MET alterations in 474 advanced non‐small‐cell lung cancer (NSCLC) patients by nCounter, an RNA‐based technique. We identified 3% with METΔex14 mRNA and 3.5% with very‐high MET mRNA expression, a surrogate of MET amplification. MET alterations identified by nCounter correlated with clinical benefit from MET inhibitors. Quantitative mRNA‐based techniques can improve the selection of patients for MET‐targeted therapies.We studied  MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA‐based technique, together with next‐generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT–PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very‐high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very‐high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very‐high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA‐based techniques can improve the selection of patients for MET‐targeted therapies.MET inhibitors have shown activity in non‐small‐cell lung cancer patients (NSCLC) with  FISHfluorescence in situ hybridizationHShistoscoreIHCimmunohistochemistrynCnCounterNGSnext‐generation sequencingNSCLCnon‐small‐cell lung cancerRT–PCRreverse transcription polymerase chain reaction1MET) gene has recently emerged as an actionable target, particularly in non‐small‐cell lung cancer (NSCLC) [MET activation, which increases cell proliferation, survival, invasion, and metastasis. MET amplification has been described in 1–6% of newly diagnosed NSCLC tumors and constitutes a frequent mechanism of acquired resistance in EGFR‐mutant (EGFR‐mut) NSCLC patients treated with tyrosine kinase inhibitors (TKI) [MET exon 14 alterations in donor and acceptor splicing sites—including point mutations, indels, and whole‐exon deletions—lead to the exclusion (skipping) of MET exon 14 at the RNA level (MET∆ex14), which has been described in 3–4% of patients with advanced NSCLC [Aberrant activation of the mesenchymal–epithelial transition ( (NSCLC) , 2. Mult (NSCLC) , 4 can lrs (TKI) . MET exoed NSCLC , 8.MET gene in NSCLC and mutations leading to MET∆ex14 were first reported in 2005 and 2006, respectively [MET∆ex14 as a potential therapeutic target in advanced NSCLC [MET inhibitors in patients with MET∆ex14 and MET amplification [MET∆ex14 [Amplification of the ectively , 10, whied NSCLC , 12. Sinfication , 16, 17,MET∆ex14 , and novMET∆ex14 .MET gene copy number variations in the clinical setting are fluorescence in situ hybridization (FISH) and next‐generation sequencing (NGS), whereas for MET∆ex14 detection, both NGS and reverse transcription polymerase chain reaction (RT–PCR) are commonly used [MET testing [MET status in advanced NSCLC and the fact that most reports evaluating the performance of different techniques have focused on a single MET alteration.The most frequent technologies used to assess nly used , 23, 24. testing  have beeMET alterations in particular types of tumors [MET alterations. To this end, we screened a large cohort of NSCLC patients and compared the nCounter results with those obtained by standard techniques. Our results indicate that multiplex, RNA‐based techniques such as nCounter have the potential to become the technology of choice to select patients for MET‐targeted therapies.The NanoString nCounter™ Analysis System is a high‐throughput, quantitative transcript‐based hybridization technology that allows for the simultaneous analysis of the expression of hundreds of target genes  and can f tumors , 29, 30,f tumors , 31. Her22.1MET alterations. Samples were collected from 10 participating hospitals (Supporting Information) with prior full informed patient consent and approval from the corresponding ethical committees. All advanced NSCLC patients arriving to our institutions and having biopsies available with sufficient tumor tissue were offered to participate in the study, which was conducted in accordance with the principles of the Declaration of Helsinki. FFPE slides (4 µm) were obtained by standard procedures and stained with hematoxylin and eosin. A pathologist determined the tumor area and evaluated the percentage of tumor infiltration. RNA was extracted with a high purity FFPE RNA isolation kit , while the GeneRead DNA FFPE Kit or the QIAamp DNA FFPE Tissue Kit  was used for DNA extraction from FFPE samples, according to the manufacturer’s instructions. DNA and RNA concentrations were measured by Qubit . Three cell lines  were used for validation purposes. The Hs746T cell line, harboring METΔex14, was purchased from the American Type Culture Collection. E98 is a patient‐derived astrocytoma cell line with amplification of MET gene. EGFR‐mut PC9 cells were obtained from F. Hoffman‐La Roche Ltd  with the authorization of Dr. Mayumi Ono . All cell lines were cultured in RPMI medium with 10% fetal bovine serum under standard conditions and counted after trypsinization. Pellets from a minimum of five T‐75 flasks were used to generate FFPE blocks.A total of 474 formalin‐fixed, paraffin‐embedded (FFPE) tumor samples from patients with NSCLC were tested to identify 2.2MET was performed with the ZytoLight® SPEC MET/centromere 7 (MET/CEP7) Dual Color Probe  according to manufacturer’s instructions. Three positivity criteria for MET amplification were used as follows: (a) a ratio (r) MET/CEP7 ≥ 2; (b) gene copy number (GCN) per cell ≥ 6; (c) ≥ 5 copies in ≥ 50% of cells; (d) or ≥ 15 copies in > 10% tumor cells. These three criteria have been employed for patient stratification in clinical trials of anti‐MET therapies (Table FISH for es Table . Immunos2.3® QIAact Lung DNA UMI Panel (Qiagen) or OncomineTM Solid Tumour (OST) DNA Panel (Thermo Fisher Scientific), according to the manufacturer’s instructions (Table DNA NGS was performed with the GeneReadns Table . Both pa® QIAact Lung RNA Fusion UMI Panel (Qiagen) according to the manufacturer’s instructions. This panel targets a fusion‐specific and splicing variants  software was employed to align the read data and call sequence variants, which were imported into the Qiagen Clinical Insight Interpret (QCI‐I) web interface for data interpretation and generation of final custom report. In the case of the OST panel, 10 ng of purified DNA was used as a template. Libraries were pooled at 20 pM and, following ion spheres, sequenced using the Ion Personal Genome Machine (Thermo Fisher Scientific). The Ion Reporter Server (Thermo Fisher Scientific) was used to align the read data to the human reference genome and call sequence variants. The MET gene copy numbers by NGS were assessed using the QCI‐A and QCI‐I software. Copy numbers provided by QCI‐I were selected according to the tumor infiltration of the sample, as assessed by an expert pathologist. The GCN ≥ 6 was chosen as a threshold based on the previous experience of our laboratory. During the validation of the NGS panel prior to its implementation in the clinical setting, this threshold had been found to show the highest correlation with FISH MET/CEP7 > 2.Qiagen Clinical Insight Analyze (2.4METΔex14 was amplified using HotStart Taq polymerase (Qiagen) in a 20 µL reaction and visualized in agarose gels. Primers used were located in exons 13 and 15, sequences were as follows: forward (exon 13) 5′‐TTTTCCTGTGGCTGAAAAAGA‐3′ and reverse (exon 15) 5′‐GGGGACATGTCTGTCAGAGG‐3′. Amplification generated a 246‐bp band for wild‐type (wt) MET RNA and a 106‐bp band for METΔex14. Positive samples were confirmed by bidirectional Sanger sequencing of RT–PCR products, using the big‐dye 3.1 sequencing kit .RNA was converted to cDNA using M‐MLV retrotranscriptase (Thermo Fisher Scientific) and oligo‐dT primers, and 2.5MET‐wt and METΔex14 target sequences. Detailed sequence information for the MET gene target regions is provided in Table ACTB; proteasome 26S subunit ATPase 4, PSMC4 and mitochondrial ribosomal protein L19, MRPL19), positive and negative controls. All processes of hybridization, capture, cleanup, and digital data acquisition were performed with nCounter Prep Station® and Digital Analyzer®  according to the manufacturer’s instructions. Reporter counts were collected with the nsolver analysis software version 2.6. Samples were considered not evaluable if the geometrical mean (geomean) of counts corresponding to the housekeeping genes was lower than 100. Counts from MET probes were normalized in two steps, as described [MET expression values. Two cutoff values were used for log‐MET results; (a) the mean plus standard deviation (SD) was used to define cases with moderately (mod.)‐elevated MET mRNA, (b) the mean plus two SD for very‐high MET mRNA levels. Regarding METΔex14 testing, log‐ratios were obtained dividing the normalized counts of the METΔex14 probe by the normalized counts for the MET‐wt probe. The cutoff for METΔex14 positivity was established as the average log‐ratio of the sample cohort plus 2 SD. Samples with no counts for the METΔex14 probe were directly considered negative.Total RNA was hybridized with a custom‐designed mixture of biotinylated capture tags and fluorescently labeled reporter probes (Elements Chemistry) that included, among others, probes for escribed , and sub2.6MET‐wt) tested negative. Next, using FFPE blocks prepared by spiking different numbers of MET‐dependent cells (Hs746T and E98) in a suspension of PC9 cells, we established that 1 mm2 of a 4‐µm section with a minimum of 10% of Hs746T cells was required for METΔex14 detection, whereas 30% of E98 cells were needed to detect high levels of MET mRNA expression by nCounter. In similar experiments, RT–PCR detected METΔex14 mRNA in mixtures with 0.1% mutant tumor cells. Finally, we performed repeatability studies using FFPE tumors from advanced NSCLC patients. In the case of METΔex14 detection by nCounter, four positive and 20 negative samples were analyzed in two independent experiments showing a 100% concordance. Regarding MET mRNA expression, 29 samples run in two independent experiments revealed concordant results in the classification for 28 of them .Using frozen pellets from Hs746T or E98 cells, we found that 5000 cells and 25 ng of RNA were sufficient for successful detection of spliced transcripts or very‐high MET mRNA levels, respectively. In contrast, pellets containing 500 000 of PC9 cells  had geomean of housekeeping gene counts below 100 and were excluded from the study. The remaining 422 evaluable samples, corresponding to 405 patients, were mostly stage IIIB/IV adenocarcinomas Table , Table 11% had ge3.2METΔex14 vs. the MET‐wt probes. A sample was considered positive if the log‐ratio was above a threshold value, established as the mean plus two times the SD of all samples analyzed  were identified, with a majority of nonsmoking females and a median age of 70 years (Table N = 4) or stabilization of the disease (N = 1) by RECIST criteria , mainly deletions in the acceptor splice‐site region (Table PIK3CA mutations while concomitant MET amplification by GCN (6–10) was