the risk of bleeding and of having children with hemophilia.Between January and December 2017, we invited all the ≥12-year-old female Ivorians, identified as HA or HB carriers, to participate in this study conducted at the Yopougon HTC. Candidates were recruited via detailed family trees of 81 PWHs followed-up at the HTC. The carriers, their parents, or legal guardians provided written informed consent for participating in the study.This was a single-center cross-sectional study conducted from January to December 2017 on the Ivorian hemophilia carrier population. Data were prospectively collected during standardized consultations held by Belgian and Ivorian teams including a hematologist and hemostasis laboratory member.Tosetto et al, 2006. Women who had one point or more for this item were considered as having menorrhagia.For each carrier, an in-depth face-to-face interview was performed gathering data on carrier status awareness, socio-demographics, educational activity, employment, medical, obstetrical, and surgical history, bleeding symptoms and circumstances, requirement and indication for whole blood transfusion, as well as co-medications including estroprogestative contraception, iron supplementation, and hemostatic and antifibrinolytic agents’ usage. A specific attention was paid to menorrhagia assessed by the bleeding questionnaire described by To identify carriers in each family, a detailed pedigree was drawn. Efforts were made to investigate as many generations as possible. Of note is that the family trees were repeated at each evaluation of several different members of the same family, whether carriers or PWHs. This allowed us to establish exhaustive and validated trees, after merging, correcting and updating steps. Mendelian laws were applied to determine the probability of being a carrier. The carriers were categorized as obligate or possible/probable carrier, based on the pedigree, the level of FVIII or FIX and according to the WFH definitions .The biological workup comprised a complete blood-count  and FVIII and FIX activity measurements by one-stage assay method on a semi-automated coagulometer  using human plasma immunodepleted for FVIII and FIX .All data were entered into a structured electronic medical record and analyzed using SigmaStat and SigmaPlot . The pedigrees were drawn using the Progeny Free Online Pedigree Tool from Progeny Software .Based on pedigree analysis of 81 Ivorian PWHs, we identified 83 obligate carriers (12 for HB and 71 for HA) and 248 possible carriers (30 for HB and 218 for HA), resulting in an average of four carriers per PHW. A total of 61 carriers for severe and moderate hemophilia A and B were included with 27 obligate carriers (3 for HB and 24 for HA) and 34 possible carriers (2 for HB and 32 for HA) , Krou (36%), Mandé , Gour (10%), or other 3,2%). Most were living in or around Abidjan (54%), had a professional activity (47.6%), were homemakers (36%), with the remainder either students or in professional training (16.4%). Their marital status was as follows: married (44%), single (28%), in couple (15%), divorced (11%), or widowed (2%). Malaria, which is endemic in Ivory Coast, was the most common medical condition . The HIV%. Most wDetailed pedigrees were obtained through 142 interviews with 61 carriers and 81 PWHs. The 61 carriers studied were issued from 32 families. A familial form of hemophilia was present in 20 families involving 42 carriers and a sporadic form, defined as cases with no other PWH – whether alive or deceased - within the family, in 12 families involving 19 carriers. In familial forms, the median number of affected relatives per carrier was 4 (range 2–11). The median (range) number of obligate carriers and possible carriers per family for familial forms was 2 (0–4) and 4 (1–14), respectively. In sporadic forms, the median (range) number of possible carriers per family was 3 (1–6). Among these 20 families, 39 PWH death cases were identified, 12 of whom were related to circumcision and 7 to intracranial hemorrhage. The median number of children per carrier was 2 (range 0–9) and that of sons affected with hemophilia per carrier was 1 (range 0–6).The most frequently spontaneous bleeding symptom was excessive or prolonged menstrual bleeding, with 31% prevalence among the whole carrier population and 26% prevalence in the obligate carrier group. Other unprovoked hemorrhagic symptoms, such as nose bleeds, cutaneous bruising, gum bleeding, or hemarthrosis, had < 5% prevalence , 1 after vaginal delivery, 1 after cesarean, and 1 after a complicated episiotomy. No transfusion was recorded after spontaneous bleeding. One carrier reported a blood transfusion for anemia in childhood. Tranexamic acid was used on-demand by only one symptomatic carrier for severe HA while experiencing menorrhagia, mucocutaneous bleeding, and postpartum hemorrhage. None of the HA carriers had previously been assessed nor treated with Desmopressin (DDAVP), or preventively treated with hemostatic therapies. Iron supplementation was recorded in 3.3%. Eighteen % were on hormonal therapy, using