Intraoperative blood salvage (IBS) recovers most lost blood, and is widely used in the clinic. It is unclear why IBS does not reduce long-term postoperative requirements for red blood cells (RBCs), and 1 possibility is that IBS affects RBC lifespan.Prospectively enrolled patients who underwent spine, pelvic, or femur surgery not involving allogeneic RBC transfusion were grouped based on whether they received IBS or not. Volumes of blood lost and of RBCs salvaged during surgery were recorded. Total blood cell counts, levels of plasma-free hemoglobin, and CD235a-positive granulocytes were determined perioperatively.P < .001), hemoglobin levels were similar between groups (P = .125) at the end of surgery. Hemoglobin levels increased in non-IBS patients (4 ± 11 g/L), but decreased in IBS patients (−7 ± 12 g/L) over the first 3 postoperative days. Nadir hemoglobin levels after surgery were higher in the non-IBS group (107 ± 12 g/L) than in the IBS group (91 ± 12 g/L). Salvaged RBC volume correlated with hemoglobin decrease . In multivariate analysis, salvaged RBC volume was an independent risk factor for hemoglobin decrease . Flow cytometry showed the numbers of CD235a-positive granulocytes after surgery to be higher in the IBS group than in the non-IBS group (P < .05).Although intraoperative blood loss was higher in the IBS group (n = 45) than in the non-IBS group (n = 52) . In contrast, another study found that IBS significantly reduced transfusions of packed RBCs within 24 hours after cardiac surgery, but not during the subsequent hospital stay. In addition, large, multicenter randomized controlled trials showed that IBS during primary hip or knee replacement surgery did not decrease the mean number of allogeneic RBCs transfused or the proportion of patients receiving transfusions after surgery.–7Intraoperative blood salvage (IBS), which allows recovery of most blood lost during surgery, is widely used in the clinic. However, whether it reduces postoperative requirements for allogeneic red blood cells (RBCs) is unclear. A multicenter cohort study it is possible that IBS damages RBCs. Indeed, our group has shown that IBS significantly reduces adenine nucleotide levels, causing RBCs to become stomatocytes and even “ghosts” at 48 hours after surgery. Damaged RBCs should be rapidly eliminated after being returned to the patient, which may explain why IBS does not reduce postoperative blood requirements in the longer term.The available evidence, then, suggests that IBS does not provide substantial benefits over longer postoperative periods. The reasons for this are unclear. Since IBS has been shown to reduce the deformability of RBCs, and also their content of 2,3-diphosphoglycerate,,11 The present study tested this hypothesis by examining whether and how IBS shortens RBC lifespan during the first 3 days after surgery.One possibility is that damaged RBCs undergo rupture (hemolysis) or are engulfed by phagocytes via receptor-mediated interactions, leading the RBCs to be cleared from the body. During engulfment, phagocytes may recognize phosphatidylserine exposed on the surface of the RBCs.22.1Patients scheduled for spine, pelvic, or femur surgery at the Affiliated Hospital of Zunyi Medical University were prospectively enrolled between January 1, 2015 and November 30, 2015 if they were between 18 and 70 years old and had American Society of Anesthesiologists physical status I to III. Patients were excluded if they had one of the following conditions: a serious disorder of the coronary, peripheral, and/or carotid arteries; low hemoglobin levels, defined as <8 g/dL; hematologic or malignant disease; diabetes; untreated hypertension, defined as diastolic blood pressure >95 mm Hg; renal or liver dysfunction; sickle cell anemia; or prophylactic anticoagulant therapy. They were also excluded if they received allogeneic RBC transfusion, or if they were undergoing repeat or emergency surgery. Patients exited the study if they withdrew consent after enrollment.This study was approved by the Ethics Committee of Zunyi Medical University, and has been registered in the China Clinical Trial Registry (ChiCTR-OCH-14005140).2.2All patients received general anesthesia involving intravenous induction with midazolam, sufentanil, propofol, and muscle relaxants, followed by endotracheal intubation and positive-pressure ventilation. Anesthesia was maintained by sevoflurane inhalation, sufentanil, propofol, and muscle relaxants. Briefly, a negative pressure system (−4.0 to −6.0 kPa) collected blood lost from the time of skin incision until wound closure. Heparin (25 U/mL) was added to 0.9% normal saline to prevent coagulation. RBCs were washed in 250-mL centrifuge bottles until the supernatant was clear, which required 1.5 to 2 L of solution. If >250 mL of RBCs were salvaged, they were re-infused into the patient as soon as possible. If <250 mL were salvaged, the RBCs were not re-infused into the patient. Patients who received transfusion of salvaged RBCs were assigned to the IBS group, and patients who did not receive salvaged RBCs were assigned to the non-IBS group. We estimated that the smallest acceptable sample size would be 36 patients in each group, to have 80% likelihood of detecting a difference of 1 g/dL in hemoglobin levels between the 2 groups on the first day after surgery. This power calculation assumed a standard deviation of 15 g/L in hemoglobin level measurements, and a 2-sided alpha level of 5%.For all patients enrolled in our study, blood was salvaged during surgery using an autologous blood recovery system , as described.2.3Blood loss in the operating room was assessed by measuring blood volume in the suction bottle , and also by weighing blood-stained absorbent gauzes generated between skin incision and skin closure (assuming blood density of 1 g/mL). Blood loss after surgery was defined as the volume of blood shed into the drainage tube over the first 72 hours after surgery.Decisions to give blood product transfusions were made in the operating room by the anesthesiologist and in the ward by the surgeon. The threshold hemoglobin concentration for performing transfusion of packed RBCs was 7 g/dL.2.4Blood cell counts and hemoglobin levels were determined using an automatic blood analyzer before surgery, at the end of surgery, and at 24, 48, and 72 hours after surgery. These time points were chosen because our preliminary studies (data not shown) indicated that RBC counts were stable by 3 days after surgery. At the same time points, levels of free hemoglobin in plasma were determined using enzyme-linked immunosorbent assay  according to the manufacturer's instructions. Counts of CD235a-positive granulocytes and monocytes were determined using flow cytometry. Requirements for blood production during surgery and during the first 3 postoperative days were recorded.2.56 cells) were incubated with 3.0 μL PE-conjugated anti-human CD14 antibody  for 30 minutes at 4°C in the dark. The mixture was centrifuged at 600g for 7 minutes at 4°C, and cells were washed twice with PBS, then resuspended in 250.0 μL fixation/permeabilization solution  at 4°C for 20 minutes. Cells were washed twice in 1 × BD Perm/Wash buffer. Fixed/permeabilized cells were thoroughly resuspended in 50 μL BD Perm/Wash buffer containing 3.0 μL fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD235a antibody (BD Bioscience) and incubated at 4°C for 30 minutes in the dark. IgG1-FITC/PE served as a negative control. Cell pellets were washed twice with 1× BD Perm/Wash buffer, suspended in PBS, then analyzed by flow cytometry.In this procedure, blood (1.0 mL) was lyzed with ammonium chloride (9 mL) and the cells were washed twice with phosphate-buffered saline (PBS), suspended in PBS, and counted using a hemacytometer. Cell suspensions , and data were analyzed using Flowjo software (Tree Star). A sequential gating strategy was used during analysis, and CD235a-positive phagocytes were confirmed using confocal microscopy Fig. .2.6g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in PBS to a concentration of 5 × 106 cells/mL. Cells (5 × 106) were seeded into three 6-cm centrifuge tubes. The cells were plated and cultured at 37°C on a platform rotating at 80 rpm. To determine what fraction of these cells bound Annexin-V, cells were harvested after 0, 24, 48, or 72 hours of culture, and diluted to 1 × 106 cells/mL using Hank balanced salt solution (Invitrogen). To the resuspended cells  was added 3.0 μL allophycocyanin-conjugated Annexin V (BD Bioscience), and the mixture was incubated at room temperature for 15 minutes in the dark. To this mixture was added 400.0 μL 1× binding buffer, and then flow cytometry of at least 50,000 events was performed.Preoperative arterial blood (fresh RBCs) and salvaged RBCs from 10 patients in the IBS group were centrifuged at 8002.7t test. Categorical measurements were expressed as counts (percentages), and Fisher exact tests were used to assess differences between groups. The paired Student t test was used to assess the significance of differences in the proportion of fresh and salvaged RBCs that bound Annexin-V after incubation. Decreases in hemoglobin levels were calculated by subtracting the nadir value (measured within 3 days after surgery) from the values at the end of surgery. Pearson correlation analysis was used to detect relationships of salvaged volume with decreases in levels of hemoglobin within 3 days after surgery. Univariate and multivariate logistic analyses were used to analyze the association of baseline or intraoperative variables with either intraoperative blood loss or decrease in hemoglobin levels. Odds ratios (ORs) and associated 95% confidence intervals (95% CIs) were calculated to predict the change in outcome variable per unit increase of the predictor. The threshold for statistical significance was defined as P < .05.Data were analyzed using SAS 9.1 . Continuous measurements were reported as mean ± standard deviation, and differences between groups were assessed for significance using Student 3In all, 107 patients were assessed for enrollment in the study, of whom 52 received transfusion with salvaged RBCs. Of these 52 patients, 2 exited the study because 1 withdrew consent and another had a postoperative accident that led to massive blood loss. Another 5 were excluded because they received transfusions of altogether 21 units of allogeneic packed RBCs, with 10 units (47.6%) transfused in the operating room, 3 units (14.3%) transfused on postoperative day 1, 4 units (19.0%) on day 2, and 4 units (19.0%) on day 3. These patients were excluded to avoid confounding effects of allogeneic RBCs on analysis of salvaged RBC survival. The remaining 45 patients were included in the final analysis . In univariate logistic analysis, surgical duration was associated with massive blood loss, defined as loss of at least 500 mL . This relationship remained significant after adjusting for age, sex, body mass index, prothrombin time, international normalized ratio, platelet count, and surgical site . Intraoperative blood loss was higher in the IBS group than in the non-IBS group .The total volume of salvaged RBCs was 633 ± 303 mL for IBS patients, corresponding to salvage of approximately 67% of lost blood. A strong correlation was found between the volume of blood lost and the volume of salvaged RBCs (3.2P = .575) and at the end of surgery (P = .317), with hemoglobin decreasing in both groups between these 2 time points (P < .001). The magnitude of this decrease was similar between the non-IBS group  and the IBS group . Hemoglobin levels in the non-IBS group returned to presurgery levels by day 1 after surgery , and they remained stable until at least 72 hours after surgery. In contrast, hemoglobin levels on day 1 after surgery in the IBS group (101 ± 14 g/L) were still significantly below the presurgery baseline (P < .001), and they decreased