identified in only one sample (20%). However, this case was negative (< 2) by MET/CEP7 ratio. Sufficient tissue to perform MET IHC was available for four of the 13 patients; MET staining was intermediate in three cases and strong in one . According to our classification algorithm (see Section 2), 15/422 samples (3.5%) presented very‐high MET mRNA levels  had MET amplification by FISH or NGS . Five patients with very‐high MET mRNA were treated with MET‐TKIs, all of them showed partial responses by RECIST criteria. Interestingly, one was negative by FISH  and epidermal growth factor receptor (EGFR), fusions in ALK receptor tyrosine kinase (ALK), and METΔex14 the most prevalent  and only three discordant samples, all of them positive by nCounter and negative by DNA‐based NGS. Regarding nCounter and RT–PCR, there was a fair agreement  and all the discordant cases (N = 11) were negative by nCounter and positive by RT–PCR. Six of those discordant samples had been analyzed by DNA‐based NGS; mutations associated with METΔex14 were not detected in any case . A moderate to substantial agreement was observed if the nCounter very‐high cutoff was employed, with the highest agreement for the FISH ratio MET/CEP7 ≥ 2  and the 14 discordant cases had moderately elevated MET mRNA but did not show copy number gains , 19 intermediate (HS 100–220), 21 weak (HS 50–99), and 17 were very weak‐negative (HS < 50). In general, a fair agreement was observed between IHC and nCounter expression levels, with IHC positivity observed among cases with both mod‐elevated and very‐high mRNA levels , but not in cases with EGFR and KRAS mutations or ALK rearrangements. Next, we compared MET mRNA expression in samples at baseline vs. progression. Overall, there were no significant differences in log‐MET values between the 306 basal and the 52 progression samples included in the study. When classified by drivers, we did observe a significant increase in MET mRNA levels in tumors at progression vs. baseline only in EGFR‐mutant samples. However, a subpopulation of samples at progression with high MET mRNA expression was apparent not only in EGFR mutant, but also in ALK‐positive rebiopsies.Finally, we compared ers Fig. . Baselin4MET alterations—amplification and exon 14 skipping—in a large cohort of 474 NSCLC samples using of a RNA‐based technology  is in line with the literature [METΔex14 transcripts by nCounter (N = 13) were also detected by RT–PCR; however, a large subset of samples positive by RT–PCR (N = 11) tested negative by nCounter. Five of these discordant samples could be submitted to an orthogonal RNA‐based NGS, which confirmed them as negative. In addition, most of them were found to harbor known drivers, particularly KRAS. Although RT–PCR is used in some laboratories to identify MET∆ex14 transcripts, these results suggest that it might not be the most adequate technique for this purpose. Low level of METΔex14 transcripts can emerge by splicing ‘mistakes’ in the cell without translating in any genomic alteration with oncogenic relevance [Our prevalence of terature , 7, partterature , 33. In elevance , 35, 36.MET∆ex14. However, this technique has been reported to detect only 63% of literature‐described splicing mutations associated with METΔex14 [METΔex14‐positive cases previously missed [METΔex14 by nCounter that were submitted to DNA‐based NGS, three (37.5%) tested negative for mutations associated with METΔex14 splicing sites. One of these discordant patients was treated with a MET‐TKI and derived clinical benefit  comprised a majority of males, former, or current smokers with a median age of 58 years. Interestingly, very‐high MET mRNA expression closely correlated with MET gene amplification by NGS or FISH and was mutually exclusive with METΔex14. Therefore, it is not surprising that the phenotype of the patients with very‐high MET mRNA agrees with the characteristics previously reported in patients with high MET amplification  [MET mRNA cases treated with MET inhibitors achieved partial responses; the majority of case baseline (7/9) did not harbor any detectable driver. In contrast, the cohort of patients with mod‐elevated MET mRNA expression did not correlate with any specific phenotype, being distributed independently of gender and smoking habits, and known drivers were detected in most cases. Although the biological oncogenic significance of MET amplification in NSCLC has been controversial [MET being a true driver in patients with very‐high expression mRNA levels. In contrast, we believe that MET is unlikely to play a clinically relevant role in tumors with mod‐elevated expression levels.In addition to the presence of or ≥ 10) , 39, 40.oversial , our resMET∆ex14‐positive NSCLC. Overall response rates of 32% have been reported for crizotinib, a nonselective inhibitor, compared to 46–68% and 40–55% in first or subsequent lines for the selective inhibitors capmatinib, tepotinib, and savolitinib [MET‐amplified patients are significantly worse, with overall response rate (ORR) 20–47% [MET/CEP7 ratios or GCN) that have been used as eligibility criteria.Several trials have recently evaluated the efficacy of MET‐TKIs in olitinib , 13, 14.) 20–47% , 40, 42.MET expression levels in NSCLC here presented provides a unique opportunity to advance in the understanding of the processes underlying MET biology. For instance, our RNA‐based assessment allowed the identification of amplification‐negative tumors with very‐high MET expression levels and also the relatively infrequent cases with gene amplification that did not express MET mRNA. Remarkably, one of the patients with very‐high MET mRNA levels but negative by FISH  was treated with a MET inhibitor achieving a partial response. These observations suggest that FISH positivity might be capturing a heterogeneous group regarding dependence on aberrant MET signaling and that RNA‐based techniques could improve the performance of FISH for patient selection. This, in turn, may help to explain the inferior response rates observed with MET‐TKIs in FISH‐amplified tumors. In order to improve outcomes to MET inhibition in this setting, it would be important to further investigate the efficacy of MET‐TKI in those tumors with gene amplification that do not express MET mRNA, and the real clinical value of high expression levels in amplification‐negative tumors where MET inhibition therapy could be considered.Our study can shed some light on this relevant issue, and the characterization of METΔex14, recent studies have suggested that MET protein expression is required for clinical benefit from MET‐TKIs [METΔex14 patient in our cohort who exhibited stable disease to MET‐targeted therapy had low levels of mRNA by nCounter, whereas all treated patients with mod‐elevated levels had partial responses. Whether mRNA expression levels can assist to predict outcome in patients with METΔex14 is currently unknown but also merits further investigation. One of the limitations of our study derives from the fact that MET is known to be expressed not only in tumor cells but also in normal epithelial [MET mRNA levels by nCounter and MET IHC staining in tumor cells indicated that, in most samples, the contribution of noncancer components to the nCounter results was not significant.Regarding MET‐TKIs . Interesithelial , dendritithelial . In cons5MET in a large cohort of advanced NSCLC and have validated the use of nCounter to identify METΔex14 in this malignancy. Our work also provides useful insights into the biology of MET as a driver in NSCLC, supporting MET very‐high mRNA expression as a surrogate of amplification, and suggesting the relevance of MET mRNA levels in patients responding to MET‐TKIs. Our results support the use of mRNA‐based techniques for multiplex, accurate, and reliable assessment of MET alterations in order to select patients for MET‐targeted therapies.We have comprehensively characterized CT has received fees for consultancy/advisory roles from Pfizer, Novartis, MSD, Roche, AstraZeneca, and Takeda, and research funding from Pfizer and Novartis. CC has received travel grants from MSD, Pierre‐Fabre Oncology, fees for consultancy/advisory from Boehringer Ingelheim, Roche, Pfizer, and Boehringer Ingelheim. LLV has received fees for consultancy/advisory roles from AstraZeneca and Roche. SV has received fees for consultancy/advisory roles from Bristol‐Myers Squibb, Roche, MSD, Abbvie, Ose Pharma, and Merck. AAH has received fees for consultancy/advisory roles from Bristol‐Myers Squibb, Roche, MSD, and Lilly. MGC has received fees for consultancy/advisory roles from Bristol‐Myers Squibb, Roche, AstraZeneca, and Pierre‐Fabre Oncology, and research funding from AstraZeneca. AP has received fees for consultancy/advisory roles from Pfizer, Lully, Nanostring Technologies, Amgen, Oncolytics Biotech, Daiichi Sankyo, PUMA, Bristol‐Myers Squibb, Novartis, and Daiichi Sankyo, and research funding from Pfizer, Amgen, Roche, Novartis, and Daiichi Sankyo. NR has received fees for consultancy/advisory roles from MSD, BMS and Pfizer, and research funding from Pfizer, Novartis, and Ministry of Health, Instituto de Salud Carlos III, Spain. CA, RR, RRe, AGC, EM, NV, SC, SM, AA, IS, EA, SR, APR, IM, AFC, RP, CS, MMG, MDL, AMB, EG, WPJL, RR, LMM, and MAMV have declared no conflicts of interest.CA, CT, RR, RRe, AGC, EM, MAMV, and NR conceived and designed the study. CA, CT, RR, RRe, AGC, EM, SM, AA, EA, SR, MAMV, and NR conducted the experiments, acquired, and analyzed data. CC, NV, SC, SM, LLV, IS, IM, SV, AFC, RP, CS, MMG, MDL, AAH, AMB, MGC, EG, WPJL, RRo, LMM, and AP provided clinical samples and collected clinical information. NR, CT, CA, and MAMV wrote the manuscript. All authors reviewed and/or revised the manuscript.https://publons.com/publon/10.1002/1878‐0261.12861.The peer review history for this article is available at Fig. S1. Heatmap displaying the molecular and clinical characteristics samples with METΔex14 discordant results between RT‐PCR and nCounter.Fig. S2.MET mRNA expression levels by nCounter in samples harboring different driver alterations.Table S1. MET amplification criteria used in clinical trials of MET inhibitors.Table S2. Description of the NGS panels used in the study.Table S3. Description of the nCounter codeset used in the study and probes design for MET wild type and METΔex14.Table S4. Characteristics of the samples with valid results, N (%). Fusions were determined by nCounter, mutations by NGS.Table S5. Results for MET exon 14 alteration splice‐site regions and mutation types.Table S6. Concordance of nCounter with RT‐PCR and NGS for METexΔ14 status.Table S7. Concordance of the nCounter categorization using the cut‐off for very high MET levels with IHC, FISH and NGS.Table S8. Concordance of the nCounter categorization using the cut‐off for moderately elevated MET levels with IHC, FISH and NGS.Click here for additional data file.