estroprogestative pills (9), implants (1), or subcutaneous injections (1). Other treatments comprised paracetamol in 14.8%, anti-malarial drugs in 4.9%, and antiretroviral therapy in 3.3%. No one reported using anti-inflammatory drugs.In 38% of participants, clotting factor activity had previously been determined in the 2016 WFH’s twinning program, with the lowest value considered for this study. The median (range) clotting FVIII level in the 56 HA carriers was 0.85 IU/mL (0.24–1.90 IU/mL). The median (range) clotting FVIII level in obligate and possible HA carriers were 0.75 IU/mL (0.24–1.90 IU/mL) and 0.92 IU/mL (0.60–1.57 IU/mL), respectively. Overall, 7 (12.5%) HA carriers had a FVIII < 0.50 IU/mL. In the 5 HB carriers, the median (range) FIX level was 0.60 IU/mL (0.42–1.76 IU/ml), with 2 (40%) displaying FIX < 0.50 IU/mL. Bleedings in correlation with factor level decreases are listed in Table The median (range) hemoglobin level was 11.9 g/dL (8.81–14.8), with a level < 10 g/dL found in 8.8%. The median (range) platelet count was 243 × 10 [Limited research has been conducted so far in Sub-Saharan countries on hemophilia carriers , 19, 4. Pedigree analysis rendered it possible to identify 331 possible or obligate carriers, of which 61 were considered for analysis. Considering the theoretical 400 carriers linked to the 81 PHWs from the HTC of Yopougon, pedigree was of great value to target carriers within PWH’s families. In the absence of molecular testing availability, pedigree is a cost-effective and useful tool for assessing the carrier status of females from PWH’s families and can be of a great help in providing genetic counselling . Family Despite numerous affected relatives or hemophilia-related deaths (31% after circumcision), at least in the familial forms, 64% of participants were unaware of their carrier status. None had ever been clinically assessed, nor had FVIII or IX level measurements been conducted prior to the twinning program. No carrier had ever had DDAVP testing, with only one carrier previously treated with tranexamic acid for bleeding. These data highlight the lack of the carriers’ knowledge of hemophilia inheritance and their bleeding risks, along with the ignorance of healthcare providers concerning their increased bleeding risk and bleeding management. This highly-relevant unawareness about carrier condition has already been reported in studies conducted in India and South Africa , 25.In our study, 44.3% of carriers reported spontaneous bleeding, with menorrhagia the most common symptom. This is in line with data from other studies focused on hemophilia carriers , 10, sevAlthough most carriers exhibited normal-range factor levels, a significant proportion displayed decreased factor activity (between 40 and 60 IU/mL in 12.5% of HA carriers and 60% of HB carriers and < 40 IU/mL in 9% of HA carriers and 0% of HB carriers). Bleeding was recorded in the three factor level categories. Our findings are consistent with literature data indicating that carriers may exhibit variable factor levels , with anOur study displays several limitations: 1) molecular testing was unavailable to formally establish or confirm the carrier diagnosis; 2) factor levels were measured only once in 62% and twice in 38%, and this measurement could not be repeated at distance of hormonal therapy; 3) the factor activity testing was performed using only a one-stage coagulation assay; 4) we had no information on blood group and von Willebrand antigen/activity; 5) anemia interpretation proves difficult in Ivory Coast where malaria is endemic .Given our study data and within the frame of the WFH’s twinning program, actions have been implemented at Yopougon’s HTC designed to improve the care of hemophilia carriers. All the participants were personally informed, using either oral or written support, about their carrier status, bleeding risk, preventive and therapeutic hemostatic measures to apply if necessary, need for a medical follow-up especially during pregnancy and upon delivery, as well as the relevance to screen all male children for hemophilia early in life and at least before circumcision. The seven carriers with a FVIII level < 50 IU/mL underwent DDAVP testing, and the use of tranexamic acid was encouraged as necessary. Educational booklets and workshops were developed and provided, highlighting the need to actively screen carriers among PWH’s families, and assess their bleeding risk by factor level measurements.Possible and obligate carriers were provided with the same information, as there is actually no regular access to DNA analysis in Côte d’Ivoire. Mother education proves to be the cornerstone towards improving PWH’s care, as gender plays a key role in shaping the burden of care in Africa, with women more likely than men to take on caregiving activities . MoreoveThe current study illustrates the lacking awareness of the hemophilia carrier condition and its implications for carriers, PWH’s families, and medical community in Côte d’Ivoire and by extension in other developing countries. The data highlight the need for