further on days 2 and 3 after surgery. On all 3 days after surgery, hemoglobin levels were significantly lower in the IBS group than in the non-IBS group   reached their nadir hemoglobin levels at the end of surgery, whereas most IBS patients (69%) reached their nadir hemoglobin levels between day 1 and day 3 after surgery . Univariate logistic regression found hemoglobin decrease to be associated with salvaged RBC volume  and surgery site (P = .005). Volume of salvaged RBCs was an independent risk factor for hemoglobin decrease , even after adjusting for age, sex, body mass index, prothrombin time, international normalized ratio, platelet, surgical time, and surgery site. Similar results were observed for RBC counts , but decreased in IBS patients (−7 ± 12 g/L) within 3 postoperative days . Incubating salvaged and fresh RBCs for 72 hours led to an increase in the percentage of Annexin-V-binding cells in a time-dependent manner. This increase was greater for salvaged cells than fresh cells by 1.48-fold at 0 hour of incubation, 3.62-fold at 24 hours, 2.73-fold at 48 hours, and 4.62-fold at 72 hours .To examine whether IBS induces phosphatidylserine exposure, salvaged and fresh RBCs were isolated in the IBS group and compared within the same patient. The percentage of salvaged cells that bound Annexin-V was higher than the corresponding percentage of fresh cells from the same patient  behind the lifespan-shortening effect of IBS on RBCs, we examined the possibility that IBS may induce hemolysis. We found that IBS was not associated with an increase in free hemoglobin levels in plasma, consistent with other studies.,11 We labeled RBCs based on the presence of CD235a, and phosphatidylserine was labeled with Annexin-V. The number of leukocytes increased after salvaged RBCs were returned to the body, and the number of RBC-engulfing granulocytes after surgery was significantly higher in the IBS group than in the non-IBS group. These findings suggest that IBS makes RBCs more likely to be engulfed after re-infusion, leading them to be cleared from the body.,11 Indeed, we found that a higher proportion of salvaged cells than fresh cells had exposed phosphatidylserine on their surface, which acts as a signal for phagocyte engulfment. The proportion of salvaged cells with exposed phosphatidylserine was as high as 11% at 72 hours after blood salvage. In addition, numbers of CD235a-positive cells increased significantly within 72 hours after surgery in the IBS group. Therefore, we propose that at least 1 of the reasons why IBS does not reduce the postoperative requirement for blood transfusions is that it shortens RBC lifespan by increasing phosphatidylserine exposure on the RBC surface, facilitating engulfment of RBCs after re-infusion.As another possibility, we explored whether injured RBCs are engulfed by phagocytes via recognition of phosphatidylserine on the RBC membrane.In our cohort, the proportion of patients undergoing surgery at sites of lumbar vertebra was higher in the IBS group than in non-IBS group, and surgery lasted slightly longer in the IBS group, with longer duration associated with massive blood loss. We found that hemoglobin decrease was associated with salvaged RBC volume, preoperative platelet count, and surgery site. Therefore, these parameters should be treated as potential confounders in studies of the impact of salvaged blood on hemoglobin levels. Finally, we did not measure ferritin, iron, erythropoietin, or reticulocyte counts, which would have indicated whether the patient had iron deficiency and would respond appropriately to blood loss.Our results should be interpreted with caution in light of several limitations. The patients in our study were not randomized, but grouped according to whether they received IBS or not. As a result, differences among the 3 groups may have introduced bias into our results. Our study was limited by small sample size, which precluded us from analyzing whether IBS is an independent risk factor for postoperative transfusion of packed RBCs. We also did not examine whether different cell saver devices affect the function of salvaged RBCs, as previously reported.5Despite these limitations, the present study provides evidence that IBS shortens RBC lifespan, at least partly by increasing phosphatidylserine exposure on the RBC surface and facilitating engulfment of RBCs after re-infusion. Future studies should examine how to prolong the lifespan of salvaged RBCs, such as through changes to the RBC washing procedure to reduce injury during salvage, and modification of the washing solution to increase adenine nucleotide levels within the RBCs.


Overall validity of existing genetic biomarkers in the diagnosis of obstructive sleep apnea (OSA) remains unclear. The objective of this systematic genetic study is to identify “novel” biomarkers for OSA using systems biology approach. Candidate genes for OSA were extracted from PubMed, MEDLINE, and Embase search engines and DisGeNET database. The gene ontology (GO) analyses and candidate genes prioritization were performed using Enrichr tool. Genes pertaining to the top 10 pathways were extracted and used for Ingenuity Pathway Analysis. In total, we have identified 153 genes. The top 10 pathways associated with OSA include (i) serotonin receptor interaction, (ii) pathways in cancer, (iii) AGE-RAGE signaling in diabetes, (iv) infectious diseases, (v) serotonergic synapse, (vi) inflammatory bowel disease, (vii) HIF-1 signaling pathway, (viii) PI3-AKT signaling pathway, (ix) regulation lipolysis in adipocytes, and (x) rheumatoid arthritis. After removing the overlapping genes, we have identified 23 candidate genes, out of which >30% of the genes were related to the genes involved in the serotonin pathway. Among these 4 serotonin receptors SLC6A4, HTR2C, HTR2A, and HTR1B were strongly associated with OSA. This preliminary report identifies several potential candidate genes associated with OSA and also describes the possible regulatory mechanisms.  According to the Centers for Disease Control and Prevention, sleep disorders now constitute a public health epidemic. The National Healthy Sleep Awareness Project estimated that at least 25 million American adults are affected by OSA for the year 2014  increasiIn-lab polysomnography (PSG) is the “gold standard” test for the diagnosis of OSA . Howeverα [Blood biomarkers may offer alternative approach for screening and identifying individuals at risk of developing OSA and related outcomes. Biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic response to a therapeutic intervention. In the case of OSA, the candidate biomarkers should be useful for the following: diagnosis, assessing disease burden and severity, and evaluating response to treatment. Several epidemiological studies suggest that the circulatory levels of natriuretic peptide (BNP) or N-terminal-pro-BNP , leptin,α , vaspin α , ghrelinα , and intα  are assoAdvances in computational approaches and experimental omics methods that allow the simultaneous analysis of multidimensional data such as DNA, RNA, and proteins in a single analysis have made these systems biology approaches feasible for biomarker discovery . Systemshttp://www.disgenet.org/web/DisGeNET/menu) [Literature search was conducted in PubMed, MEDLINE, and Embase search engines for studies published for “genes, polymorphisms, and biomarkers” using the terms “Apnea, Obstructive Sleep” or “Apneas, Obstructive Sleep” or “Obstructive Sleep Apnea” or “Obstructive Sleep Apneas” or “Obstructive Sleep Apnea Syndrome” or “OSAHS” or “Sleep Apnea Hypopnea Syndrome” or “Sleep Apneas, Obstructive” or “Sleep Apnea Syndrome, Obstructive” or “Syndrome, Obstructive Sleep Apnea” or “Syndrome, Sleep Apnea, Obstructive” or “Syndrome, Upper Airway Resistance, Sleep Apnea” or “Upper Airway Resistance Sleep Apnea Syndrome” for obstructive sleep apnea (MeSH® ID: D020181). Furthermore, we queried the DisGeNET database (ET/menu)  that inthttp://amp.pharm.mssm.edu/Enrichr/) [z-score of the deviation from the expected rank; and the third method  is a combination of both P value computed using the Fisher exact test and the z-score. For this analysis, we have employed third approach for gene ontology analysis.The gene ontology analysis was performed using ToppGene suite . The Topnrichr/) , 25. Enrhttps://www.ingenuity.com/) for mapping to canonical pathways and identifying upstream regulators using a set of criteria: genes and endogenous chemicals, direct and indirect interactions, maximum molecules per network, networks per analysis, humans as the selected species, and all tissues and primary cells. The resulting networks were scored based on the fold change provided by Cuffdiff as log2 (fold change) for each gene. The obtained P values correspond to Fisher's exact test, with the null hypothesis that the molecules within the networks are connected based on chance.Finally, the shortlisted genes were submitted to Ingenuity Pathway Analysis ). The top 10 pathways and their corresponding genes revealed by Enrichr tool are given in Figures P = 2.84E−24), obesity (P = 1.65E−23), obsessive-compulsive disorder (P = 8.77E−21), dyslipidemia (P = 5.87E−20), social anxiety disorder (P = 1.28E−18), T2DM (P = 5.14E−18), blood pressure (P = 3.52E−17), eating disorders (P = 3.75E−15), depression (P = 3.61E−14), and metabolic syndrome (P = 1.10E−13) , for further analysis. The analysis of statistically overrepresented pathways in the shortlist of genes revealed 28 canonical pathway maps (by ToppGene suite) with confidence level .10E−13) . Among tP < 1.05E−09), melatonin (P < 1.41E−09), glucocorticoid (P < 5.83E−09), lithium (P < 1.45E−08), and RAB1A (P < 5.48E−08).For network analysis The overall validity of existing biomarkers in the diagnosis of obstructive sleep apnea (OSA) remains unclear. Although the existing adipokines and inflammatory markers seem to have a favorable potential to become a good biomarker to identify OSA they are not specific for the disease. In this study, we have performed the gene mining analyses and found 23 candidate genes that are likely associated with OSA. Functional enrichment analysis suggested that most of these genes are directly involved in the above-mentioned biological processes , which aC, HTR2A, and HTR1B which associated with OSA.The disease pathway analysis showed that the selected candidate genes were also associated with the OSA comorbid conditions such as anxiety disorders, obesity, and dyslipidemia. For the subsequent analysis, we focused only on serotonin pathway because majority of serotonin genes make up this pathway. Dysfunction of the serotoninergic system has long been suspected in sleep disorders and respiratory diseases , 36. 5-HThe genomewide association studies demonstrated the association of 5-HTR 2A/2C polymorphisms with OSA. Furthermore, by regulating the magnitude and duration of serotonergic responses, the SLC6A4  is pivotal to the fine-tuning of brain serotonergic neurotransmission and of the peripheral actions of 5-HT .It is clear that there is an intricate network of interacting genes regulating sleep and the derangement in them leads to OSA. In this preliminary report, we identified 23 novel biomarkers especially from the serotonin pathway which might associate with OSA. Consequently, we demonstrated network-level analysis of potential OSA candidate genes and the plausible regulatory mechanisms. Limited evidence from the clinical trials showed that 5-HT has a critical role influencing respiratory control during sleep. 5-HT acts as a potent central ventilatory stimulant and serves to maintain upper airway patency and maintain eucapnia via the chemoreceptor properties. Relative reductions in 5-HT may lead to the development and worsening of OSA. Further biological experiments are warranted to study the role of peripheral 5-HT and its receptors on the pathogenesis of OSA.