Elevated body mass index (BMI) is related with reduced mortality in various cardiovascular diseases.Gender‐specific association between BMI and mortality exists in atrial fibrillation (AF).In this multicenter observational study with a mean follow‐up of 1 year, a total of 1991 AF patients were enrolled and divided into two groups based on the gender. The primary endpoint was all‐cause mortality while the secondary endpoints were defined as cardiovascular mortality, stroke, and major adverse events during 1‐year follow‐up. Cox regression was performed to identify the association between BMI and clinical outcomes according to gender.P = .027) and thinner (P < .001) than male patients with AF. They were more likely to have heart failure, hyperthyroidism, and valvular AF , but less likely to have coronary artery disease and prior myocardial infarction . Multivariate analysis revealed that overweight (HR(95%CI): 0.55(0.41‐0.75), P < .001) and obese patients (HR(95%CI): 0.56(0.34‐0.94), P = .028) were associated with significant lower all‐cause mortality compared with normal weight patients for the entire cohort. Similar association between elevated BMI and reduced all‐cause mortality were only identified in female patients with AF : 0.43(0.27‐0.70); obesity vs normal weight: HR(95%CI): 0.46(0.22‐0.97)), but not in male patients with AF.Female patients with AF tended to be older (This study indicates that overweight and obesity were related with improved survival in patients with AF. The association between elevated BMI and reduced mortality was dependent on gender, which was only significant in female patients, rather than male patients. Twenty representative hospitals around China  have participated in this study. The diagnosis of AF was confirmed by reviewing clinical records, electrocardiographic evidence, and electronic databases according to International Classification of Disease, 9th Revision, Clinical Modification Diagnostic Code 427.31 or 427.32. The study was approved by the ethics committee of each center and obeyed the Declaration of Helsinki. All patients have provided written consent to participate in this study.2.2Baseline data about demographics information, medical histories, admission vital signs, and treatments were collected by interviewing the patients, consulting their treating physicians and reviewing their medical records. BMI was recorded on admission and calculated by dividing weight in kilograms by the square of height in meters. AF was classified to paroxysmal, persistent, and permanent according to guidelines.2.3Follow‐up was carried out by trained research personnel via telephone interview, outpatient visit, or medical records procurement, with a mean duration of 1 year. The primary endpoint was all‐cause mortality, including cardiovascular and non‐cardiovascular mortality. The secondary endpoints were defined as cardiovascular mortality, stroke, and major adverse events (MAEs) during 1‐year follow‐up. All outcomes were adjudicated by an independent committee blinded to the patients according to standardized definitions. Deaths and its causes were determined by medical records obtained and reports of the participants' relatives or physicians. Cardiovascular deaths included deaths due to heart failure, myocardial infarction, sudden/arrhythmic death, stroke, pulmonary embolus, peripheral embolus, aortic dissection, or other cardiovascular disorders. MAEs referred to composite endpoint events of all‐cause mortality, stroke, non‐central nervous system (CNS) embolism, and major bleeding. Stroke was defined as focal neurological deficits lasting more than 24 hours and confirmed by imaging. Non‐CNS embolism was defined as a vascular occlusion due to embolism confirmed by imaging or surgery. Major bleeding was defined as life‐threatening bleeding, and/or symptomatic bleeding in a critical area or organ, such as intra‐cranial, or pericardial, or intra muscular with compartment syndrome, and/or bleeding causing a fall in hemoglobin level of 20 g/L (1.24 mmol/L) or more, or leading to transfusion of two or more units of whole blood or red cells.2.4U test for the data were not normally distributed. Categorical variables were presented as frequencies and percentages and compared by Chi‐square test. BMI was evaluated both as a continuous variable and as a categorical variable. According to Chinese criteria of weight for adults, patients were divided into four categories: underweight (BMI < 18.5 kg/m2), normal weight (BMI 18.5‐23.9 kg/m2), overweight (BMI 24‐27.9 kg/m2), and obesity (BMI ≥ 28 kg/m2).P value <.10 in the univariate analysis and factors relevant with outcomes were entered into the multivariate Cox models for adjustment. All statistical tests were two‐sided, and a P value <.05 was considered significant. SPSS 25.0  was used for statistical analysis.Continuous variables were presented as medians with interquartile ranges and compared by Mann‐Whitney 3Between September 2008 and April 2011, 2016 patients presenting to emergency department with documented AF were recruited. Among them, 25 patients were excluded due to incomplete data. Consequently, a total of 1991 patients with a median age of 71 years and 1093 females (54.9%) were included in the final analysis and divided into the male group and the female group.P = .027) and thinner (P < .001). They were more likely to have heart failure, hyperthyroidism and valvular AF , and less likely to have coronary artery disease and prior myocardial infarction . As to medications, there was no significant difference according to gender, except that female patients had a higher rate of digoxin use (P = .034). When CHA2DS2‐VASc score was calculated, 623(69.4%) male patients and 987(90.3%) female patients had a CHA2DS2‐VASc score ≥ 2 (P < 0.001). However, among the patients with prior diagnosis of AF and anticoagulant indications, only about 20% of them have received anticoagulant therapy at enrollment.The baseline characteristics and treatments of patients with AF are shown in Table P = .510), cardiovascular mortality  and MAEs , but a significantly higher incidence of stroke .The 1‐year outcomes of patients with AF are shown in Table P < .001) and cardiovascular mortality (P = 0.003) decreased significantly with the increase of BMI.Figure P < .001) and obese patients (HR(95%CI): 0.56(0.34‐0.94), P = .028) were associated with significantly reduced all‐cause mortality for the entire cohort. Moreover, the overweight group had notably lower risk of cardiovascular mortality (HR(95%CI): 0.54(0.37‐0.81), P = .003) and MAEs (HR(95%CI): 0.79(0.63‐0.99), P = .042). In female patients with AF, overweight (HR(95%CI): 0.43(0.27‐0.70), P = .001) and obesity (HR(95%CI): 0.46(0.22‐0.97), P = .042) were still remarkable predictors for decreased all‐cause mortality. However, all‐cause mortality was comparable across BMI strata in male patients with AF , but not in male patients (P = .748). However, multivariate regressions in male and female subgroup have not been performed in that study.Several studies have demonstrated that there exist sex‐related differences in the phenomenon of obesity paradox. Vest et al found that overweight was associated with significant survival benefit only in female patients with heart failure,There might be several explanations for the sexual dimorphism in obesity paradox. First, previous studies suggested that female patients were associated with more severe AF symptom and worse quality of life, which might result in earlier diagnosis and better treatment.The study had several limitations need to be pointed out. First, as one of the most common parameter of obesity, BMI has defects in reflecting the percentage and distribution of adipose tissues. However, our study lacked data of determinants for central obesity, such as waist circumference and waist‐to‐hip ratio, which have been confirmed to be better predictors of clinical outcomes.5Our study has demonstrated that overweight and obesity were related with decreased mortality in patients with AF. The association between elevated BMI and reduced mortality was dependent on gender. The phenomenon of obesity paradox could only be detected in female patients with AF. The exact mechanisms of the gender‐specific association between BMI and mortality have not been fully elucidated and require further investigation.The authors declare that they have no competing interests.Si‐qi Lyu: collected the data, performed the statistical analysis, drafted, and wrote the manuscript. Yan‐min Yang and Jun Zhu: designed and revised the manuscript. Shuang Wu, Juan Wang, Han Zhang, Xing‐hui Shao, and Jia‐meng Ren: collected the data. All the authors read and approved the final manuscript.


These subjects have a higher morbidity and mortality rate, but there are no effective treatments to reverse this situation so far. This study used data from an interrupted phase I/II clinical trial to evaluate safety and immune recovery after INRs were given four infusions, at baseline and at weeks 4, 8, and 20, with human allogeneic mesenchymal stromal cells from adipose tissue (Ad‐MSCs). Based on the study design, the first 5 out of 15 INRs recruited received unblinded Ad‐MSC infusions. They had a median CD4+ nadir count of 16/μL  and CD4+ count of 253 cells per microliter (171‐412) at baseline after 109 (54‐237) months on antiretroviral treatment and 69 (52‐91) months of continuous undetectable plasma HIV‐RNA. After a year of follow‐up, an independent committee recommended the suspension of the study because no increase of CD4+ T‐cell counts or CD4+/CD8+ ratios was observed. There were also no significant changes in the phenotype of different immunological lymphocyte subsets, percentages of natural killer cells, regulatory T cells, and dendritic cells, the inflammatory parameters analyzed, and cellular associated HIV‐DNA in peripheral blood mononuclear cells. Furthermore, three subjects suffered venous thrombosis events directly related to the Ad‐MSC infusions in the arms where the infusions were performed. Although the current study is based on a small sample of participants, the findings suggest that allogeneic Ad‐MSC infusions are not effective to improve immune recovery in INR patients or to reduce immune activation or inflammation. ClinicalTrials.gov identifier: NCT0229004. EudraCT number: 2014‐000307‐26.Between 15% and 30% of HIV‐infected subjects fail to increase their CD4 Between 15%‐30% of HIV‐infected subjects, do not achieve a significant immune recovery despite continuous viral suppression . These subjects have an aberrant state of immune activation and inflammation. The treatment with adipose tissue allogeneic adult mesenchymal stromal cells does not neither improve immune recovery, nor has it succeeded in reducing the state of immune activation and inflammation. Antiretroviral therapy suppresses viral replication and reduces this aberrant state, leading to an immune recovery in a high proportion of subjects. However, between 15% and 30% of patients fail to achieve sufficient immune reconstitution, triggering a rise in the rate of morbidity associated with both AIDS and non‐AIDS events. These patients are called immune nonresponders (INRs), and no current therapies are available to treat them. Mesenchymal stromal cells have been shown to have immunomodulatory properties because they interact and modulate the function of multiple immune cells. For that reason, this clinical trial was planned to evaluate the efficacy and safety of infusions of human allogeneic mesenchymal stromal cells from adipose tissue (Ad‐MSCs) in INRs. However, results suggest that Ad‐MSC infusions are not effective to improve immune recovery in INRs or to reduce immune overactivation or inflammation state.HIV infection is characterized by a progressive CD4Adipose tissue allogeneic adult mesenchymal stromal cells infusions are not effective to improve immune recovery or to reduce immune overactivation or inflammation state in immunologically nonresponding HIV‐infected patients.Donor‐associated factors and manufacturing procedure could affect efficacy and safety.