implementing dedicated care and developing guidelines taking in account local conditions and priorities, enabling carriers to be identified, their factor levels to be assayed, and counseling on the possible chances of bleeding and having children with hemophilia.This work underlines the significance of repeated and detailed pedigrees in carrier screening, especially in countries with limited molecular testing. Providing access to individual assessment, establishing appropriate hemostatic treatment plans, and using adjunctive hemostatic drugs  should be actively promoted. Education and information of the carriers, their families, and healthcare givers form the basis enabling us to carry out this major project in Côte d’Ivoire.This research demonstrates the benefits and strengths of a multidisciplinary and comprehensive approach in holistic hemophilia care involving all family members including females in the WFH twinning program setting.


Within the B–C–IM group, preoperative ECHO raised suspicion of type A in 13 patients , whereas non-A CA was suspected in 12 patients . With the exception of reoperation, which was seen only in the ID subgroup , the intraoperative (cardiopulmonary bypass time and cross-clamp time) and postoperative morbidity indices were comparable in both ID and CD subgroups (p > 0.1). Although there is a significant risk for early postoperative morbidity in TGA patients with single, interarterial, and intramural CA, there seems to be relatively limited influence of preoperative ECHO assessment of coronary anatomy on this morbidity burden.In transposition of the great arteries (TGA), certain coronary patterns have been associated with major adverse events early after the arterial switch operation (ASO). We sought to determine the impact of preoperative echocardiographic (ECHO) diagnosis on the intra and postoperative morbidity. All patients with TGA born between June 2001 and June 2017 and who underwent ASO were reviewed. Data on presumed coronary anatomy (CA) preoperatively were obtained from the preoperative ECHO report. Intraoperative CA was categorized according to Yacoub classification. Major postoperative morbidity included at least one of the following: delayed sternal closure (DSC), prolonged (> 72 h) mechanical ventilation, reintubation, peritoneal dialysis (PD), ECMO, reoperation, and readmission within 30 days after surgery. 240 patients with median age of 5 days (range 1–614) and mean weight at surgery was 3.6 kg (1.8–8.4) were included. Preoperative ECHO assessment of CA was available in 228 patients. Intraoperatively, 181 patients (75%) were found to have type A, 25 patients had type B or C or intramural , and 34 patients had type D or E . Patients with types B, C, and intramural coronary (B–C–IM) had increased risk for delayed sternum closure (9/25 vs. 20/181 in type A and 8/34 in type D–E;  Transposition of the great arteries (TGA) is a congenital heart defect that, in terms of both diagnosis and surgical management, has undergone important advances with significant improvement in mortality and morbidity. A major step in this regard has been the adoption of the arterial switch operation (ASO) instead of the previously used atrial switch . ASO is To understand and improve outcome of TGA patients, several studies have investigated risk factors for postoperative mortality and morbidity. Thus, recognized factors for mortality and morbidity include institutional and surgeon’s inexperience, prematurity, lower weight at ASO, presence of ventricular septal defect, and left ventricle outflow obstruction including arch anomaly –9. One aThere are a few ways to categorize the CA in TGA. A commonly used classification is based on Yacoub`s studies where the anatomy is divided in 5 subgroups; type A to E . The mosThe preoperative diagnosis of TGA is typically based on transthoracic echocardiography (ECHO) and includes assessment of coronary anatomy , 17, 18.Whether an accurate CA diagnosis improves ASO outcome has, to the best of our knowledge, not been investigated. We therefore sought to determine the impact of preoperative ECHO diagnosis on the intra and postoperative morbidity.Clinical records of all patients with TGA, with or without ventricular septal defect (VSD), born between June 2001 and June 2017 and treated at Skane University Hospital were reviewed. Medical data until discharge, including readmission within 30 days, were retrieved from the electronic patient chart system or from the paper-based medical records available in the hospital archive. Inclusion criteria were patients with TGA who underwent primary ASO at Skane University Hospital, and born during the above time interval. Exclusion criteria were double outlet right ventricle, outflow tract obstruction, chromosome abnormalities, and associated non-cardiac diseases.Hemodynamically significant VSD was defined as either surgically closed VSD or by need of banding of the pulmonary artery at the time of ASO. Restrictive atrial septal defect was defined by need for balloon atrioseptostomy prior to ASO. Hospital mortality was defined as