Correlation analysis revealed a singularity of shNT blastocysts as they separately gathered from the others. Analysis of developmentally important genes revealed that, in shNTs, the stemness-associated differentially expressed genes (DEGs), including OCT4, were mostly underrepresented. Overrepresented epi-driver genes were largely associated with heterochromatin establishment and maintenance. By multilateral comparisons of their transcriptomes, we classified DEGs into three groups: 561 manipulation-associated DEGs (MADs) common to shNTs and scNTs, 764 donor genome-associated DEGs (DADs) specific to scNTs, and 1743 zygote manipulation-associated DEGs (zMADs) specific to shNTs. GO enrichment analysis generated various terms involving “cell-cell adhesion,” “translation,” and “transcription” for MADs and “cell differentiation” and “embryo implantation” for DADs. Because of the transcriptomic specificity of shNTs, we studied zMADs in detail. GO enrichment analysis with the 854 zMADs underrepresented in shNTs yielded terms related to protein and mitochondria homeostasis, while GO enrichment analysis of 889 shNT-high zMADs yielded terms related to endoplasmic reticulum stress and protein transport. We summarized the DEGs, which, with further investigation, may help improve our understanding of molecular events occurring in cloned embryos and our ability to control clonal reprogramming.Somatic cell nuclear transfer (scNT) embryos suffer from damage caused by micro-operation (manipulation) and inefficient genome reprograming that hinder their normal development at different levels and in distinct ways. These two effects are inseparable in the nature of the scNT embryo, although methods to separately measure them are needed to improve scNT technology and evaluate incoming reprogramming tools. As an attempt to meet these demands, we made bovine sham nuclear-transfer (shNT) blastocysts, special embryos made with a standard nuclear-transfer procedure at the zygote stage, while retaining an intact genome. We compared their transcriptomes with those of other blastocysts derived by  Somatic cell nuclear transfer (scNT) is a powerful technique to produce genetically modified animals that can be used as industrial bioreactors or models for biomedical research  and scNT embryos, we expect to obtain valuable information on variable sets of differentially expressed genes (DEGs) involving those in association with oocyte manipulation, zygote manipulation, or the donor genome. These lists of DEGs will serve as valuable resources to help advance our understanding of reprogramming in scNT embryos and our ability to control genome reprogramming for more efficient cloning.A special kind of cloned embryo derived from a sham nuclear transfer (sham NT) can be taken as alternative. It can be prepared by manipulating a bovine zygote, not a mature oocyte to remove a part of its cytoplasm, while keeping its genomic material intact in the pronucleus. Early studies reported the developmental arrest of cloned embryos prepared from zygote enucleation in mice  supplemented with 10% (v/v) fetal bovine serum , 10 μg/ml FSH-P , 0.6 mM cysteine, 0.2 mM sodium pyruvate, and 1 μg/ml estradiol-17β together. To generate IVF embryos, the matured oocytes were fertilized by incubating with 2 × 106 sperms/ml in fertilization medium at 38.5°C in 5% CO2 for 20–22 h  for 5 min, followed by treatment of 2.5 mM DMAP in CR1aa culture media supplemented with 0.3% BSA for 3.5 h. Blastocysts were generated 7 days post-IVF or -NT. The quality of each blastocyst was assessed by Hoechst staining, and only high-quality ones at mid blastocyst stage possessing 60–80 blastomeres were chosen for transcriptomic analysis.2 in air  according to the manufacturer's recommendation. The ultra-pure mRNAs were reverse transcribed using a custom pico-profiling adapter as previously described  and our previous publication (PMID: 28443134).A total of 35 RNA-seq libraries  were constructed using TruSeq DNA Sample Preparation kit (Illumina)  from the HiSeq2000 system were preprocessed to remove NGS artifacts such as adapter sequences and low quality bases by “trimgalore,” and the trimmed reads were aligned on the reference genome (UMD3.1) using TopHat  among the three groups. From principal component analysis (PCA), we found that the shNTs separately grouped from the IVFs and the scNTs, while the latter two associated together  and the next highest from IVF-shNT (0.812) and shNT-scNT matches  between shNTs and IVFs .” In contrast, those DEGs present in the IVF-scNT DEG set but absent from the IVF-shNT set may be donor genome-dependent. These present as a signature of inefficient reprogramming, which we call the “donor genome-associated DEGs (DADs).”p < 0.05) were identified as MADs. Heat-map analysis shows a collection of 261 overrepresented and 300 underrepresented MADs, in which the levels differed from those of the IVFs , “translation” (p = 2.55 × 10−3), and “transcription” (p = 0.031) are shown as expression heat-maps with gene symbols in Figure As shown in the Venn diagram in Figure s Figure . GO enrip = 0.027; Figure p = 0.014). Further information on the MADs and DADs is presented in Supplementary File The DADs outnumbered the MADs Figure . Seven hn = 2,921, p < 0.05) were found as shNT-specific DEGs . Under these new criteria, 889 shNT-high and 854 shNT-low DEGs were identified , “protein folding” (p = 1.58 × 10−5), and “protein stabilization” (p = 2.98 × 10−3). DEGs implicated in “rRNA processing” (p = 7.24 × 10−5) and “double-strand break repair” (p = 2.12 × 10−3) functions were characterized by higher expression levels in the shNTs. “Apoptosis pathway”  and “I-κB kinase- and NF-κB signaling”  functions were also detected with statistical significance.A relatively large number of genes  and the corresponding genes had 3.5-fold higher expression compared to those in IVFs  during ER stress . However, the expression level of ATF6B, which functions to reduce expression of ER stress proteins, was not different (p = 0.276) between shNTs and IVFs  to Golgi vesicle-mediated transport” and “intracellular protein transport” Figure , Table 2s Figure . In linep > 0.822, paired sample t-test; Figure KDM5A, KDM5B, SETDB1, SUV420H1, HDAC8, DNMT3A, and KDM2A). This suggests that shNTs treat heterochromatin favorably. In addition, several protein arginine methyltransferase genes  and sirtuin genes (SIRT4 and SIRT7) were found in that section of the heat-map. Interestingly, of the seven PRMTs that were expressed in cow blastocysts, six were overrepresented in shNTs  development  or underrepresented  in shNTs compared to that in IVFs  were detected even at a lower stringency (p < 0.05). Thus, GATA2 and PITPNC1 were shared as DEGs in common between shNTs and scNTs. Of the four scNT DEGs, the latter three coincided with those previously identified as TE-related DEGs in scNT blastocysts  between IVFs and shNTs, but not in other comparisons  and KLF5 expression levels were significantly reduced in shNTs but not in scNTs  compared to that in the IVFs, about 75% (24/32) were underrepresented, while only 43% (16/37) of epi-driver genes and 34% (60/178) of chromosome 1 (chr1) genes were underrepresented  underrepresented in shNTs. Because of the small number of DEGs, a comparison of gene expression between scNTs and shNTs was not possible. Meanwhile, there were some stemness genes, such as BMPR1A (p = 2.43 × 10−8) and DPPA3 (p = 1.61 × 10−4), that were significantly overrepresented in shNTs  and summarized in Figure n = 1873) from the IVF-shNT comparison exceeded the number of DEGs (n = 439) from the IVF-scNT comparison  and half scNT  characteristics, we had initially thought that the nature of shNTs and their transcriptomes would be midway between those of IVFs and scNTs. This speculation was proven incorrect. shNTs did not resemble IVFs or scNTs as much as we had expected and appeared as a unique entity. Looking at the results, there are also other aspects to be considered. The manipulation experience of shNTs was unlike IVFs, and they did not undergo nuclear reprogramming of the somatic cell genome as did the scNTs. Nevertheless, the inclusion of shNTs in the otherwise simple comparison of transcriptomes of IVFs and scNTs enabled the identification of valuable DEG resources, such as MADs, DADs, and zMADs. Closer inspection of these defined DEGs and related GO terms will significantly improve our understanding of the cellular and molecular events occurring in scNT embryos and our ability to control genome reprogramming for efficient cloning. Lastly, the irrelevance of zygotes as an NT recipient in cloning has been suggested experimentally (McGrath and Solter, BM: performed all the molecular works and data analysis, and wrote the paper; JP: performed all the embryo manipulations including SCNT and sham NT; Y-KK: designed and supervised the project, and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


How weight perception influences weight-related knowledge, attitudes, and behaviors in Chinese children is unknown. We investigated self-perception of body weight and its correlates, and analyzed the relationship between weight perception and weight-related knowledge, attitudes, and behaviors in children in Guangzhou, China.We assessed self-reported weight perception, weight-related knowledge, attitudes, and behaviors in 3752 children aged 7–12 years. Underweight or overweight was defined using the Chinese criteria based on body mass index (BMI). Binary logistic regression analyses were performed to assess correlates of weight underestimation.In total, 27.3% of children underestimated and 6.7% overestimated their weight status. Weight underestimation was common among normal-weight (34.1%) and overweight children (25.3%). Older age, female sex, and child BMI z-score were negatively associated with normal-weight children's underestimation, whereas older age, paternal obesity, maternal obesity, and child BMI z-score were negatively associated with overweight children's underestimation. Correct answers on weight-related knowledge questions ranged from 81.5% to 98.6% and did not differ by weight perception within BMI categories. Although negative perceivers  had a higher intention to change weight, they behaved more unhealthily on fruit intake, breakfast, screen time, and daily moderate-to-vigorous physical activities time than counterparts.Weight underestimation was prevalent in normal-weight and overweight children in Guangzhou. Negative perceivers had stronger willingness to change weight but tended to behave more unhealthily on certain behaviors than positive perceivers. Childhood obesity interventions should incorporate health education and practical support to promote healthy eating and physical activity. •Over one third of children misperceived their weight status in Guangzhou, China.•Underestimation of body weight was much more prevalent than overestimation.•Underestimation was common in younger children, girls, and those with obese parents.•Children perceived as underweight/overweight had a higher intention to change weight.