+ T‐cell depletion and an immune overactivation and inflammation state. Antiretroviral therapy suppresses viral replication and reduces this aberrant state, leading to an immune recovery in a high proportion of subjects. However, between 15% and 30% of patients fail to achieve sufficient immune reconstitution, triggering a rise in the rate of morbidity associated with both AIDS and non‐AIDS events. These patients are called immune nonresponders (INRs), and no current therapies are available to treat them. Mesenchymal stromal cells have been shown to have immunomodulatory properties because they interact and modulate the function of multiple immune cells. For that reason, this clinical trial was planned to evaluate the efficacy and safety of infusions of human allogeneic mesenchymal stromal cells from adipose tissue (Ad‐MSCs) in INRs. However, results suggest that Ad‐MSC infusions are not effective to improve immune recovery in INRs or to reduce immune overactivation or inflammation state.HIV infection is characterized by a progressive CD41+ T‐cell depletion and the functional impairment of the immune system caused by HIV infection.+ T‐cell recovery despite successful viral suppression; these subjects are known as immunological nonresponders (INRs).+ T‐cell counts above different thresholds in a given time period.+ T cells, with a disturbed maturational profile, a reduced thymic function, and increased levels of activation and apoptosis, among other characteristics.Chronic immune activation and inflammation is considered today as the main driving force of CD4reg) cells results in a change from a proinflammatory state to an anti‐inflammatory state.+ T lymphocytes and a decrease of the activation of T lymphocytes and soluble inflammation mediator levels without significant adverse effects or loss of viremia control,On the other hand, several studies, both in vitro and in vivo, have shown that mesenchymal stromal cells (MSCs) can modulate the function of T helper cells and B lymphocytes, natural killer (NK) cells, and dendritic cells, whereas stimulating regulatory T , which also acted as sponsor.This was originally planned as a phase I/II, randomized, placebo‐controlled, clinical trial designed to evaluate the safety and efficacy of adipose tissue allogeneic adult MCSs (Ad‐MSCs) in INRs. The design was carried out jointly with the Andalusian Network for the Design and Translation of Advanced Therapies  performed a preliminary analysis of safety and efficacy data. In the second phase, (permanently suspended), 10 additional subjects would have been randomized to receive Ad‐MSC infusions or placebo.2.2trials.gov number NCT0229004, and it was conducted according to the principles of Good Clinical Practice (GCP) at Virgen del Rocío University Hospital in Seville (Spain).The clinical trial was approved by the National Health Authority and the Ethics Committee for Clinical Research of the participating site. All of the subjects provided informed consent. It was registered at the European Medicine Agency with EudraCT number 2014‐000307‐26 and Clinical 2.3Ad‐MSCs were provided by the Cell Production and Tissue Engineering Unit of Virgen de las Nieves University Hospital (Granada). Ad‐MSCs were obtained from subcutaneous adipose tissue samples from four donors obtained by surgical exeresis in aseptic conditions fulfilling the provisions of the Spanish Royal Decree 1301/2006.Treponema pallidum. Only one donor was diabetic and a smoker  at a dose of 1 × 102.5+ T‐cell count ≤350 cells per microliter whose CD4+ T‐cell count increased <75 or <150 cells per microliter after 1 or 2 years with undetectable viral load, respectively, and/or a CD4+ T‐cell count increase <350 cells per microliter after 3 years on treatment. Exclusion criteria included opportunistic infections in the previous 12 months, active coinfections with hepatitis B virus or hepatitis C virus, Child‐Pugh class C liver cirrhosis, portal hypertension or hypersplenism, malignant tumors, or treatment in the previous 12 months with immunomodulators, interferon, chemotherapy, or any other drug that might alter CD4+ T‐cell count. Pregnant or breastfeeding women and subjects refusing to use accepted contraceptive methods throughout follow‐up were also excluded from participation in the trial. All subjects agreeing to participate in the clinical trial provided written informed consent before undergoing any study‐related procedure.The subjects were selected among the HIV‐infected adults followed at the Virgen del Rocío University Hospital. An INR was defined as an HIV‐infected subject with a basal CD42.6The main aims of the study were to assess the safety and efficacy of four infusions of Ad‐MSCs. In accordance with GCP, all adverse events (AEs) observed by the investigator or reported by the subjects, whether or not attributed to the investigational medicinal product (IMP), were carefully monitored and recorded. AEs were summarized and classified on the basis of MedDRA terminology and their relationship to Ad‐MSC administration and categorized via the standardized toxicity‐grade scale used by the AIDS Clinical Trials Group.+ T‐cell counts, percentage of CD4+ T cells, and CD4+/CD8+ ratios after infusions and throughout 96 weeks after the first Ad‐MSC dose. The subjects had a total of 12 visits: at baseline and on weeks 3, 4, 7, 8, 12, 19, 20, 24, 36, 48, and 96, when adverse events, adherence to ART (subjects' self‐report and pharmacy records), hematology and chemistry tests, CD4+ and CD8+ T‐cell counts, and plasma HIV‐RNA levels were assessed. The CD4+ and CD8+ T‐cell counts were determined in fresh blood with an FC 500 flow cytometer . Plasma HIV‐1 RNA levels were measured by quantitative polymerase chain reaction  with a lower detection limit of 20 copies per milliliter, according to the manufacturer's instructions.Efficacy was measured as the changes in CD42.72.7.1+ (CD3+ CD4+) and CD8+ (CD3+ CD8+) T cells, and the different subsets were defined as follows: naïve (TN) CD45RA+ CD27+, CD4+ recent thymic emigrants (RTEs) CD45RA+ CD27+ CD31+, central memory (TCM) CD45RA− CD27+, effector memory (TEM) CD45RA− CD27−, and terminally differentiated (TEMRA) CD45RA+ CD27−. Other cellular subsets were defined as follows: myeloid dendritic cells , plasmacytoid dendritic cells , NK cells (CD3− CD56+ CD16+), and Treg cells (CD3+ CD4+ CD25++ Foxp3+). Moreover, the frequency of T cells expressing markers of cellular proliferation (Ki67), apoptosis (Annexin V), exhaustion (PD‐1), activation (HLA‐DR/CD38), and replicative senescence (CD28− CD57+) were also quantified in both total CD4+ and CD8+ T cells. Samples were acquired on a Fortessa LSR II instrument , discarding dead cells, and analyzed using FlowJo 9.3.2 software.Peripheral blood mononuclear cells (PBMCs) were isolated using a gradient technique with Ficoll and stored with fetal bovine serum and 10% dimethyl sulfoxide in liquid nitrogen until assay time. On the day of the assay, frozen PBMCs were thawed at 37°C and washed with phosphate‐buffered saline. Afterward, cells were stained with different fluorochrome‐conjugated antibodies to assess the expression of surface markers, including CD3‐BV786, CD4‐APC‐Cy7, CD8‐BV510, CD45RA‐Pe‐Cy7, CD27‐BV421, HLA‐DR‐PE‐CF594, Lineage Cocktail 1‐FITC, CD56‐PE‐CF594, CD16‐AF700, CD25‐BV421, Foxp3‐PE, PD1‐BV605, Ki67‐FITC, CD38‐BV605, HLA‐DR‐FITC, CD57‐APC, CD28‐PE, Annexin V‐APC, and CD31‐PE  and CD11c‐PE and CD123‐APC . Viable cells were identified using 7AAD‐PerCP‐Cy5.5 (BD Biosciences) or LIVE/DEAD fixable Yellow Dead Cell Stain  for intracellular staining. The cellular markers were analyzed by multicolor flow cytometry on total CD42.7.2Total cellular associated HIV‐DNA  extracted from PBMCs using Blood DNA Mini Kit  was assayed by real‐time polymerase chain reaction using specific primers  and a probe within the selected gag long terminal repeat region (5′‐TACCGACGCTCTCGCACCCA‐3′) labeled with FAM.2.7.3Plasma samples were aliquoted and stored at −20°C until subsequent analysis of the following biomarkers: high‐sensitivity C‐reactive protein (hsCRP), interleukin (IL)‐6, TNF‐α, and soluble CD14 (sCD14). The levels of hsCRP were determined with an immunoturbidimetric serum assay using Cobas 701 . Commercially available enzyme‐linked immunosorbent assays were used for the assay of IL‐6 , TNF‐α , and sCD14  following the manufacturers' instructions.2.8P value was <.05. The statistical analyses were performed using IBM software .Results were expressed as median values with interquartile ranges (IQRs) for continuous variables and as numbers and percentages of cases for categorical variables. The Wilcoxon signed rank test was performed to compare changes in continuous variables over time. Differences were considered statistically significant if the 33.1+ T‐cell nadir 16 (2‐108) cells per microliter, CD4+ T‐cell count 253 (211‐340) cells per microliter, CD4+/CD8+ T‐cell ratio 0.42 (0.19‐0.48), percentage of CD4+ T cells 24.1% (11.4‐z25.5), months on ART 109 (73‐210), and 69 (53‐84) months of continuous undetectable plasma HIV‐RNA levels before their inclusion in the study  basal age was 53 (45‐58) years, CD43.2In total, 10 venous thrombosis events, 24 to 48 hours after Ad‐MSC infusion, were observed in three out of the five subjects in the arms where Ad‐MSCs were infused, which required low molecular weight heparin treatment. A decrease in the emergence of thrombotic events was observed when low molecular weight heparin (80 mg of enoxaparin per day) was given 1 day before, on the day of the infusion, and during the following 2 days, together with a retraining course on the cell infusion procedure, as it was detected that the infusion rate was significantly higher than that established by protocol in one case. All other adverse events (n = 5) were considered unrelated to the Ad‐MSCs  except for an increase in the TEM CD8+ T subset  and a trend in PD1+ CD8+ T cells  at week 96  of Ad‐MSCs, but no changes were observed in the CD4+ T‐cell counts, percentages, or CD4+/CD8+ ratios, and no consistent changes were observed in the different subsets and phenotypes of the CD4+ and CD8+ T cells or percentage of mDCs, pDCs, NK cells, or Treg cells. Likewise, the infusions of Ad‐MSCs had no effects on sCD14, IL‐6, TNF‐α, and hsCRP plasma levels. Overall, we did not find a decrease in activation, exhaustion, apoptosis, and senescence at week 96. Only a significant decrease in the PD1+ CD4+ T cells was found, but its meaning is uncertain, and this change did not influence CD4+ T‐cell recovery.In our study, the subjects received four doses  observed could have a complex multifactorial causality. On one side, the study subjects may be at increased risk of VTEs because of the patients' history of parenteral drug use (2/5), higher risk of recurrence in cases of previous thrombotic episodes,Our study has several limitations. Regarding the safety of Ad‐MSCs, it would have been desirable to determine their tissue factor expression before their administration, although in more than 150 patients treated with this same product in different clinical trials sponsored by the Andalusian Network for the Design and Translation of Advanced Therapies, thrombotic events occurred exceptionally. Furthermore, a full characterization of the infused MSCs was carried out, but neither in vitro functional assay to determine their immunomodulatory potency nor biodistribution analysis was performed. However, previous studies have shown that after intravenous infusion, cells were accumulated in lung, spleen, liver, and bone marrow.On the other hand, a higher number of patients and a control group, planned in the second phase of the trial, would have allowed us to better assess the fluctuations that some patients presented in several immunological parameters. Furthermore, the analyses are based on PBMC samples, which may not always reliably reflect tissue‐related processes during HIV infection. The use of lymph node samples instead of PBMCs would have needed periodical biopsies, lowering the feasibility of the study.5The Ad‐MSC infusions have not proven to be effective to improve the immune recovery, nor have they succeeded in reducing immune activation or levels of inflammatory markers in INRs, at least with the dosage schedule selected.The authors declared no potential conflicts of interest.M.T.‐R.: collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; P.V., A.R.‐G., O.E.‐I., S.A.‐S.: provision of study material or patients, final approval of manuscript; I.R.‐J.: administrative support, collection and/or assembly of data, final approval of manuscript; A.I.Á.‐R.: data analysis and interpretation, final approval of manuscript; J.M.‐A., R.M., O.F.‐L.: financial support, administrative support, final approval of manuscript; E.R.‐M.: data analysis and interpretation, final approval of manuscript; A.G.‐V.: conception/design, data analysis and interpretation, manuscript writing, final approval of manuscript; L.F.L.‐C.: conception/design, provision of study material or patients, final approval of manuscript.Data S1. Supporting Information.Click here for additional data file.