death within 30 days of the ASO. Major postoperative morbidity (MPM) was defined as the presence of 1 or more of the following: MV > 72 h, delayed sternum closure, reoperation, need of non-invasive ventilation post extubation, use of extracorporeal membrane oxygenation (ECMO) after ASO, peritoneal dialysis, and readmission within 30 days after discharge. Reoperation was defined as any surgical reintervention within the first month after ASO.One hundred seventy-seven of 240 patients were diagnosed at hospitals other than Lund and seven patients were transferred from the Reykjavik University Hospital, Iceland. Median age at admission to Lund was 1 day. Fetal diagnosis of TGA was available in 59 cases. Prostaglandin infusion of 10–100 ng/kg/min was started either immediately after birth, when fetal diagnosis was available, or after postpartum diagnosis. ECHO of CA was attempted in all patients, as part of routine preoperative assessment. If unclear CA, ECHO was repeated typically by a senior cardiologist. All patients were admitted to either the pediatric or the neonatal ICU.ASO was performed through median sternotomy and under cardiopulmonary bypass with hypothermia in all patients. In two cases, coronary arteries could not be transferred to the neo-aorta due to complex coronary anatomy. Eventual additional cardiac or extracardiac defects were repaired at the same stage. Three patients underwent a pulmonary banding during ASO due to a complex VSD and with a significant shunt that could not be surgically closed. Assessment of surgical result was performed by the pediatric cardiologist using transoesophageal or epicardial ECHO. Standard care during the first days after surgery included mechanical ventilation (MV), inotropic, diuretic, and analgesic support. New ECHO examination was routinely performed prior to extubation, and, if stable hemodynamics and spontaneous breathing were attained, patients were transferred to the pediatric cardiac ward for further care.Aorta was transected 0.5–1 cm above the commissures and the coronary ostia were inspected and investigated for any abnormal anatomy. The coronary ostia were excised with a large D-shaped cuff. The pulmonary artery was transected and medial hinged trapdoor flaps were cut at the site of previously placed marking stitches. If the coronary ostia were placed eccentrically or were intramural, the valve commissure was excised and later resuspended to the pericardial patch that was implanted to cover the defects in the aortic sinuses. The coronary artery cuffs were transferred and re-implanted into the incisions producing the trapdoor flaps without creating any kinking, rotation, or inappropriate stretching. Techniques used to deal with non-A CA are described elsewhere .Data on presumed CA preoperatively were obtained from the surgeons preoperative evaluation and eventually compared with the pediatric cardiologist`s ECHO report. Intraoperative CA was categorized according to Yacoub classification in types A–E , 21. DemThe preoperative ECHO diagnosis was assessed by the ability to distinguish type A from non-A CA. Secondly, preoperative ECHO was evaluated by the accuracy to predict the intraoperative CA, which was regarded as reference standard.p value of less than 0.05 was considered statistically significant. SPSS Statistics version 22.0 for Mac  and StatView 5.0 were used for data analysis.Categorical data are presented as counts and percentages, whereas numerical data are given as mean ± SD or, in case of variables with non-Gaussian distribution, as median and range. Either non-parametric test  or ANOVA were used to compare numerical variables, whereas Chi-square test with Yates correction for continuity was used for categorical dichotomous variables. A n = 61) were reviewed. Median age at ASO was 5 days (range 1–614) and mean weight at surgery was 3.6 kg (1.8–8.4). Preoperative characteristics are summarized in Table Two hundred forty-five patients matched the inclusion criteria. Of these, two patients had missing clinical records and were therefore excluded. Two patients were excluded due to total anomalous pulmonary venous drainage and Goldenhar syndrome , respectively. One premature baby born week 34 with birth weight 2 220 g and CA type A, developed severe myocardial failure shortly after ASO and died several days later on ECMO due to severe cerebral bleeding. This patient was also excluded from further analysis. Consequently 240 patients (girls Fourteen (5.8%) patients had extracardiac defects; in 13 patients , correction was achieved during ASO. In one patient, the coarctation was repaired at a later stage as it was initially judged as non-significant. Eighty-three (34.6%) had a VSD requiring repair. The intraoperative characteristics are summarized in Table Preoperative ECHO data of CA were available in 228 patients; the remaining 12 patients had missing or unclear preoperative CA assessment. Type A was suspected in 196 patients and non-A CA in 32 patients.Intraoperative findings identified 181 patients (75.4%) with type A, 25 patients (10.4%) type B or C or intramural (IM), and 34 patients (14.2%) with type D or E. Among the 196 patients with preoperative ECHO suspicion of type A, 33 patients (17%) had non-A CA. Among the 32 patients with suspect non-A CA, 8 patients (25%) were found to have type A. For type A, the preoperative ECHO had a sensitivity of 95.3% and a specificity of 42%.p = 0.3).Preoperative ECHO assessed CA was incorrect in 40 out of 228 cases. When comparing first and second half of the study period, there was no significant difference regarding the accuracy of diagnosing CA with preoperative ECHO , peritoneal dialysis , and ECMO . The remaining postoperative indices, including non-invasive ventilation, reoperation, and MPM-score, showed no significant difference (p ≥ 0.1). Similarly, there was no difference in intraoperative variables including cardiopulmonary bypass and cross-clamp time. Postoperative data are summarized in Table Patients with types B, C, and intramural coronary (B–C–IM) had increased risk for delayed sternum closure . Regarding postoperative morbidity indices, reoperation was needed in 4/12 in the ID group versus 0/10 in the CD group (p = 0.04). The preoperative and postoperative characteristics are summarized in Tables Within the B–C–IM group, preoperative ECHO raised suspicion of type A in 13 patients , whereas non-A CA was suspected in 12 patients . When comparing these two subgroups for intraoperative variables (cardiopulmonary bypass time and cross-clamp time), no difference was found , interarterial (type C), and intramural (IM) CA, with increased early postoperative morbidity. Even though the accuracy of preoperative ECHO diagnosis for non-A CA was fairly modest, ECHO appeared to have only minor effect on postoperative outcome. Thus, with the exception of the risk for reoperations, which was mildly increased, no other index of early postoperative morbidity was associated with failure to correctly diagnose non-A CA by ECHO.Given the complexity of ASO as surgical method, the preoperative assessment includes thorough investigation of CA. With the advance of ultrasound technique, echocardiography is increasingly used in the preoperative diagnosis of CA in neonates with TGA. Coronary ECHO is though time-consuming and requires in addition to modern ultrasound equipment a high level of training and expertise. Thus, we sought to assess the accuracy and importance of preoperative coronary ECHO on several indices of intra- and postoperative morbidity. Since only one patient died during the studied time interval, no conclusions can be drawn in with regard to the impact of coronary ECHO on mortality.As some types of CA have been linked to both immediate and late complications after ASO, the purpose of preoperative coronary ECHO is to increase the awareness of surgical team with respect to eventual high-risk CA. Therefore, we divided the cohort based on preoperative CA echo in type A and non-A. ECHO diagnosis of CA has highest sensitivity for type A (18). Assessment of CA is far more difficult in non-A types, especially in patients with intramural and single coronaries .Previous TGA studies of CA assessment with ECHO have reported a sensitivity for CA diagnosis of 60–90% , 22, 23 The present findings indicate increased early postoperative morbidity in patients with types B–C–IM but similar intraoperative indices compared with patients with type A. Most of the previous reports of CA have mainly focused on postoperative mortality, however, such analysis was not possible in our study due to only one death. Previous meta-analysis of CA and outcome showed that intramural coronaries have an approximately six-fold higher mortality (OR 6.5), single coronary with a loop has threefold increased risk (OR 2.9), whereas CA with looping coronaries around the great vessels from different ostium were not linked to increased hospital death . This obFrom a morbidity perspective, intramural coronaries have been correlated with higher incidence of intra- and postoperative coronary problems requiring interventions  and inveAs with all retrospective studies, there are inherent limitations that caution carefulness in interpretation. Although the main cohort has a relevant size, the subgroup B–C–IM is small and could neutralize any significance even though we failed to find any. Clearly, further large prospective studies are warranted.Although there is a significant risk for early postoperative morbidity in TGA patients with single, interarterial, and intramural CA, there seems to be relatively little influence of preoperative ECHO assessment of coronary anatomy on this morbidity burden. Refining of ECHO technique for better delineation of especially non-A CA is nevertheless important. Further prospective studies are warranted to better investigate the association of preoperative ECHO diagnosis with postoperative morbidity and mortality. Until future studies have elucidated the impact of ECHO, surgeons need to be aware of the relative inaccuracy of ECHO in CA assessment.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. For this type of study, formal consent is not required.