•Children perceived as normal weight behaved more healthily on certain behaviors. The prevalence of childhood overweight and obesity has increased dramatically worldwide in recent years.Although accurate self-perception of weight status is thought to be an important aspect of successful childhood obesity prevention and management, weight misperception has been repeatedly documented,13Children's views about their weight status might influence their dietary intake and physical activity. Overweight or obese children who accurately perceived their weight status tended to report higher levels of weight loss behaviors, such as exercising to lose weight or dieting, than the misperceivers.China has the largest population of children in the world, and self-perception of weight status and its correlates in Chinese children may differ from those found in the West due to different socio-cultural backgrounds. A few studies have investigated the self-perception of weight status in Chinese younger-aged populations.NCT02343588).The cross-sectional data were retrieved from the baseline survey of a school-based obesity intervention program among Chinese children .A multistage random cluster sampling method was used to recruit the subjects in Guangzhou, a city in southern China. In the first stage, we selected four urban districts from the 10 districts of Guangzhou using the judgment sampling method. In the second stage, one or two primary schools were randomly selected from each of the four districts. Probability proportional to size sampling method was adopted in this stage, and a total of seven schools were chosen. In the third stage, all students in grades 2–5 were sampled from the selected schools. The principals were sent invitation letters and the research details. With the principals' permission, all students in grades 2–5 were invited to participate. Children were excluded if they were aged <7 years, or they were diagnosed with visceral diseases, abnormal growth and development, physical abnormality, or obesity caused by endocrine diseases or drugs. The response rates for schools and students were 100% (7/7 schools) and 78.4% (4942/6300 students), respectively. All individuals were asked to take a physical examination and answer a questionnaire. Participants who had no record of questionnaire (n = 690) or physical examination (n = 473), or had missing information about their age (n = 1), body mass index , or self-reported weight perception (n = 14) were excluded, leaving 3752 children in the final analysis.Student and parent questionnaires were administered to the whole class by well-trained investigators before the physical examination. One teacher supervised the survey procedure in each class. Students in grades 4–5 filled in the student questionnaire on their own in class, whereas students in grades 2–3 finished the student questionnaire under parents' guidance at home. Parent questionnaires were delivered by children to their parents and returned in 3 days. All questionnaires were checked for integrity and logicality upon return.Demographic characteristics included children's date of birth, gender, number of siblings, parents' educational level, parents' occupations, and monthly household income. Parents self-reported their height (cm) and weight (kg). Potential correlates of children's weight underestimation were examined among these factors.Children's self-perception of weight status was assessed by asking: “How do you feel about your current weight status?”, with five response options: “very thin”, “rather thin”, “average”, “rather fat”, and “very fat”. The responses were categorized into: perceived underweight (“very thin” and “rather thin”), normal weight (“average”), and overweight (“very fat” and “rather fat”). Children's self-perception was compared with actual weight status to assess underestimation, accurate estimation, and overestimation of body weight.2) was calculated by dividing weight (kg) by height (m) squared. Underweight was defined based on the age- and gender-specific BMI cutoffs proclaimed by the National Health and Family Planning Commission of the People's Republic of China.2, whereas obesity was defined as BMI ≥ 28 kg/m2, in accordance with criteria for Chinese adults.Children's height (cm) and weight (kg) were measured by trained technicians following a standardized procedure. Height was measured using metal column height-measuring stands (200 cm long with 0.1 cm precision), and weight was measured using lever scales (weights to 120 kg with 0.1 kg precision). BMI  “To what extent do you think obesity is bad for health?” (2) “Are you satisfied with your weight status?” (3) “Do you want to change your present weight status?” and (4) “Do you believe you can achieve an ideal weight status through effort?”. The responses were: “little”, “rather little”, “not sure”, “rather greatly”, or “greatly” for the first question and “no”, “rather no”, “not sure”, “rather yes”, or “yes” for the other three questions.Weight-related behaviors included 10 items regarding dietary intake, physical activity, and screen time. Children reported frequency (days) and amount of fruit, vegetable, meat products, and sugar-sweetened beverages (SSBs) intake over the preceding 7 days. Average daily intakes of these foods were calculated as follows: average daily intake = [days × (amount in each of those days)]/7. Daily intake of fruit, vegetable, and meat products were then dichotomized according to dietary guidelines for Chinese children (2007).26P < 0.05 indicated statistical significance.Calculations were conducted using IBM SPSS software version 19.0 . Continuous variables are presented as mean (standard deviation), whereas categorical variables are presented as number (percentage). For children's actual weight status, overweight and obesity were combined into a single category (overweight). Differences of continuous and categorical variables between different perceptions were evaluated using one-way ANOVA and Pearson Chi-Square tests, respectively. Binary logistic regression analyses were performed to assess the correlates of weight underestimation among normal-weight and overweight children. Potential correlates included child age, gender, and number of siblings; parents' educational level, occupations, monthly household income, and weight status; and child age- and gender-specific BMI z-score. In the regression analyses of normal-weight children, the overestimators were excluded because of small numbers . A two-sided P < 0.001). Underestimation was also prevalent among overweight children (25.3%). P < 0.05). In normal-weight children, overestimators had the highest knowledge score (P < 0.01).P < 0.001).P < 0.05 and P = 0.056, respectively). Normal-weight children who overestimated themselves had a lower reaching rate of breakfast and screen time than their counterparts (P < 0.05). In overweight children, the accurate perceivers had a higher reaching rate of vegetable intake but a lower reaching rate of MVPA than the underestimators (P < 0.05).Only 25.6% and 19.9% of participants met the recommendation for fruit (≥2 servings/day) and vegetable consumption (≥3 servings/day), respectively. Less than half did not consume SSBs (43.9%) and high-energy snacks (33.1%). A majority of children met the recommendation for screen time (84.0%), but only one-third reached the recommendation for MVPA (35.2%). We found no statistical difference of the reaching rates of most behaviors between different perceptions. However, underweight children with accurate perception had a lower reaching rate of fruit intake and a critical lower reaching rate of MVPA than the overestimators  misperceived their body weight, especially among normal-weight individuals. Nearly 41% of normal-weight children misperceived themselves as thin or fat, while about 25% of overweight children considered their weight as average or thin. Results were similar to observations reported by Zhao et al,Factors associated with weight misperception are not well-understood among Chinese children. In the present study, child age, gender, BMI z-score, and parental obesity were identified as important correlates of weight underestimation. Younger children were more likely to underestimate their body weight, which was in line with somePrevious studies have linked weight perception to weight control behaviors in children,In agreement with other studies,Like previous studies,This study has some limitations, due to which the findings must be interpreted carefully. First, the cross-sectional design precluded the identification of casual associations. Second, a substantial proportion of children did not participate in the study or were excluded from the final analysis, which might result in selection bias in this study. However, no difference was found in the distribution of age or gender between the participants and non-participants, indicating that selection bias stemming from non-participation might be small. Third, the children in grades 2–3 completed the student questionnaire under parents' guidance, so their response might be influenced by parents. Furthermore, we did not take into account other possible correlates of weight perception, such as exposure to social media,This study showed that underestimation of weight status was common among normal-weight and overweight children in Guangzhou, China. Children who negatively perceived themselves had stronger weight dissatisfaction and willingness to change their body weight. However, they tended to behave more unhealthily on certain weight-related behaviors than positive perceivers. Intervention for childhood obesity should incorporate education about accurate identification of actual weight status and practical strategies for promoting healthy eating and physical activity.None declared.


AbstractEntomologie, ou histoire naturelle des insectes was issued in two parts, one probably in 1795 and the second in 1800. All new taxa made available in this work have previously been dated 1795 in the literature. A list of new species described in 1795 and a list of those that have to be dated 1800 are appended. The genus Necrobia should be credited to Latreille, 1797, not Olivier, 1795.Despite the title page is dated 1795, the fourth volume of Olivier’s  PageBreakOlivier’s connection with the Entomologie ou histoire naturelle des insectes. For this project, Olivier travelled to Britain and the Netherlands to describe the insects and have them illustrated. At about the same time, Olivier was approached to contribute to the natural history of the insects for Charles-Joseph Panckoucke’s  Encyclopédie méthodique, one of the major scientific publication achievements of all time . In 1814, he went to his native town to rest and on his way back stopped at Lyon where he was found dead, from an aortic aneurism, in his bed on October 1. He was 58 years old. Olivier was a close friend to Johan Christian Fabricius and a patron PageBreakto Pierre André Latreille particularly during the French Revolution. This account of Olivier’s life is derived from Cuvier (1818) and Born in the commune Les Arcs , a small village near Toulon in the Var department, southeastern France, on 19 January 1756, Guillaume-Antoine Olivier  issued in 30 livraisons (One of the two major publications of Olivier is his vraisons .Prionus (41 pp.); No 67, Capricorne / Cerambix [Cerambyx] (132 pp.); No 68, Saperde / Saperda (41 pp.); No 69, Stencore / Stenocorus (30 pp.); No 70, Callidie / Callidium (72 pp.); No 71, Spondyle / Spondylis (4 pp.); No 72, Calope / Calopus (4 pp.); No 73, Lepture / Leptura (34 pp.); No 74, Nécydale / Necydalis (10 pp.); No 74bis, Cucuje / Cucujus (10 pp.); No 75, Donacie / Donacia (12 pp.); No 75bis, Lupère / Luperus (4 pp.); No 76, Clairon / Clerus (18 pp.); No 76bis, Nécrobie / Necrobia (6 pp.); No 77, Bostriche / Bostrichus (18 pp.); No 78, Scolyte / Scolytus (14 pp.); No 79, Bruche / Bruchus (24 pp.); No 80, Macrocéphale / Macrocephalus (16 pp.). Its title page is dated 1795  in the Gazette Nationale ou Le Moniteur Universel and in the Fructidor an VIII (= 18 August–22 September 1800) issue of the Journal Général de la Littérature de France, both journals recording new books published in France. The citation mentioned that the continuation of Olivier’s work was postponed because of the six-year voyage of the author in the Orient, and that the present livraison contains about three-quarters of the fourth volume, including the explanatory text of 56 plates. There is other evidence that part of Olivier’s fourth volume of his Entomologie was issued after 1795. The work contains five explicit references to Fabricius’ Supplementum entomologiae systematicae which was published in 1798: “Lamia bicincta. Fab. suppl. Ent. Syst. pag. 145” under Capricorne continu , “Lamia marmorata. Fab. Suppl. Ent. Syst. pag. 144. no.1” under Capricorne bigarré , “Cucujus rufus. Fab. Suppl. Ent. Syst. emend. pag. 123” under Cucuje fauve , “Lema flavipes. Fab. Suppl. Ent. Syst. pag. 93. no. 21” under Lupère flavipède , and “Anthribus niveirostris rostro latissimo plano elytrorumque apicibus anoque albis. Fab. Ent. Syst. Suppl. pag. 160” under Macrocéphale nivéirostre . In addition, on page 121 , Olivier mentioned “Ce genre ayant été imprimé pendant mon voyage dans les contrées orientales, on a omis quelques descriptions que je m’empresse de donner ici” [This genus was printed during my voyage to the oriental region and some descriptions were omitted which I hasten to present here]. As mentioned previously, Olivier returned from his trip in December 1798. Finally, Vorrede, dated 15 April 1800, of the first volume of his German translation of Olivier’s work “Entomologie” that the fourth volume of the series was not yet published [Der vierte noch nicht erschienene Band wird wahrscheinlich der Werk schliessen].The fourth volume treated 18 genera: No 66, Prione / ambyx] 13 pp.; No PageBreaklivraison 23 since the recording journals cited above simply mentioned that it included about three-quarters of the fourth volume? Cerambix, while those on the following pages have the name correctly spelled Cerambyx. This is circumstantial evidence that a break occurred in the printing of the text. So, as far as I am concerned all new species described from page 81 (No 67) onwards should be dated 1800. A list of them is given in Appendix One problematic question remains. What exactly is the content of Necrobia has been attributed to “Olivier 1795” from this work by almost all authors seen. The name is so entered in the Official List of Generic Names in Zoology following Opinion 604  wrote under Necrobia “Le cit. Latreille est le premier qui ait senti que ces insectes [Clerus] devoient être séparés des uns et des autres, et former un genre particulier, auquel il a donné le nom de Nécrobie...” [Latreille