Adolescence as one of the most challenging periods of humans’ growth is accompanied with major physical, behavioural, social-emotional, and neuroendocrine changes. Early maturation and eating disorders (EDs) have been reported to be associated with adverse health conditions. Therefore, the present study was conducted to assess age of onset of menarche (AM), EDs, and their possible relationships with weight, body mass index (BMI), waist circumference (WC), and socioeconomic status (SES) in the Iranian female adolescents.p < 0.05). There was a negative association between weight and anorexia nervosa (AN), BMI and AN, also WC and AN (p < 0.001). A significant positive correlation was found between SES and AM, also EDs and AM (p < 0.05) then between weight and bulimia nervosa (BN) & binge-eating disorder (BED), BMI and BN & BED, also WC and BD & BED (p < 0.001). Our results also revealed that AM in mothers had a significant positive relationship with AM in their daughters .In the study population, mean ± standard deviation (SD) of AM was 12.95 ± 1.14 years. Inverse significant relationships were found between weight and AM, also BMI and AM ( Menarche is often considered as a hallmark of puberty among females . The ageStrong evidence shows that the increased rate of obesity in children is a significant factor contributing to early onset of menarche . MatkoviAccording to review of the literature, there was different and somewhat contradictory evidence regarding AM and EDs and their relationship with anthropometric parameters, and socioeconomic status (SES) so, herein, it was attempted to study AM and EDs and their relationship with weight, BMI, waist circumference (WC), and SES in the Iranian female adolescents.This cross-sectional study included female students aged 14–18 years that were selected randomly from public and private high schools in all five educational districts during academic year of 2015–2016 in Tabriz, Iran (n = 725). Students with any endocrine disorders, hormonal disorders, or delayed menarche were excluded from the study.Purpose of the study and instructions for completing questionnaires were explained for the selected students in a training session. An informed written consent was obtained from all the participants and their guardian.2). WC; the distance around the narrowest area of the waist between the lowest rib and iliac crest at the end of a gentle expiration; was measured using a measuring tape. The age- and sex-specific BMI cut-off points were used, provided by the Centre for Disease Control and Prevention (CDC) according to which the BMI between 85th and 95th percentiles was classified as overweight and BMI above than 95th percentile was considered as obese [All the participants were asked to complete a researcher-made questionnaire containing demographic and SES characteristics. SES was assessed by collecting data regarding parents’ educational and occupational situation, number of family members, and type of school, and was categorized into “low”, “moderate”, and “high” subgroups. An Eating Disorders Diagnostic Scale (EDDS) was used based on diagnostic and statistical manual of mental disorders, fourth edition (DSM-IV) for diagnosis of EDs . Validitas obese . AM was p value < 0.05 was considered as statistically significant.Results were expressed as mean ± standard deviation (SD) for continuous variables and frequencies (%) for categorical ones. Data normality was checked using Kolmogorov–Smirnov test. The association between AM and anthropometric parameters was assessed using linear regression. Analyses of Variance (ANOVA) and Tukey’s post hoc were used to compare the mean ± SD of anthropometric parameters between AM groups as well as ED’s subgroups. The distribution of socioeconomic characteristics in EDs subgroups was determined by the chi-square test. SPSS software  was used for all statistical analyses, and p < 0.05, 131 r = 0.1). This trend was shown in Additional file p < 0.05) . Distribution of EDs among all participants was as follow: AN: 11.7% (n = 85), BN: 5.2% (n = 38), and BED: 0.7% (n = 5). Because of the low prevalence of BED, we supposed BED and BN subgroups as one group. Table Mean age was 15.83 ± 0.99 years and 75.7% of the students were 15 years old. Mean of AM in girls was 12.95 ± 1.14 years (n = 724). After the classification of participants into three groups based on the AM: (1) 12 < AM ≤ 14, (2) 14 < AM ≤ 16 and (3) 9 ≤ AM ≤ 12, the mean of weight, WC, and BMI in group one were significantly higher than group three (Table p = 0.03). Most of the participants’ fathers had middle income. The relationship between normal and EDs groups with regards mother’s education was also significant (p = 0.01). Most of the participants’ mothers belonged to “Diploma” and “Elementary and illiterate” subgroups in normal and EDs groups, respectively. SES in the most of the participants was determined as “middle” and with regards to this parameter, the relationship between normal and EDs groups was close to significant (p = 0.05).Distribution of socioeconomic characteristics in EDs subgroups has been shown in Additional file In the present study, mean ± SD of AM was 12.95 ± 1.14 and 13.5 ± 1.27 154 years for girls and their mothers, respectively. Ghergherechi and Shoari in a study conducted in Tabriz (2011) showed that mean ± SD of AM was 12.58 ± 1.30 and 13.22 ± 1.22 years for girls and their mothers, respectively . ResultsIn the present study, AM was inversely correlated with weight and BMI, which was similar to the study by Tehrani et al. , who fouOur results demonstrated no significant associations between height and AM, which was in line with other studies , 16. UnlOur results showed a significant positive correlation between AM and SES, which was in contrast to the studies by Mee-Hwa Lee et al. , GharaviOur results demonstrated significant relationships between weight, BMI, WC, and EDs subgroups so that, weight, BMI, and WC were lower in AN group and higher in BN and BED groups. These associations have been also found in earlier studies , 32. In No significant relationship was observed between height and EDs subgroups. According to review of the literature, there was no study investigated the effects of EDs on height of the people. Since, this study was a cross-sectional study and height changes are time-dependent, thus prospective studies are needed to understand whether EDs can lead to short stature. Results of the current study revealed that AM occurred later in EDs subgroups, which was in contrast to the studies by Yannakoulia et al. , and AlmThe findings of this study indicated an inverse association between father’s income, mother’s education, and SES with EDs as EDs were found to be lower in 197 subjects with higher father’s income, mother’s education and SES strata. Yannakoulia et al. , did notDue to the reduction of AM and its relationship with unfavourable psychopathological health conditions like EDs, determining factors affecting AM, control related risk factors and reproductive health education should be considered as important health system priorities.The role of pre-existing psychiatric disorders or family history of a psychiatric disorder was not considered in this study, and it was not possible to determine its effects on the results, thus it was regarded as the main limitation of this study.Additional file 1: Table S1. Distribution of socioeconomic characteristics in Eating Disorders (EDs) subgroups (n = 725).Additional file 2: Figure S1. (A) Age at menarche (AM) depending on body mass index (BMI). AM had an inverse significant relationship with BMI, as menstruation was occurred earlier in girls with higher BMI. (B) Age at menarche (AM) depending on socio-economic status (SES). AM had a statistically significant positive correlation with SES, as with increment in the level of SES, AM increased.


Similarly, eutrophic water bodies (n = 552) exhibited higher DC concentrations than mesotrophic (n = 215) and oligotrophic water bodies (n = 85); DC in lakes  was significantly higher than DC in reservoirs . All data used in this investigation is accessible online.A dissolved carbon (DC) data set, including dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC), from 224 lakes (513 stations) and 141 large reservoirs (337 stations) across China is presented in this study. In addition to DC, the data set also includes results for electrical conductivity (EC), total phosphorus (TP), chlorophyll-a and transparency. The impact of trophic status and EC gradient on DC concentration in water bodies are discussed. Results from our investigation indicate that DC in saline (EC > 1000 μS cm Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11890596 The predominant terrestrial carbon input to most lakes and reservoirs is dissolved carbon (DC), followed by particulate carbon, and the proportion of inputs varies with lake location and hydrology2. The dominant form of aquatic carbon in some temperate regions, high northern latitudes and boreal forests in a carbonate terrain is dissolved inorganic carbon (DIC)4. In contrast, dissolved organic carbon (DOC) is the dominant form in humid tropical areas and in noncarbonate boreal forests2. Spatial DC measurements are useful for understanding carbon sources and cycling in aquatic systems, and it is dependent on an array of factors6, e.g., catchment soil and landscape, hydrology, trophic status and anthropogenic discharge8. In general, DC in aquatic systems mainly comes from decomposition of organic matter, i.e., plants in the catchment and algae and macrophytes within the water body, and the metabolic secretion of phytoplankton and microbes. However, inland waters in China, particularly those situated in the Northeast, East and South regions, are severely polluted with high nutrients concentration and eutrophic status, which also changes the concentration and constituent of DOC from algae9. In addition, anthropogenic discharge also provides a large volume of dissolved organic matter into lakes and reservoirs in these regions11.Although inland water bodies occupy a small fraction of the earth’s surface, they play a major role in the global carbon (C) cycle17. However, investigations of DC patterns are imbalanced with respect to geography5. Lakes in Europe, particularly in Scandinavian countries where lakes are more abundant, have been intensively investigated18. Likewise, lakes in the USA, Canada and Japan have also been extensively examined for DC spatiotemporal variation21. Comparatively, fewer investigations have been undertaken to investigate DC variation for inland waters in China17.Numerous investigations have been carried out to examine the variations in DC in inland waters24. It was estimated that by 2002, China contained 85,288 reservoirs with a surface water area of 23,000 km2, and almost 2700 lakes (area >1 km2) with a total area of 81,414.6 km2 26, with saline lakes accounting for about half of the lake area27. Previous investigations have also indicated that DC concentration significantly differs between fresh water and saline water bodies28. The trophic status of inland water bodies in China ranges from oligotrophic to hypereutrophic. Previous investigations indicated that lakes with eutrophic status in various climatic regions may exert a strong impact on DC of inland waters20. Thus, a systematic examination of DC characteristics in inland water bodies in China is urgently needed.Due to differences in climate across the country, and different land-use types surrounding water bodies, China contains a large