is the first that saw the necessity to separate these insects (referring to those of the genus Clerus) and formed a peculiar genus to which he gave the name Necrobie 1]. Necrobia and made it available. This was recognized by Précis des caractères génériques des insectes while Necrobia “Olivier 1795 [?], Entomologie 4, no. 76 (bis); Latreille 1796, Préc. Car. Ins., 35.” Although Latreille described the genus, he did not include any species under it. The nominal species first subsequently and expressly included in the genus Necrobia are the three cited by N.violacea, N.rufipes, and N.ruficollis. In Opinion 604 , 23 for Cerambix / Cerambyx (No 67), 4 for Saperda (No 68), 3 for Stenocorus (No 69), 8 for Callidium (No 70), 1 for Spondylis and Calopus (Nos 71 and 72); 4 for Leptura (No 73); 1 for Necydalis (No 74); 1 for Cucujus (No 74bis); 1 for Donacia and 1 for Donacia and Luperus (Nos 75 and 75bis); 1 for Clerus and 1 for Clerus and Necrobia (Nos 76 and 76bis); 3 for Bostrichus (No 77); 2 for Scolytus (No 78); 3 for Bruchus (No 79); 2 for Macrocephalus (no 80). There are no scientific names on the plates2, except for the respective genus at the top. At the recommendation of the editor, these plates were usually placed in the eighth volume (the second of the plates) of the series. The title page is dated 1808 but it is obvious that most, if not PageBreakall, of the plates were issued prior to this date. For example, Prionus), 1–12, 14–19 (Cerambyx), 1 and 2 (Saperda), 2 (Stenocorus), 1–7 , 1 and 2 (Leptura).There are 72 plates associated with the genera treated in volume 4 of Olivier’s livraison in which this part was published nor did I find a date of publication in a recording journal for livraison 22 of the work, which could deal with the first part. Livraison 21 was noticed in 1796 in the third volume of the second year of the Magasin Encyclopédique ou Journal des Sciences, des Lettres et des Arts (p. 558) but no indication was provided as to its content. What is puzzling is that the first author I found giving reference to any of the species included in the entire fourth volume is Coleoptera known at the time, did not include any of the new species described in Olivier’s entire fourth volume of his Entomologie. Since the title page is dated 1795  was actually published. I have been unable to find the PageBreak


Scientific Reports 10.1038/s41598-017-12981-5, published online 03 October 2017Correction to: In this Article, some of the wording in the Results section is ambiguous and should be corrected as follows:18,29, the Purkinje isoform Dp427p was virtually absent in the brain throughout development, with expression levels even lower than muscle dystrophin Dp427m.”“In contrast to previous reportsshould read:18, 29, the Purkinje isoform Dp427p was virtually absent in human brain throughout development, with expression levels even lower than muscle dystrophin Dp427m.”“In contrast to previous reports on mouse brainIn addition30,31, Dp427p was expressed in the mouse cerebellum and not in the mouse cerebral cortex.”“Yet in line with previous studiesshould read:29,30,31, Dp427p was expressed in the mouse cerebellum and not in the mouse cerebral cortex.”“Yet in line with previous studies


Little is known about oral health related to electronic-cigarette (EC) use, even though EC use is increasing rapidly. The aim of this study is to assess the relationship between EC use and oral health, including ‘gingival pain and/or bleeding’, ‘tongue and/or inside-cheek pain’, and ‘cracked or broken teeth’ among adolescents.A total of 65,528 students in 2016 were included in this cross-sectional study.For EC use, 0.5% (n = 297) students were daily users, 1.9% (n = 1259) were ‘1 to 29 days past month users’, and 5.9% (n = 3848) were former users. Overall, 18.5% students reported they had experienced ‘gingival pain and/or bleeding’, 11.0% reported ‘tongue and/or inside-cheek pain’, and 11.4% reported a ‘cracked or broken tooth’ within the past 12 months. When comparing ‘daily EC users’, ‘1 to 29 days past month EC users’, and ‘former EC users’ with ‘never EC users’, the adjusted ORs for ‘cracked or broken tooth’ were 1.65 (95% CI: 1.19–2.27), 1.26 (95% CI: 1.06–1.51), and 1.16 (95% CI: 1.04–1.30), respectively. Comparing ‘daily EC users’ with ‘never EC users’, the adjusted OR for ‘tongue and/or inside-cheek pain’ was 1.54 (1.05–2.26). However, EC use among adolescents was not associated with ‘gingival pain and/or bleeding’ when adjusted for the potential confounders.Based on the results, the odds of cracked or broken teeth among daily, ‘1 to 29 days past month’, and former EC users were significantly higher than those among never EC users. The odds of tongue and/or inside-cheek pain among daily EC users were significantly higher than those among never EC users. In conclusion, the results suggest that daily EC use among adolescents may be a risk factor for cracked or broken teeth and tongue and/or inside-cheek pain. Electronic cigarettes (EC) are battery-powered electronic devices, which aerosolize liquid that contains nicotine, humectants, and flavors . EC use alkaline phosphatase activity significantly decreased in nicotine-treated human dental pulp cells, and mineralized nodule formation was also inhibited [Oral disease is one of the most common public health issues worldwide and constitutes a significant socio-economic burden . Oral henhibited . Namely,nhibited . Moreovenhibited . A previnhibited .Even though EC use is increasing rapidly, little is known about oral health related to EC use. There has never been a representative population study assessing the association of EC use with oral health among adolescents or among adults. Therefore, the aim of this study was to assess the association between EC use and oral symptoms that includes ‘gingival pain and/or bleeding’, ‘tongue and/or inside-cheek pain’, and ‘cracked or broken teeth’ among adolescents in South Korea.th to 12th school grade students. The population was sampled from 400 middle and 400 high schools. Out of 67,983 students, 65,528 students responded, an overall response rate of 96.4% from 798 schools. Out of 33,251 middle school students, 32,219 students responded, an overall response rate of 96.9%. Out of 34,732 high school students, 33,309 students responded, an overall response rate of 95.9%.The Twelfth Korean Youth Risk Behavior Web-based Survey (KYRBWS) was approved by an institutional review board of the Korean Center for disease Control and Prevention (2014-06EXP-02-P-A). This study was reviewed by the Institutional Review Board of Hanyang Women’s University and complied with ethical requirements (AN01-201504-HR-010-01). Data used was from the Twelfth KYRBWS, 2016, Ministry of Education, Ministry of Health and Welfare, and Korean Center for Disease Control and Prevention , 20. TheOral symptoms were defined as an outcome on a student’s self-report. Students were asked the question: “Within the past 12 months, have you experienced gingival pain and/or bleeding?” (yes/no). Students were also asked the question: “Within the past 12 months, have you experienced tongue and/or inside-cheek pain?” (yes/no). Students were lastly asked the question: “Within the past 12 months, have you experienced a cracked or broken tooth?” (yes/no).th– 12th grade’, ‘7th -9th grade’, ‘1st - 6th grade’, and ‘<1st grade.’ We also assessed the reasons for using ECs applying the questions “What is the main reason for using ECs?” The response options were ‘it seems to be healthier than CCs’, ‘to quit smoking CCs’, ‘to use them indoors’, ‘it is easier to get ECs than CCs’, ‘good taste’, ‘good flavors’, ‘doesn’t smell bad’, ‘curiosity’, and ‘other.’ We assessed sources from which EC users acquire EC-liquids using the questions “How do you usually get EC-liquids?” The response options were ‘from friends’, ‘purchase from an EC shop’, ‘purchase through the internet’, ‘other’, and ‘only purchase nicotine free EC-liquids.’ The response options were also re-classified into two groups: ‘nicotine-free EC user in the past 30 days’ and ‘nicotine-containing EC user in the past 30 days.’ Unless explicitly specified, all EC fluids in Korea contain nicotine due to their popularity. Also, only ECs which contain nicotine have been classified as tobacco products by the Ministry of Finance (MOF) since 2010 in Korea [EC use was defined by the question, “Have you ever used an EC in your life, even one or two puffs?” (yes/no). A no answer was categorized as ‘never user.’ Respondents who answered in the positive were asked the next question: “During the past 30 days, on how many days have you used ECs?” Respondents answering ‘none’ were categorized as a ‘former user.’ Positive responses were re-categorized into two groups: ‘1 to 29 days past month user: 1 to 29 days use’ and ‘daily user: all 30 days use.’ A report of the Surgeon General on smoking assessed current CC smoking prevalence for youth and young adults based on having smoked all or part of at least one cigarette in the past 30 days . Similarin Korea . Length etc.).” Overweight status was calculated using the body mass index . Overweight status was classified into two groups: ‘BMI ≥ 25 kg/m2’ and ‘BMI < 25 kg/m2.’ CC smoking was assessed by the question, “Have you ever smoked a CC in your life, even one or two puffs?” (yes/no). A no answer was classified as ‘never smoker.’ Respondents who answered in the affirmative were asked a follow-up question: “During the past 30 days, on how many days have you smoked CCs?” Respondents answering ‘none’ were classified as a ‘former smoker.’ Affirmative responses were re-classified into two groups: ‘1 to 29 days past month smoker: 1 to 29 days use’ and ‘daily smoker: all 30 days use.’ Attempt to quit smoking was assessed by the question: “During the past 12 months, have you ever attempted to quit CC smoking?” (yes/no). A yes answer was classified as ‘attempt to quit smoking.’ Second hand smoking at home was assessed by the question: “During the past week, on how many days were you exposed to second hand smoke at home?” All response options are shown in detail in Age, gender, school grade, city size, economic status, stress, overweight status, carbonated drinks, alcohol, vigorous sports activity, CC smoking, attempt to quit CC smoking, and second hand smoke at home were assessed. The questions were as follows: For economic status, “What is your household’s economic status?” The response options were ‘very high’, ‘high’, ‘middle’, ‘low’, and ‘very low.’ Stress was also investigated , as a stThe data were described by descriptive statistics and analyzed using binary-logistic regression analyses (IBM SPSS Version 23.0). First, frequency analyses were conducted to assess the rate of oral health outcome and EC use, including sociodemographic variables. Second, to assess the unadjusted odds ratios (ORs) indicating association between EC use and oral health outcome, we applied binary-logistic regression analyses. Third, to assess the adjusted ORs, we applied multiple logistic regression analyses including the potential confounders; age, gender, school grade, economic status, and city size. Third, to assess the final adjusted ORs, we performed multiple logistic regression analyses including the above in addition to carbonated drink, overweight status, stress, alcohol, vigorous sports activity, CC smoking, attempt to quit smoking, and second hand smoking at home. Logical errors  and outlier values , are treated as missing values according to the statistical analysis guidelines of the KYRBWS. The proportion of respondents treated as missing value was below 2%. Missing data were handled by using pairwise deletion.The frequencies of the adolescent characteristics are shown in st grade and 7.5% (n = 401) had experienced ECs between 1st and 6th grade. With relation to the main reason for EC use, ‘to use ECs indoors’ was the most common reason among daily EC users  of students were daily EC users, 1.9% (n = 1259) were ‘1 to 29 days past month EC users’, and 5.9% (n = 3848) were former EC users during the past month. About 91.8% (n = 60124) were never EC users. For first EC experience time, amongst ever EC users, 2.9% (n = 159) had experienced ECs before 1EC users .As a reason, ‘curiosity’ tapered off as frequency of use increased. ‘Curiosity’ accounted for 28% of former EC users, 11% among ‘1 to 29 days past month EC users’, and 5% among daily EC users.Among ‘1 to 29 days past month EC users’, 35% (n = 443) reported friends as a primary source . Among dThe ORs for CC smoking were used for a preliminary comparison. The adjusted ORs indicating association between CC smoking and oral symptoms among adolescents are shown in For ‘cracked and/or broken tooth,’ comparing ‘daily CC smoker’ with ‘never CC smoker’, the adjusted OR was 1.33 (1.08–1.63). For ‘cracked and/or broken tooth,’ comparing ‘former CC smoker’ with ‘never CC smoker’, the adjusted OR was 1.25 (1.14–1.37). Other oral health measures