number of inland water bodies which have a wide variation in water qualityThe objective of this study is to describe a DC data set collected by field surveys undertaken in lakes and reservoirs across mainland China. Our investigation provides a record of DOC and DIC measurements in 288 lakes across five lake regions, and 141 large reservoirs spanning complex topography, landscape, hydrology, eutrophic status, and salinity. Finally, we discuss DC concentrations for lakes and reservoirs with respect to the impact of eutrophic status and a saline gradient in different lake regions in China. We also provide full details for data access.29.Surface features in China can be grouped into three levels based on elevation: the first level is represented by the Tibet Plateau; the second level contains areas lying north of the Kunlun Mountains, including several basins and plateaus; the third level consists of hills and plains Fig. . AlthougLake and reservoir density across China vary significantly, largely controlled by terrain, hydrology and population density Fig. . In addi22  were determined using a YSI 600 XLM Sonde with multiple probes . Water transparency was determined using Secchi disk depth  for each station for most of the water bodies investigated. All water samples for DOC and DIC analysis were kept in a portable refrigerator at 4 °C for no more than 7 days before they were returned to the laboratory.Water samples were typically collected at 2–3 sampling stations, on average, from every reservoir or lake with a surface area <100 kmp < 0.05 represents a significant correlation.A Kruskal-Wallis test was employed to test the difference in the DC concentrations among waters with different trophic status using SPSS 22.0. A Two-Sample Kolmogorov-Smirnov Test was employed as an analysis method using SPSS 22.0 to test the difference in DC concentrations between lakes and reservoirs, between saline and fresh lakes, −1 (median/mean ± SD: 23.65/32.5 ± 38.44 mg L−1), indicating a relatively low DC concentration in this region. Lakes in the NLR recorded higher DC concentrations (range of 8.65–2155.25 mg L−1), and lakes in the MXR also exhibited a high DC concentration with large variations. Lakes in the YGR recorded average DC concentrations comparable to those in the ELR. A large DC concentration range (2.3–1860.72 mg L−1) was recorded for lakes in the TQR, as well as the highest DC concentration (115.66/289.80 ± 408.26 mg L−1). This finding was associated to a close link with evapo-concentration for endorheic lakes28. As shown in Table 29.DC concentrations in ELR water bodies ranged from 1.63 to 287.81 mg Lp < 0.001, p = 0.020, p < 0.001, p < 0.001, p = 0.003). This trend was also recorded in the fresh water lake data set when the five lake regions were combined  were also significantly higher than that in fresh water bodies   Higher DC concentrations in saline lakes are associated to evapo-concentration effects28. As shown in Fig. DC concentrations in saline water bodies (186.53/297.13 ± 356.14 mg L−1), followed by TQR, NLR and ELR; lakes in the YGR recorded the lowest DOC concentration (3.5/5.4 ± 4.4 mg L−1). DOC concentrations in fresh lakes significantly differed with trophic status: DOC concentrations of eutrophic lakes were the highest, and concentrations in mesotrophic lakes were notably higher than those in oligotrophic lakes  was significantly higher than that in reservoirs  (p < 0.001)  Fig. . Detaile−1, n = 513) was significantly higher than DOC in reservoirs   Fig. , and DIC−1 17. Samples exceeding this limit were diluted using Milli-Q water to ensure concentrations were within the measurement range of the analyzer . Analysis of blanks and replicates showed a detection limit of 0.3 mg L−1 and a precision of 5% at a concentration of 4 mg L−1.Water quality parameters were measured within 24 h after arriving in the laboratory. To determine dissolved carbon (DC), water samples were initially filtered through pre-combusted 0.45 μm glass-fiber filters  before analysis using a Total Organic Carbon Analyzer . All water samples were measured using the same instrument with the same protocol for DC determination. In addition, results from our previous study indicated that DC concentrations for some saline lakes exceeded 100 mg L−1) was measured at room temperature (20 ± 2 °C) using a DDS-307 EC meter. Chlorophyll-a (Chl-a) was extracted from water samples using a 90% buffered acetone solution, and the concentration for each water sample was determined using a Shimadzu UV-2660 PC spectrophotometer30. Total phosphorus (TP) concentration in water samples was determined using a Continuous Flow Analyzer , using the molybdenum blue method after samples had been digested using potassium peroxydisulfate31.In the laboratory, electrical conductivity  to represent the nutrient classification state of the lakes: TSI ≤ 30 indicated an oligotrophic state, Supporting Information


Consumption of a prudent dietary pattern rich in healthy nutrients is associated with enhanced cognitive performance in older adulthood, while a Western dietary pattern low in healthy nutrients is associated with poor age-related cognitive function. Sex differences exist in dietary intake among older adults; however, there is a paucity of research examining the relationship between sex-specific dietary patterns and cognitive function in later life.Mage = 74 years, n = 664 women, n = 612 men) from the Quebec Longitudinal Study on Nutrition and Successful Aging (NuAge). A 78-item Food Frequency Questionnaire was used to estimate dietary intake over the previous year. Sex-specific dietary pattern scores were derived using principal component analysis. Global cognition was assessed using the Modified Mini-Mental State Examination (3MS).The current study aimed to investigate sex differences in the relationship between sex-specific dietary pattern adherence and global cognitive function at baseline and over a 3-year follow-up in 1268 community-dwelling older adults . No association was found between prudent or Western dietary patterns and cognitive change over time in men or women.Adjusted linear mixed effects models indicated that a healthy, prudent dietary pattern was not associated with baseline cognitive performance in men or women. No relationship was found between Western dietary pattern adherence and baseline cognitive function in women. Among men, adherence to an unhealthy, Western dietary pattern was associated with poorer baseline cognitive function (These findings highlight the importance of conducting sex-based analyses in aging research and suggest that the relationship between dietary pattern adherence and cognitive function in late life may be sex-dependent. Women were also asked whether they currently or have ever received estrogen treatment.The following non-dietary variables were collected during a structured, computer-assisted interview at baseline: age, sex, education, body mass index [BMI; measured weight (kg)/measured height  which estimates usual intake of sample portions of 78 different food items over the previous 12 months on a 6-point scale from ‘never or rarely’ to ‘2 times or more per day’ . ParticiThe Modified Mini-Mental State Examination (3MS) was administered at baseline and annually for 3 years to evaluate global cognitive function and detect possible cognitive impairment. The 3MS is an extended version of the Mini Mental State Examination  assessinn = 664; men: n = 612). Dietary pattern scores for each participant were calculated by converting the average score for each category belonging to the given pattern to a z-score.Sex-specific dietary patterns were derived using varimax rotated principal component analysis (PCA) with the FFQ food items. Prior to deriving dietary patterns, the frequency of consumption of each food item was multiplied by 0.5, 1, or 1.5 if the portion size was reported to be smaller, similar, or larger than that presented in the FFQ, respectively. The number of factors retained in the PCA was determined by considering eigenvalues > 1, the scree plot, and interpretability of the resulting patterns. Factor loadings with an absolute value > 0.209 for men and > 0.201 for women were considered significant contributors to the interpretation of the factors as per guidelines by Norman and Streiner . These ct-tests for continuous variables, and χ2 for categorical variables. Associations between sex-specific dietary patterns, baseline cognitive function, and cognitive change over 3 years were determined by linear mixed effects models using the “nlme” [, slope). Positive estimates indicate that greater adherence to the dietary pattern is associated with less decline during follow-up. Dietary intake and covariates were entered as fixed effect factors and participants were specified as a random effects factor. All models were adjusted a priori for age, education , BMI, diabetes, daily energy intake , and hypertension. All models were also adjusted a priori for past or current estrogen use in women. Statistical significance for all analyses was set at p < 0.05.Sex differences in baseline characteristics were calculated using e “nlme”  and “inte “nlme”  statistie “nlme” , with anp < 0.001), were more likely to have hypertension (p = 0.003) and less likely to have diabetes (p < 0.001). No sex differences were found for age, education, or BMI. Participant demographic and health-related characteristics, including cognitive performance on the 3MS, for the total sample and stratified by sex are shown in Table The majority of participants were Caucasian (98.7%). Participants ranged in age from 67 to 84 years with 44.7% aged 75 years and older. Mean scores on the 3MS at baseline and at each subsequent annual visit were: 94.5 ± 4.1, 93.8 ± 5.1, 93.1 ± 5.7, and 93.2 ± 6.3 for women, and 93.5 ± 4.5, 91.9 ± 6.1, 91.9 ± 6.5, and 91.6 ± 6.2 for men. On average, women scored higher than men on the 3MS at baseline ; whereas greater adherence to the Western dietary patterns was associated with poorer baseline cognitive performance . In women, higher adherence to the prudent dietary pattern was also associated with better baseline cognitive performance , while the Western dietary pattern was not associated with baseline cognition .Unadjusted linear mixed effects models revealed that, in men, higher adherence to the prudent dietary pattern was associated with better baseline cognitive performance ; however, the association between prudent dietary pattern adherence and baseline cognition was no longer statistically significant following adjustment for covariates . In women, the association between prudent dietary pattern adherence and baseline cognitive function was no longer statistically significant following adjustment for covariates . Linear mixed effects models showed significant decline in 3MS performance over time in men  and women , which remained significant after controlling for covariates. However, unadjusted and adjusted models revealed that the slope of cognitive decline did not change as a function of degree of adherence to the prudent or Western dietary patterns in men and women (ps > 0.10). See Table Adjustment of the linear mixed effects models for covariates in men did not significantly change the association between Western dietary pattern adherence and baseline cognition  represent correlation of an FFQ item with dietary pattern score.Additional file 2: Supplementary Table 2. Factor loadings for dietary patterns in women. Note. Loadings (>|0.201|) represent correlation of an FFQ item with dietary pattern score.