were statistically insignificant or showed statistically significant differences with ORs close to 1.0.The ORs indicating association between EC use and ‘gingival pain and/or bleeding’ among adolescents are shown in 2-test were produced in Model 2  and in Model 3 .Comparing ‘daily EC users’ with ‘never EC users’, the adjusted OR was 1.41 (95% CI: 1.05–1.90) in Model 2 and 1.00 (95% CI: 0.72–1.41) in Model 3. Good fits to the data according to the Hosmer-Lemeshow χThe ORs indicating association between EC use and ‘tongue and/or inside-cheek pain’ among adolescents are shown in 2-test were produced in Model 2  and in Model 3 .Comparing ‘daily EC users’ with ‘never EC users’, the adjusted OR was 2.04 (95% CI: 1.48–2.81) in Model 2 and 1.54 (95% CI: 1.05–2.26) in Model 3. Namely, when adjusting for the potential confounders, the odds of ‘tongue and/or inside-cheek pain’ among daily EC users was over 50% higher (p = 0.028) than that among never EC users. Further, good fits to the data according to the Hosmer-Lemeshow χThe ORs indicating association between EC use and ‘cracked or broken tooth’ among adolescents are shown in 2-test were produced in Model 2  and in Model 3 . Just for supporting information, using the weighted no. and the weighted %, we assessed multivariable adjusted odds ratios showing association between EC use and oral symptoms among adolescents  in Model 2 and 1.65 (95% CI: 1.19–2.27) in Model 3. Finally, in Model 3, the odds of ‘cracked or broken tooth’ among daily EC users was over 60% higher (p = 0.003) than that among never EC users. In addition, the odds of ‘cracked or broken tooth’ among former EC users  and ‘1 to 29 days past month’ EC users  were significantly higher than those among never EC users. Furthermore, good fits to the data according to the Hosmer-Lemeshow χlescents . The weiThe adjusted ORs indicating association between nicotine-free or nicotine-containing EC use and oral symptoms among adolescents are shown in 2-test for this analysis the model provided a good fit to the data . For ‘tongue and/or inside-cheek pain,’ the adjusted OR was 1.56 (1.07–2.28) when comparing ‘nicotine-free EC users in the past 30 days’ with ‘never EC users.’ According to the Hosmer-Lemeshow χ2-test for this analysis the model provided a good fit to the data . For ‘gingival pain and/or bleeding,’ there were no significant associations between EC use according to nicotine content and oral symptoms. Therefore, of the studied oral conditions, ‘cracked and/or broken tooth’ was significantly associated with the use of nicotine-containing ECs, and ‘tongue and/or inside-cheek pain’ was significantly associated with the use of nicotine-free ECs.For ‘cracked and/or broken tooth,’ the adjusted OR was 1.37 (1.15–1.63) when comparing ‘nicotine-containing EC users in the past 30 days’ with ‘never EC users.’ According to the Hosmer-Lemeshow χThis study evaluated the association between EC use and oral symptoms among adolescents. It found that EC use was associated with a significantly increased chance of either cracked or broken teeth or tongue and/or inside-cheek pain among adolescents. However, the results did not find an association between EC use and gingival pain and/or bleeding among adolescents.alkaline phosphatase activity were significantly decreased in nicotine-treated human dental pulp cells of smokers, and mineralized nodule formation was also inhibited by nicotine in human dental pulp cells [Streptococcus mutans biofilm formation and biofilm metabolic activity, increasing the development of caries [Nicotine may be a contributing factor in cases of cracked or broken teeth. This hypothesis is supported by the significant association between nicotine-containing EC users and cracked or broken teeth. This hypothesis is also supported by the report that the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and lp cells . Namely,lp cells . Dental lp cells . Nicotinf caries . Acid prf caries . EC inhaf caries . In addif caries . Xerostof caries . Xerostof caries . In genef caries . The hypf caries . Lead, df caries . In addif caries .Regardless of nicotine, the thermal degradation byproducts formed from glycerin, propylene glycol, and flavorings during vapor generation may be a cause of tongue and/or inside-cheek pain. This hypothesis is supported by the additional result of this study that tongue and/or inside-cheek pain was significantly associated with the use of nicotine-free ECs. Also, this hypothesis is endorsed by an in-vitro experimental study, conducted to explore the effects of ECs on the oropharynx that, independently of nicotine, ECs induced DNA strand breaks and cell death . AldehydConversely, we were not able to identify the association between EC use and ‘gingival pain and/or bleeding’ among adolescents in this study. Our results for ‘gingival pain and/or bleeding’ do not coincide with the research that ECs with flavorings cause increased oxidative/carbonyl stress and inflammatory cytokine release in human periodontal ligament fibroblasts . BesidesGiven the cross-sectional study design, these findings need to be interpreted with caution. The results cannot establish a causal relationship between EC use and oral health. Furthermore, due to a reliance on self-reported oral symptoms without oral examination, we should admit recall bias. Thus, dental surgeon’s medical records for oral health including cracked or broken teeth and tongue and/or inside-cheek pain need to be evaluated to confirm the association between EC use and oral health. Also, diet, oral hygiene status, oral hygiene regimen and overjet of teeth were not taken into consideration in this study, even though diet and oral hygiene could be confounding factors in causing caries, broken teeth, and bleeding or pain. In addition, the strength of EC use, for example nicotine dose per day of EC use, was not considered. As some EC users started because they perceived it as less harmful or helpful when quitting CCs. These health-oriented EC users may have experienced complications  as a result of smoking CCs, before trying ECs. At the same time, those users who only smoke CCs but not ECs may have been less susceptible to complications from CC use. Another limitation is the difference in referenced timeframes between ‘EC use during the past 30 days’ and ‘oral health within the last 12 months’, which do not exactly represent current health status. However, this study is supplemented by detailed information on EC users, such as exposure time from first experience with EC and the main reason for EC use by type of EC user. The numbers of EC users in the study, in particular, daily users, were relatively small. The statistical reliability of the study would have been better if the sample size of EC users were larger. In future EC research, the main cause of oral health problems needs to be identified by oral examination. Despite the potential limitations, it is notable that this is the first large representative population study to evaluate the association of EC use with oral health, using daily EC use and recent data from 2016.Based on the results, after adjusting for potential confounders, EC use was associated with a significantly increased chance of either cracked or broken teeth, or tongue and/or inside-cheek pain among adolescents. The odds of cracked or broken teeth among daily, ‘1 to 29 days past month’, and former EC users were significantly higher than those among never EC users. The odds of tongue and/or inside-cheek pain among daily EC users were significantly higher than those among never EC users. In conclusion, the results suggest that EC use among adolescents may be a risk factor for tongue and/or inside-cheek pain and cracked or broken teeth.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Pparγ, Acot1/2, Fgf21, Pdk4 and Inhbb were upregulated in the liver of the offspring in DHEA group compared with those in controls, whereas Cyp17a1 expression was significantly decreased. However, the expression of these genes was not detected in male offspring. Our results show that female offspring in DHEA group exhibit perturbed growth and glucolipid metabolism that were not observed in male offspring.Polycystic ovary syndrome (PCOS) is a common reproductive disorder that has many characteristic features including hyperandrogenemia, insulin resistance and obesity, which may have significant implications for pregnancy outcomes and long-term health of women. Daughters born to PCOS mothers constitute a high-risk group for metabolic and reproductive derangements, but no report has described potential growth and metabolic risk factors for such female offspring. Hence, we used a mouse model of dehydroepiandrosterone (DHEA)-induced PCOS to study the mechanisms underlying the pathology of PCOS by investigating the growth, developmental characteristics, metabolic indexes and expression profiles of key genes of offspring born to the models. We found that the average litter size was significantly smaller in the DHEA group, and female offspring had sustained higher body weight, increased body fat and triglyceride content in serum and liver; they also exhibited decreased energy expenditure, oxygen consumption and impaired glucose tolerance. Genes related to glucolipid metabolism such as  Polycystic ovary syndrome (PCOS) is a common reproductive disorder with many characteristics, including hyperandrogenemia, insulin resistance and obesity, which may have significant implications for pregnancy outcomes and long-term health of women. Familial history of PCOS represents a risk factor for development of the disorder, as first-degree relatives of patients with PCOS have higher prevalence of PCOS , its sulfoconjugate and androstenedione  mice of the BALB/c strain  were randomly divided into the DHEA group and control group. The PCOS-like mouse model was established by subcutaneously injecting DHEA (6 mg/100 g body weight dissolved in 0.05 mL sesame oil) once daily for 20 consecutive days as described , followed by 10 IU of human chorionic gonadotropin  48 h later. On the same day, equine chronic gonadotropin was given, DHEA administration was stopped. DHEA and control mice were paired with BALB/c males (6–8 weeks of age) in the evening of HCG injection, and the females with confirmed vaginal plugs the next morning were considered pregnant. Spontaneous delivery was allowed, and offspring were weaned at age 3 weeks. Female and male offspring were fed separately until age 12 weeks and weighed weekly. All offspring were maintained under controlled temperature and lighting conditions and allowed free access to food and water. Metabolic status and reproductive function were assessed at age 12–13 weeks, with the exception of estrous cycle evaluation, which was performed at age 6 weeks.To determine body fat mass composition, offspring were placed in a clear plastic holder without anesthesia or sedation and inserted into an EchoMRI device  at age 12–13 weeks. Eight female and eight male mice were assessed per group.2) and carbon dioxide production (VCO2) were monitored for 3 min every hour in each cage for 24 h. At the same time, locomotor activity was monitored by Activity Sensor (Oxylet), and these data were collected every 2 min. Energy expenditure  and respiratory quotient (RQ = VCO2/VO2) were calculated using METABOLISM software (Oxylet). Eight female and eight male mice per group were assessed at age 12–13 weeks.Energy expenditure was measured using an indirect open-circuit calorimeter . Mice were placed individually into metabolic cages for 48 h. After a 24-h acclimation period, oxygen consumption (VOThe blood pressure of offspring (eight mice per group) was measured with a CODA noninvasive blood pressure meter  at age 12–13 weeks.An oral glucose tolerance test was performed in mice following an 8 h fast. Glucose levels were measured in tail-vein blood samples using a blood glucose meter . After the fast, glucose levels were measured, and then glucose (2 g/kg body weight dissolved in 10 mL water) was administered to mice by oral gavage, and tail-vein samples were obtained at 30, 60, 90 and 120 min after administration.At age 12–13 weeks, eight female and male offspring in each group were killed. Blood was drawn from the inner canthus after the mice were fasted for 8 h. The serum was immediately separated and stored at −80°C for subsequent analysis or hormone determinations. Leptin and fasting insulin levels were measured with radioimmunoassay kits . Lipid profiles were determined using biochemical analysis kits . After blood sample collection, each liver was immediately removed and stored at −80°C. Total cholesterol (CHO) and triglycerides (TG) were extracted from liver and measured with a Tissue Total Cholesterol Assay Kit and Tissue Triglyceride Assay Kit .−ΔΔCT method. All primers were synthesized by Sangon Company, and the sequences are shown in Supplementary Table 1, see section on Quantitative real-time PCR was used to determine the levels of the expression of glucolipid metabolism–related genes in liver. Total RNA from the liver was isolated with TRIzol reagent (Invitrogen). cDNA was synthesized using the First Strand cDNA Synthesis kit (Thermo Fisher). For quantitative PCR, amplification was performed with an ABI7500 (Applied Biosystems) using the SYBR Green kit (Applied Biosystems). The cycling conditions were 2 min at 95°C, followed by 40 cycles of 30 s at 95°C and 1 min at 60°C for annealing. GAPDH served as the internal control for gene expression normalization. The relative expression levels of the glucolipid metabolism–related genes were calculated using the 2s.e.m. Statistical significance was determined as indicated by two independent sample t-tests, Mann–Whitney U test and χ2 tests, using SPSS 16.0 software appropriately. A P value of <0.05 was considered to reflect a statistically significant difference.Measurement data were presented as the mean ± DHEA-induced PCOS female mice and control female mice were mated with normal BALB/c male mice . The pregnancy rate in the DHEA group was only 8% (compared with 67% for the control group), which was too low to obtain sufficient offspring; hence, in subsequent experiments, hormone therapy was used to stimulate ovulation.P < 0.05).In the control group, a vaginal plug was observed in 10 mice after stimulating ovulation and mating, and 7 mice produced offspring (70%); average litter size was 7.6 ± 1.0. In the DHEA group, a vaginal plug was observed in 13 mice, and 6 mice produced offspring (46%); average litter size was 4.2 ± 2.3, which was significantly smaller than that of the control group , VO2 ; total CHO, however, was similar in the two groups  and B. FAcc, Acs, CD36, Cpt-2, Cyp17a1, Fas, Fatp, Fgf21, Hmgcl, Hmgcs2, Inhbb, Lepr, Pparα, Pparβ, Pparγ, Scd and Vnn1; Aqp3, Glut4, Gyk, Pck1, Pdk4 and Pepck; Acaa1a, Acot1, Acot2, Acox1, Aldh3a1, Cpt1A, Cyp4a1, Cyp4a31, Etfdh, Hadha, Hsd17b4, Ucp2 and Ucp3; Acot1, Acot2, Cyp4a31, Cyp17a1, Fgf21, Inhbb, Pdk4, Pparγ and Vnn1) were expressed differently between the DHEA and control groups. To verify the expression of these 9 genes in the liver of female and male offspring, the sample size was expanded to six mice per group. As assessed by mRNA level, the expression of each of Pparγ, Fgf21, Pdk4, Inhbb, Acot1 and Acot2 was significantly upregulated in DHEA female offspring, whereas Cyp17a1 was significantly downregulated . Among these genes, 17 were associated with lipid synthesis, transport and metabolism , a member of the nuclear hormone receptor superfamily, is a class of ligand-activated transcription factors that play a key role in glucose homeostasis and maintaining lipid metabolism. It was traditionally believed that one subtype of PPARγ is expressed in adipose tissue, in which it regulates preadipocyte differentiation and promotes adipocyte maturation . FGF21, CYP17A1, encodes an enzyme of the cytochrome P450 superfamily, and it plays a role in cortisol production in humans and animals , ‘Reproductive health and major birth defects prevention and control research’ Key Special Fund (2016YFC1000601), the National Natural Science Foundation General Program , the Beijing Nova Program (xxjh2015011), the Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and special projects of the 2015 Science and Technology Innovation Base Cultivation and Development (Z151100001615023).The principal investigators Y Zhao, R Li, Y Yu and J Qiao take primary responsibility for the paper and contributed to the conception, design and coordination of the research. Y Huang and J-M Gao performed the experiment and data analysis. C-M Zhang and H-C Zhao contributed to the acquisition of reagents/materials/analysis tools. Y Huang and J-M Gao contributed to writing the manuscript. Y Zhao, R Li and Y Yu contributed to the revision of the article, and the final version was approved by all the authors.


Early recognition and diagnosis of Wernicke encephalopathy is pivotal for the prognosis of this medical emergency, especially in patients with liver failure which predisposes individuals to develop hepatic encephalopathy. For these patients, distinguishing between hepatic encephalopathy and Wernicke encephalopathy is a challenge in real-world clinical practice.A male patient with 21-year medical history of liver cirrhosis presented diarrhea and ascites. One month before this visit, he was noted to have poor appetite and progressive fatigue. After admission, although several major symptoms, including diarrhea, ascites, hyponatremia, and hypoproteinemia, were greatly improved through appropriate treatments, his laboratory indicators were not changed much. His appetite was not reversed at discharge. On the 5th day after discharge, the patient suddenly became reluctant to speak and did not remember the recent happenings. Simultaneously, unsteady gait and strabismus occurred. On the basis of clinical manifestations and brain magnetic resonance imaging scan results, the patient was diagnosed as Wernicke encephalopathy and these relative symptoms were resolved after intravenous vitamin B1.To our knowledge, this is the second case report of Wernicke encephalopathy developing in a critically ill cirrhotic patient without hepatocellular carcinoma or operative intervention. Wernicke encephalopathy may be underdiagnosed in these patients and this case raises physicians’ awareness of its possible onset. Several case reports describe Wernicke encephalopathy as a rare clinical condition; however, it is suggested, by guidelines from the European Federation of Neurological Societies, that Wernicke encephalopathy is not a rare disorder, but rather a rare diagnosis. Here, we present a critically ill patient with decompensated liver cirrhosis who developed Wernicke encephalopathy.Wernicke encephalopathy is an acute neurological disorder resulting from thiamine (vitamin B1) deficiency and occurs frequently in patients with compromised absorption, increased metabolism, or increased carbohydrate intake.2Written informed consent was obtained from the patient for the publication of this case report. A copy of the written consent is available for review by the editor of this journal.39; neutrophil percentage, 83.0%; red blood cell count, 3.60 × 1012; platelet count, 35 × 109; hemoglobin, 99 g/L; international normalized ratio, 1.51; blood ammonia, 24.7 μmol/L ; serum HBV DNA, <40 IU/L; serum alanine aminotransferase, 29 U/L; serum aspartate aminotransferase, 40 U/L; serum alkaline phosphatase, 122 U/L; serum gamma glutamyl transferase, 42 U/L; serum total bilirubin, 27.2 μmol/L ; serum albumin, 26 g/L ; serum creatinine, 92 μmol/L ; serum sodium, 130 mmol/L , serum kalium, 3.6 mmol/L ; and serum alpha fetoprotein 2.66 ng/mL . Results of other virologic workups, including tests for hepatitis A, C, D, and E virus, human immunodeficiency virus, and cytomegalovirus, were unremarkable. Abdominal ultrasonography reported liver cirrhosis, portal hypertension, ascites, and splenomegaly. The patient refused to undergo the endoscopy. The neurological examination did not reveal abnormalities.A 61-year-old man was admitted to the hospital for diarrhea and newly onset ascites. The patient had a 21-year medical history of liver cirrhosis associated with hepatitis B virus (HBV) infection and did not have a habit of alcohol consumption. His serum HBV DNA was not detected 15 years ago. After that, he did not receive any relative examinations or treatments due to economic reasons. One month before the current admission, he was noted to have poor appetite and progressive fatigue. Admission laboratory evaluation yielded the following: white blood cell count, 1.71 × 109; neutrophil percentage, 70.3%; red blood cell count, 3.53 × 1012; platelet count, 32 × 109; hemoglobin, 101 g/L; international normalized ratio, 1.45; blood ammonia, 17.5 μmol/L; serum HBV DNA, <40 IU/L; serum alanine aminotransferase, 19 U/L; serum aspartate aminotransferase, 39 U/L; serum alkaline phosphatase, 72 U/L; serum gamma glutamyl transferase, 35 U/L; serum total bilirubin, 26.8 μmol/L; serum albumin, 29 g/L; serum creatinine, 96 μmol/L; serum sodium, 134 mmol/L; and serum kalium, 3.9 mmol/L. No mental alteration was observed.After admission, his diarrhea was cured by levofloxacin (500 mg daily) at the 3rd day, and ascites was removed through repeated puncture and diuresis at the 6th day. The symptoms of hyponatremia and hypoproteinemia were improved with intravenous supplements; however, the levels of serum sodium and albumin did not arrive at the normal lower limit. At the 7th day, the patient claimed to discharge because of economic reasons and these treatments were thus discontinued. His appetite was always poor from the admission to the discharge. At discharge, his laboratory data were as follows: white blood cell count, 1.23 × 10At the 5th day after discharge, the patient suddenly became reluctant to speak and did not remember the recent happenings. Simultaneously, unsteady gait and strabismus occurred. Therefore, he was immediately sent to the local hospital. A brain magnetic resonance imaging (MRI) scan was rapidly performed and the results showed symmetric increased T2 signal in the tectal plate and periaqueductal gray matter. After an urgent telephone consultation to a neurologist and thorough a literature review, Wernicke encephalopathy was eventually diagnosed based on his clinical manifestations and MRI scan results. Intravenous vitamin B1 was therefore prescribed, with 200 mg daily for 5 days. In the days that followed, his memory loss, unsteady gait, and strabismus gradually improved and at the end of the vitamin B1 treatment, these symptoms were resolved. Finally, the patient died of bleeding due to rapid deterioration of his liver function.4 Although Wernicke encephalopathy is a disease of known etiology, the above triad is not always present, which leads to difficulties in the differential and confirmed diagnosis. It is reported that the diagnosis is confirmed in 0.4% to 2.8% of autopsies, yet may be overlooked in 68% of patients with alcoholism and 94% of patients without alcoholism. The Caine criteria for diagnosis of Wernicke encephalopathy have been demonstrated to be optimal and are summarized in Fig.  Till now, only 4 Wernicke encephalopathy cases associated with liver diseases  and abnormal glucose metabolism caused by impaired liver function contribute to the development of Wernicke encephalopathy. For patients with underlying liver cirrhosis, distinguishing between hepatic encephalopathy and Wernicke encephalopathy sometimes becomes a tough problem, especially in an emergency.,11 while mental alteration is usually a most noticeable symptom of Wernicke encephalopathy. Early recognition and diagnosis of Wernicke encephalopathy is pivotal for the prognosis of this medical emergency. If overlooked, it can develop into Wernicke–Korsakoff syndrome, coma, or even death.–15 It is vitally important that on observing a patient's early symptoms the physician immediately suspects that the symptoms may point to Wernicke encephalopathy. Factually, distinguishing between hepatic encephalopathy and Wernicke encephalopathy in real-world clinical practice is very difficult. First, an absolute majority of hepatologists are not familiar with Wernicke encephalopathy. Second, no mental alteration is unique in the 2 disorders. Third, distinctions in the neuroimaging findings between the 2 disorders are not easily identified. Like MRI findings in Wernicke encephalopathy, MRI scans in several cases with hepatic encephalopathy also present with symmetrical T2 high signal intensities in the bilateral cerebellar hemispheres and brachium pontis. Regarding the present case, some physicians who participated in the discussion insisted on their diagnosis of hepatic encephalopathy and they did not give up their opinions until the condition of the patient was reversed by intravenous vitamin B1. Therefore, when difficulties exist in distinguishing between hepatic encephalopathy and Wernicke encephalopathy, intravenous vitamin B1 (200 mg) can be considered as a discriminative method or a preemptive treatment.As is known, hepatic encephalopathy, which may be associated with the increased ammonia or endozepine and is characterized by a wide spectrum of psychiatric and behavioral disturbances and motor disorders, is a common complication of liver failure,In conclusion, to our knowledge, this is the 2nd case report of Wernicke encephalopathy developing in a critically ill cirrhotic patient without hepatocellular carcinoma or operative intervention. Wernicke encephalopathy may be underdiagnosed in these patients because it is not easily distinguished from hepatic encephalopathy. This case can also raise physicians’ awareness of the risk of incident Wernicke encephalopathy in critically ill patients with liver diseases.The authors thank National Natural Science Foundation of China (No. 81501652) for the support.