Most of the patients with type 1 and V hyperlipoproteinemia (HLP) present with symptoms and signs of acute pancreatitis due to marked elevation of triglycerides, but this baby presented with a chest infection, which was later diagnosed as type V HLP on laboratory workup.We report a case of a 1 month and 15 days old baby boy, product of 2-nd degree consanguinity admitted to a nearby hospital with complaints of refusal to feed, cough and excessive crying. On examination his heart rate was 102 beats/min, respiratory rate was 55 breaths/min and temperature was within the normal range, provisional diagnosis of Pneumonia was made. His samples were tested at our laboratory, the lipid Profile at age of 1 month 15 days showed total cholesterol (TC) of 1400 mg/dl reference range (RR < 200 mg/dl), triglycerides (TG) of > 885 mg/dl after dilution it was 31,400 mg/dl (RR < 150 mg/dl), High density Cholesterol (HDL) of 35 mg/dl (RR > 40 mg/dl) and low density cholesterol (LDL) of 200 mg/dl (RR < 100 mg/dl). The patient’s blood sample was grossly milky and lipemic in appearance. A “Refrigerator test” was performed after overnight storage of the sample in refrigerator at 4 °C, which gave a creamy layer at the top and clear infranatant due to caking of the Chylomicrons. Lipoprotein electrophoresis performed 1 month later showed Chylomicrons of 4.7% (RR 0–2%), Pre-beta lipoproteins of 51.5% (RR 5–22%), beta lipoproteins of 16.5% (RR 39–70%) and alpha of 27.3% (RR 23–53%). Initially he was diagnosed as type 1 HLP, but later on he was correctly diagnosed as type V HLP. Cholestyramine (Questran sachet) powder was started at a dose of 100 mg/kg on t.i.d basis with NAN-1 formula Milk at the age of 1 month and 15 days. On follow up, detailed advices regarding the weaning food was given to the mother . His lipid profile was repeated at age of 3 months, which showed some improvement, his TGs were 1986 mg/dl and TC 105 mg/dl.There is no universal diagnostic criterion for diagnosing Type V HLP, most likely, due to a scanty literature on this disorder. It stimulated us to report this case so that our findings may help for a timely diagnosis of the affected patients. Most of the patients with type 1 and V hyperlipoproteinemia (HLP) present with symptoms and signs of acute pancreatitis due to marked elevation of triglycerides levels, but this baby presented with a chest infection instead which was later diagnosed as type V HLP on the laboratory workup.Lipids, such as cholesterol and triglycerides, are insoluble in plasma and are therefore transported in the plasma via Lipoproteins. Lipoproteins consist of esterified and unesterified cholesterol, triglycerides, phospholipids, and proteins . LipoproLipid disorders are of two types, primary and secondary. Primary dyslipidemias occur due to an increase production or decreased removal of lipoproteins; whereas secondary dyslipidemias occur due to an abnormality in either the lipoprotein itself or in the lipoprotein receptors. A well recognized classification system of Fredrickson and World Health Organization (WHO), International Classification of Diseases (ICD) classifies Hyperlipoproteinemia (HLP) phenotypes based on the pattern of the lipoprotein fractions. Fredrickson type V HLP is also called as hyperchylomicronemia late onset, Hyperchylomicronemia with hyperprebetalipoproteinemia, familial Hyperlipemia combined fat and carbohydrate induced hyperlipemia, mixed Hyperlipidemia type V and Type V HLP. Increased Chylomicrons levels are present in Frederickson type 1 and type 5 HLP ; these gWe report a case of a 1 month 15 days old baby boy, he was admitted to a nearby hospital with the provisional diagnosis of Pneumonia and his sample was sent to our hospital for laboratory testing. The blood sample on gross examination appeared milky, and we therefore took a detailed history from the parents with their consent. He was the product of 2nd-degree consanguinity, first child with no sibling’s, born full term via spontaneous vaginal delivery (SVD) with a normal antenatal history. His developmental history was normal and was up to date on all the immunizations. The past medical and the family history were not significant for Hyperlipidemia.He was admitted to the emergency ward of a nearby hospital with a history of refusal to feed, cough and excessive cry. On examination his heart rate was 102 beats/min, respiratory rate was 55 breaths/min, temperature was 37 °C and oxygen saturation was 98%.The laboratory testing revealed the following: Complete blood count (CBC) done on XN-1000 Sysmex hematology analyzer by flow cytometry technique showed hemoglobin of 6.8, White blood cell count of 15.8 and platelets of 161. The sample that was obtained.appeared milky and lipemic and therefore, he was also screened for Hyperlipidemia. Lipid profile was done by enzymatic method on Cobasc-311 analyzer; its analytical measuring range (AMR) for triglycerides is 8.85 mg/dl to 885 mg/dl. Analyzer gave the result of > 885 mg/dl, since the patients’ physician requested for the exact levels, the sample was rerun, and the results were reported after manual serial dilutions of 1: 50 and multiplication with factor. HDL and LDL levels were performed by the Homogeneous enzymatic colorimetric method on Cobas c-311 analyzer with Analytical Measurement Range (AMR) of 3.09–150 mg/dl and 2–548 mg/dl respectively. Serum amylase and lipase was done on Erba XL-200 by enzymatic method, while liver function tests were done by IFCC kinetic method on the Cobas c-311 analyzer as shown in Table Electrocardiography showed severely hypertrophied normal sized left ventricle with a normal systolic function likely due to non-obstructive hypertrophic cardiomyopathy. Age wise lipid profile is shown in Table Type V hyperlipoproteinemia (HLP) has many names including hyperchylomicronemia late onset, Hyperchylomicronemia with hyperprebetalipoproteinemia, familial Hyperlipemia combined fat and carbohydrate induced, Hyperlipemia mixed, Hyperlipidemia type V and Hyperlipoproteinemia type 5.In this case, a ratio of triglycerides to total cholesterol (TG/TC) was 22.4 mg/dl (reference range 5 mg/dl). A ratio of total triglycerides to total cholesterol (TG/TC) above 2.2 (if measured in mmol/l) or above 5 (in mg/dl) is a sign of a high level of circulating Chylomicrons and VLDL. “Refrigerator test” was also performed which yielded a creamy layer at the top and clear infranatant . LipoproOur patient had a very severe hypertriglyceridemia, his TG levels were 31,400 mg/dl and total cholesterol were 1400 mg/dl at the age of 1 month and 15 days; he was started Cholestyramine therapy at that age. His lipid profile was repeated at 3 months of age, which showed TGs reduced to 1986 mg/dl and TC to 105 mg/dl. The endocrinologists have planned to start satins if lipid profile (TGs and VLDL) worsens on the next follow-up.A case by Manzor et al. in 2015 reported a 10 days old baby girl with symptoms of refusal to feed and irritability. She was a product of 3rd-degree consanguinity, diagnosed based on altered lipid profile which showed total cholesterol of 1236 mg/dl (reference range 170 mg/dl) and triglycerides of 2132 mg/dl. Genetic testing was not done; statins were started along with dietary modification .Pugni et al. in 2014 described the case of a newborn presenting with severe hypertriglyceridemia (> 10 mmol/L), diagnosed with monogenic lipoprotein lipase deficiency, who benefited by exchange transfusion, as a safe alternative to plasmapheresis, in order to prevent acute pancreatitis in young infants with this condition .The type V hyperliproteinemia could be related to genetic factors and acquired or environmental factors . The possible explanation for dyslipidemia development in our case was most likely genetic and not related to environmental factors as the patient was otherwise healthy.Gotoda T et al. in 2012 stated that environmental factors such as diabetes, drinking hormonal therapy, drugs and myeloma present in only 2/3 of patients with type V hyperlipoproteinemia, while 1/3 patients does not have these factors . The envThis disorder is caused by mutations in different genes such as LPL, Apo B, APOC2, APOA5, GPIHBP1, and LMF1 genes. These mutations are inherited in an autosomal recessive fashion. Our limitation was that we did not perform genetic testing due to non-availability of these tests in the country and due to the financial constraints associated with sending the samples abroad. As per Gotoda T et al., the possibility of LPL deficiency is high if the serum TG level is 1500 mg/dl or higher, the serum total cholesterol level is about 1/10 the serum TG level or lower . Our patA case by Cristina Oana et al. in November 2017 reported a 16-year-old female teenager presenting with severe abdominal pain prominently in the lateral left quadrant, nausea, vomiting and constipation for 2 days. On examination, she had ascites and paralytic ileus. She also had increased level of triglycerides 555.1 mg/dl with normal total cholesterol of 180.5 mg/dl. Lipoprotein electrophoresis showed low level of alpha-lipoproteins (7.6%), increased pre-beta lipoproteins (71.2%), lack of beta-lipoproteins and raised Chylomicrons (21.2%). They found low level of Apolipoproteins A (0.82 g/L), and a normal level of apolipoproteins B. She was diagnosed with type V hyperlipoproteinemia based on lipoprotein electrophoresis and Apo A .In conclusion the literature and case reports on type v hyperlipoproteinemia is scarce. Bianshly Rivera et al. in May 2019 stated that there are no specific diagnostic and management guidelines for this disorder due to insufficient number of cases [


We aimed to investigate the therapeutic mechanism of Yinzhihuang (YZH) liquid, a traditional Chinese medicine mainly composed of extracts of four components, on nonalcoholic steatohepatitis (NASH) induced by a high‐fat, high‐cholesterol diet (HFHCD) in rats.Altogether 30 Sprague‐Dawley rats were randomized into three groups: control, the model group  and the treatment group (HFHCD + YZH). Liver histological features and serum biochemical parameters were assessed by the end of the 16th week. RNA sequencing and protein mass spectrometry detection were performed. The genes and proteins expressed differentially were subjected to KEGG pathway enrichment analysis and included in a network‐based regulatory model.P < 0.05). The network‐based regulatory model showed that these deregulated genes and proteins were mainly involved in oxidative phosphorylation, Toll‐like receptor, nucleotide‐binding oligomerization domain‐like receptor, peroxisome proliferator‐activated receptor signaling, nuclear factor‐kappa B tumor necrosis factor signaling pathways and fatty acid metabolism.The weight, liver and fat indices and serum alanine transaminase, aspartate transaminase and total cholesterol levels of the HFHCD + YZH group were all significantly lower than those of the HFHCD + saline group. Moreover, their hepatic steatosis, ballooning and lobular inflammation were relieved, and 64 hepatic genes and 73 hepatic proteins were found to be reversed in their expression patterns after YZH treatment (YZH could alleviate NASH in HFHCD‐fed rats by inhibiting lipogenesis, accelerating lipid β‐oxidation, alleviating oxidative stress and relieving necroinflammation in the liver. The reasons may include the nature of the diseases, natural evolution of feedback loops and pathway redundancy. Traditional Chinese herbal medicines are rich in numerous bioactive substances with several biological properties, including anti‐inflammatory, anti‐infection and immunoregulatory properties.In clinical practice, YZH oral solution can be used in the treatment of neonatal jaundice and cholestasis. It can significantly reduce serum bilirubin levels, shorten the time for jaundice recovery, and ultimately achieve a high rate of therapeutic efficacy.Complex multicomponent nature may make these traditional Chinese herbal medicines valuable resources due to their synergistic effects. In addition, multiple systems biology platforms have been shown to be the most powerful technologies available to uncover molecular mechanisms and connections between drugs and their multitargeted networks.22.1Herba Artemisiae Capillariae, 6.4 g of Fructus Gardeniae and 8 g of L. japonica were mixed and dissolved by adding 300 mL of double‐distilled water. The pH value was adjusted to 6.5 by adding 10% sodium hydroxide solution and the solution was filtered and reserved. S. baicalensis (40 g) was then dissolved in 300 mL of double‐distilled water and the pH value was adjusted to 6.5‐7.0 with a 10% sodium hydroxide solution. After filtration, the filtrate was mixed with the reserved solution. The compound was thoroughly mixed with 0.5 g of citric acid, 100 g of sucrose, 50 g of honey, 2 g of aspartame and 3 g of sodium benzoate. The mixture was refrigerated at 4–8 °C for 24 hours and the pH value was adjusted to neutral. An appropriate amount of double‐distilled water was added to this solution to adjust the total amount to 1000 mL. Finally, the solution was left to stand, filtered and sterilized to finally obtain the YZH liquid.The YZH liquid was purchased from China Resources Sanjiu Medical & Pharmaceutical . The identification and quantification of the marker compounds in the YZH liquid were performed according to the methods described in the Updated Pharmacopeia of the People's Republic of China.Marker compounds were analyzed using high‐performance liquid chromatography  with a Venusil MP C18 column , as described in a previous study.2.2In total 30 male Sprague‐Dawley rats (6 weeks old) obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences  were kept (5 rats per cage) under controlled temperature (24 °C ± 2 °C) and humidity (50% ± 5%) with free access to food and water. The animal experiments were performed with reference to the National Research Council's Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee of Society for Human Resource Management (no. SHRM‐IACUC‐001).Following a week of acclimation, the rats were randomly divided into three groups using random number table. The control group was given a normal diet composing of 64.5% carbohydrates, 13.3% fat, and 22.2% protein. The model group  was given a HFHCD , and an addition of saline by gavage at a dose of 15 mL/kg once daily beginning at the end of the 8th week. The treatment group (HFHCD + YZH) was given a HFHCD and YZH liquid by gavage at a dose of 15 mL/kg once daily beginning at the end of the 8th week  from the livers of 12 rats (four rats per group). The RNA sequencing experimental process is shown in Figure 2.4Protein was extracted from the remaining liver tissues of these 12 rats using a lysis buffer and two magnetic beads (diameter 5 mm). The main procedures of the iTRAQ quantitative proteomics experiment are shown in Figure The exported mascot generic format (MGF) files were transformed based on the raw tandem mass spectrometry (MS)/MS data, and they were searched in the NCBI nr database (ftp://ftp.ncbi.nih.gov/blast/db). The peptides were labeled with isobaric tags and then quantitatively analyzed with the automated software IQuant . All proteins with a false discovery rate of less than 1% were subjected to downstream analysis. Furthermore, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the differentially expressed proteins.2.5https://string-db.org) and web resource. The default confidence cut‐off value was set as 400; solid lines were used between genes and proteins when interactions with scores were over 400, while dashed lines were used in interactions with scores lower than 400. Genes/proteins are represented with circular nodes, while KEGG pathways are indicated with rectangles. The pathways are colored in a gradient from yellow, indicating a larger P value, to blue, indicating a smaller P value. Genes/proteins are colored red (upregulated) and green (downregulated) in fold‐change analysis.The generation of a network‐based regulatory model was based on the Cytoscape web application  with information obtained from several levels of functional analysis: the fold‐change analysis of genes or proteins, the protein–protein interaction analysis, the KEGG pathway enrichment analysis and the biological process enrichment analysis. The protein‐protein interaction analysis was accomplished by selecting genes and proteins expressed differentially via the widely used STRING biological database . Graphs were generated using GraphPad Prism Software version 6 . Differences were considered statistically significant at 33.1P < 0.01), while the HFHCD + YZH group had a significantly lower body weight, liver index and fat index than the HFHCD + saline group ‐like receptor signaling pathway, biosynthesis of unsaturated fatty acids, the nuclear factor‐κB signaling pathway, fatty acid degradation, fatty acid metabolism, glycolysis and gluconeogenesis and bile secretion  signaling pathway, alpha‐linolenic acid metabolism, and metabolic pathways Table .3.4Fads1, Fads2, Pck1, Me1, Adh4 and Adh6 were upregulated, and Scd1, phospholipase A2 (Pla) family members (including Pla2g2d and Pla2g6), Prkcd, Fabp5, Plin2, Apoa5, Aldh1b1, and Acyp2 downregulated and were involved in the PPAR signaling pathway, biosynthesis of unsaturated fatty acids, fatty acid degradation, fatty acid metabolism, alpha‐linolenic acid metabolism and pyruvate metabolism, which were mostly associated with fatty acid metabolism. Meanwhile, the upregulated Ndufa4, Ndufa9, Ndufa12 and Cox15 were involved in oxidative phosphorylation, which is a pathway regulating oxidative stress. Genes including Lbp, Stat1, Ifi47, Ripk2, Gbp2, Irf1, Ccl2, Ccl3, Ccl4, Ccl21, Cxcl1, Cxcl9, Cxcl10 and Cxcl11 were all downregulated. They were mostly involved in inflammation and immune response pathways, including the NF‐κB, Toll‐like receptor, NOD‐like receptor, and TNF signaling pathways. Overall, the network‐based regulatory model indicated that YZH liquid could alleviate NASH in HFHCD‐fed rat models by regulating fatty acid metabolism, oxidative stress, inflammation and immune responses.The 64 differentially expressed genes and 73 differentially expressed proteins after YZH administration were selected to establish a gene–protein interaction network (Figure 4The mechanism remains unknown although our previous studies have demonstrated the effectiveness of YZH liquid on MCD‐ and HFHCD‐induced NASH in animal models. In the current study HFHCD‐induced NASH rat model was used, which is more characteristic of the natural course of human NASH compared with MCD‐induced model. We found that YZH liquid not only reduced the body weight, liver and fat index of the NASH rats but also improved hepatic histological features and decreased serum transaminase levels. Importantly, for the first time, we combined RNA sequencing with protein profiling to explore the mechanism by which YZH liquid alleviated NASH and constructed a network‐based regulatory model. Based on this model, YZH liquid might inhibit lipogenesis and accelerate lipid β‐oxidation by regulating fatty acid metabolism and the PPAR signaling pathway, alleviate oxidative stress by upregulating oxidative phosphorylation and relieve intrahepatic inflammation through downregulating Toll‐like receptor, NOD‐like receptor, NF‐κB and TNF signaling pathways.Fads expression has been found to be negatively associated with hepatic fat content, and FADS can suppress the accumulation of hepatic lipids.Pla2 family members, which are upstream checkpoints regulating fatty acid uptake that are upregulated in patients with NASH.Pla2 has been proven to achieve a 56.5% decrease in fatty acid influx in hepatocytes.Resulting from the imbalance between lipid deposition and removal, hepatic fat accumulation can be regulated by nuclear receptors and cytoplasmic transcription factors, including PPAR family members.Ndufa4, Ndufa9, Ndufa12 and Cox15. The impairment of mitochondrial oxidative phosphorylation can decrease adenosine triphosphate production and inhibit lipid β‐oxidation, which leads to hepatocellular steatosis, thus initiate oxidative stress and subsequent development of necroinflammation.The accumulation of TG in hepatocytes is caused by increased lipogenesis with decreased fatty acid β‐oxidation, which, combined with increased levels of reactive oxygen species mainly generated in mitochondria, contributes to the development of NASH.Lbp, Stat1, Ripk2, Irf1, Gbp2 and several chemokines, were suppressed after YZH liquid therapy. Chemokines are chemoattractants for leukocyte trafficking, growth and activation at sites of injury and inflammation, and in the livers of patients with NASH, the expression of chemokines and their receptors was increased based on accumulated data.Ccl2 or Cxcl10, which links hepatocyte lipotoxicity to macrophage‐associated liver inflammation in NASH as a potent chemotactic ligand.Lbp is increased in patients with NASH and this correlates with lobular inflammation,Lbp in mice can decrease indices of liver damage determined by liver histology and serum transaminases.Stat1 serves as a mediator in interferon γ and chemokine expression induced by lipopolysaccharide in macrophages, and the inhibition of Stat1 can inhibit the release of chemokines and suppress the amplification cascade of inflammatory signals.Hepatocyte injury is the key feature that differentiates NASH from isolated steatosis.There were some limitations to our study. First, TG levels were measured only in the serum, while its accumulation in hepatocytes is actually the main component of hepatic steatosis. Second, the therapeutic mechanism of YZH liquid was evaluated in NASH rat models. The efficacy and functional mechanism of this herbal medicine need further evaluation in clinical studies. Third, the specific regulatory relationships between genes and proteins in this network‐based regulatory model have not been fully elucidated in our study. Therefore, the verification of RNA sequencing in several selected pathways is needed and functional experiments need to be specifically designed to support our claims.5In conclusion, YZH liquid was effective in reducing body weight and serum aminotransferase levels and alleviating pathological liver injury in HFHCD‐fed NASH models. The network‐based regulatory model of YZH liquid on NASH shows that YZH liquid can reduce lipid deposition in the liver by inhibiting lipogenesis and accelerating lipid β‐oxidation, and can alleviate oxidative stress by upregulating the expression of mitochondrial respiratory chain complex. Finally, YZH liquid relieves intrahepatic inflammation by downregulating chemokines and other inflammation‐related factors. These results provide a theoretical basis for undergoing a randomized clinical trial of YZH liquid on patients with NASH in the near future.The authors declare that they have no competing interests.Figure S1. Marker compounds in yinzhihuang liquid measured by high‐performance liquid chromatography at 327 nm. A, Negative control. B, Mixture control. C, yinzhihuang liquid sample. 1, chlorogenic acid; 2, geniposide; 3, hyperoside; 4, baicalin; 5, luteolin.Click here for additional data file.


BPDCN shows clinically heterogeneous characteristics. And as other hematological malignancies, symptoms of BPDCN suggesting a high tumor burden, such as high white blood cell count or splenomegaly, should be carefully considered to prevent TLS. BPDCN shows clinically heterogeneous characteristics. And as other hematological malignancies, symptoms of BPDCN suggesting a high tumor burden, such as high white blood cell count or splenomegaly, should be carefully considered to prevent TLS. We report a case of a 63‐year‐old man with BPDCN showing severe tumor lysis syndrome (TLS). BPDCN with high tumor burden should be carefully considered to prevent TLS.CALR) mutation arising from myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN‐RS‐T) without cutaneous lesion, showing severe tumor lysis syndrome (TLS) immediately after the induction therapy.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy with dismal prognosis. With difficulty in diagnosis, BPDCN was used in the 2008 World Health Organization (WHO) classification for the first time and became a distinct entity in 2016 WHO classification.29/L with 88% blasts; hemoglobin, 5.5 g/dL; platelet count, 17.0 × 109/L; and serum lactate dehydrogenase (LDH), 300 IU/L. Genetic analyses of his peripheral blood cells detected type 1 CALR mutation without Janus kinase 2 mutation. Markedly hypercellular marrow occupied by agranular blasts with small cytoplasm and fine chromatin were detected in the bone marrow aspiration (Figure A 63‐year‐old man previously diagnosed with MDS/MPN‐RS‐T 6 years before the presentation was referred to our hospital for rapid progression of leukocytosis and anemia. Physical examination and computed tomography revealed splenomegaly and inguinal lymphadenopathy without skin involvement Figure  A, B. Hen Figure , and bonn Figure . Flow cy9/L and worsening malaise, the dose‐reduced SMILE regimen without methotrexate which contained etoposide 70 mg/m2 day1–3, ifosfamide 1050 mg/m2 day 1‐3, dexamethasone 30 mg/body day 1‐3, and L‐asparaginase 4000 U/m2 day 7, 9, 11, 13, 15, 17, and 19 was immediately initiated without accurate diagnosis.Due to rapidly increasing WBC count of 106 × 103SF3B1 mutation.SF3B1 mutations were not examined in this case, morphologically apparent dysplasia and the presence of ringed sideroblasts strongly supported the diagnosis of MDS/MPN‐RS‐T. On the other hand, while there is one case report about a patient with MDS/MPN‐RS‐T that was positive for both CALR and SF3B1 gene mutations,CALR mutation has not yet been reported to the best of our knowledge. Therefore, in this case, MDS/MPN‐RS‐T might already have been accompanied with CALR mutation.In this case report, the patient was diagnosed with BPDCN arising from MDS/MPN‐RS‐T that progressed into clinical TLS immediately after the initiation of chemotherapy. Majority of patients with MDS/MPN‐RS‐T have a TdT negativity was reportedly associated with inferior survival in BPDCN;Previous studies showed that BPDCN with leukemic presentation without skin manifestation rarely occurs and tends to present with cytopenia rather than leukocytosis.Although the risk prediction of TLS in BPDCN is unknown because of the rarity and heterogeneity of this disease, the high‐disease burden may indicate the risk of TLS as same as other hematological malignancies, and the evaluation of peripheral blood WBC counts and serum LDH concentration may be useful for risk classification of TLS in BPDCN as in acute leukemia.In conclusion, BPDCN shows clinically heterogeneous characteristics, and patients with clinical symptoms suggesting an aggressive clinical course with high tumor burden, including high WBC count or splenomegaly, should be carefully considered to prevent TLS.There are no conflicts of interest to report.KS: selected the patient and designed the manuscript. KS, MI, NF, and AK: wrote the manuscript. RF: performed the immunohistochemistry. All the authors: reviewed the paper and agreed with the final version.This study was approved by the institutional ethical committee of the Konan Kosei Hospital. Written informed consent was obtained from the family of the patient for publication of this case report.