Organic dyes generally suffer from small Stokes shift that usually leads to self‐quenching and ‐gaining errors during the fluorescent imaging process. Here, a through‐bond energy transfer (TBET) cassette is developed with large Stokes shift to pursue precise cell imaging. The TBET is constructed by covalently conjugated tetraphenylethene (acts as donor) and rhodamine (acceptor) through an acetylene bond. The constructed TBET cassette distinctly behaves as dual‐Stokes shifts, including a large pseudo‐Stokes shift caused by energy transfer, from donor's absorption to acceptor's emission (up to 260 nm) and a smaller Stokes shift of acceptor molecules itself. Due to the intrinsic dual‐Stokes shifts, TBET cassette exhibits specific “dual distinct absorbances, single shared emission” properties, which can be excitated under two different laser channels. By colocalization of the imaging readouts of these two channels, the precisely “double checked” fluorescent imaging is achieved in living cells. Fluorescent imaging techniques have been broadly applied for biological and medical research, due to their high sensitivity, superior biocompatibility, and great versatilities for cell investigation, but minimally disturbing cells proliferations.Since it is still challenging to develop single organic dyes with intrinsic large Stokes shift, scientists tend to seek multichromophore with donor–acceptor architectures as alternatives. Based on the principle of energy transfer, fluorescence resonance energy transfer (FRET) has been widely utilized to obtain large pseudo‐Stokes shift.For biological fluorescent imaging, undesired spontaneous fluorescence inevitably accompanies with targeted fluorescent signal, such as autofluorescence.To realize more specific biological imaging, fluorescent molecules are supposed to be with large Stokes shift to deplete spectra crosstalk caused imaging interference; moreover, they are requested to possess multiple absorbance wavelengths, which can realize multichannel imaging, so that the fluorescence readouts can be screened. Hence, we constructed a TEBT cassette, which not only behaved as two distinct absorbances, that is, the absorbances of donor and acceptor respectively, but also was born with large pseudo‐Stokes shift and perfectly meets the requirements for specific biological fluorescent imaging.FigureIn TBET cassette, molecular structures of donor and acceptor cannot be planar, in case the newly constructed fluorescence system behaves as a single conjugated dye.[[qv: 10a]] Hence, a typical aggregation‐induced emission (AIE) molecule, tetraphenylethylene (TPE), was served as energy transfer donor, due to the nonplanar molecular structures of AIE molecules.−6m) were dissolved in DMSO/water mixtures with different fractions of water (fw%) for AIE investigation. As shown in Figure S5 in the Supporting Information, TPE molecules showed less fluorescence when they were well dissolved in dimethyl sulfoxide (DMSO), and the fluorescence became distinguishable when the water fractions were ≥60%. Hence, the DMSO/H2O  mixture was employed as solvent for the optical measurements. TRc molecules were supposed to behave two distinct absorptions, which were originated from donor and acceptor, respectively. As shown in FigureTPE was typical AIEgen and did not show fluorescence in the dissolved state, due to the nonradiative decay. To make the energy transfer easily to be observed, we checked the AIE behaviors of TPE and tried to testify the energy‐transfer related optical behaviors of TRc when the donor was luminant. TPE molecules 10 × 10−6m were diSchemeTo manifest this hypothesis, fluorescence behaviors of TRc molecules were investigated. In the fluorescence spectra Figure b, TPE moFigure−6 to 10 × 10−6m, indicating that our TBET cassette performed good biocompatibility and was suitable for biological applications.TBET cassette was implemented for “double checked” imaging in living cells. We first incubated TRc with cells for biocompatibility evaluation. As shown in −6m of TRc were incubated with MCF‐7 breast cancer cells for 30 min. As control, same molar of TPE (donor) and Rho (acceptor) was also incubated with cells under identical procedures. To investigate the subcellular distributions of these different fluorescent molecules, MCF‐7 cells were observed by confocal laser scanning microscopy (CLSM) under three different laser channels, which corresponded to donor channel , acceptor channel , and energy transfer channel . In TPE treated group  by compared with their precursors (TPE and Rho). Meanwhile, we found that TRc exhibited different subcellular behaviors, comparing to its donor and acceptor molecules: they preferentially accumulated in a particular region. We then costained the cells with a commercial lysotracker (LysoTracker Deep Red) and found that the fluorescence from dual channels of TRc was showing excellent colocalization with commercial lysotracker, indicating that our TRc molecules were potentially capable of lysosomes tracking. On the contrary, neither TPE nor Rho exhibited better lysosomes colocalization pattern. The colocalization efficiency of TPE, Rho, and TRc treated cells were evaluated in Figure To evaluate the fluorescence behaviors of the TBET cassette at cellular level, 2.5 × 10FigureX, Y, and Z axes, respectively  mode for pinpointing the undesirable fluorescence during the imaging process. The results were shown in In this study, we developed a novel through‐bond energy transfer fluorescent dye with unique “dual distinct absorbances, single shared emission” characteristics for “double checked,” precise cell imaging. In TBET cassette, fluorescence can be excitated either at the range of donor's absorbance or acceptor's absorbance and thus giving two distinct fluorescent readouts. By colocalizing of two groups of fluorescence signals, undesirable fluorescence that always misleads the imaging results can be sorted out. After colocalization, the fluorescence does not merge with their counterparts is considered as undesirable fluorescence emitted from cells or during the CLSM observation process. The fluorescence signals that can be perfectly merged were considered as the real subcellular positions of the fluorescent dye. The “double checked” TBET cassette provides a straightforward solution for depletion of undesirable fluorescence which may confound fluorescent imaging in vitro. Our strategy smartly achieves precise cell imaging only based on fluorescence dye and does not need to spend huge expenses on tackling undesirable fluorescence disturbance by improving the instruments and thus offering great promise for inspiring scientists to develop more sophisticated and specific imaging materials.Chemicals and Instruments: All chemicals and regents were purchased and used as received without further purification. 1H nuclear magnetic resonance (NMR) spectrum was measured on a Bruker AV 300 spectrometer in chloroform with tetramethylsilane (δ = 0) as an internal reference. Mass spectrum was taken on a high‐resolution mass spectrometry . UV absorbance was evaluated by a UV–vis spectrometer ; fluorescence behaviors of the materials were tested by a fluorescence spectrometer  and cellular fluorescence distributions were observed by confocal laser scanning microscope .Synthesis of TRc: TPE and Rho were prepared following the literature method.3)2Cl2 , and PPh3  was dissolved in a solvent mixture of dry triethylamine (3 mL) and anhydrous tetrahydrofuran (THF) (3 mL) under nitrogen (N2 ) protection. The mixture was stirred at 70 °C for 24 h. After removal of solvent under vacuum, the residue was purified by column chromatography  to give a purple solid; yield 26.0 mg (10%). 1H NMR  δ8.79 , 8.39 , 7.76 , 7.42 , 7.4 , 7.37 , 7.35 , 7.19–7.13 , 7.12–7.04 , 6.98 , 6.95 , 6.92 , 3.7 , 1.38 . HRMS: m/z 753.3835 .Transmission electron microscopy (TEM) Observation of TRc Nanoaggregates: 1 µL TRc DMSO solution (100 × 10−3m) was dropped into 999 µL Milli Q water, with sonication for 30 s, the 100 × 10−6m TRc nanoaggregates were obtained. The TRc nanoaggregates were then diluted to 10 × 10−6m and dropped on copper grid for TEM observation after naturally dried.Calculation of the Energy Transfer Efficiency (ETE%): The ETE% was calculated by the equation: ETE% = [1 − (IDA/ID)] × 100. IDA denotes the integral of the emission spectra of the donor in the presence of an acceptor; ID is the integral of the emission spectra of donor molecules without an acceptor presence.Cell Viabilities Evaluation: MCF‐7 cells were seeded at 5 × 103 cells well−1 in a 96‐well plate, preincubated for 24 h, then incubated with TRc for 24 h with concentrations ranging from 0.1 × 10−6 to 10 × 10−6m. The medium was then replaced with 100 µL fresh medium containing 0.5 mg mL−1 MTT and after 2 h, the MTT solution was replaced with 150 µL DMSO solution. The absorbance was measured at 570 nm with a reference wavelength of 630 nm using an Infinite M200 microplate reader . Untreated cells in medium were used as controls.Fluorescence Distributions of TPE, Rho and TRc in Living Cells: 105 MCF‐7 cells were seeded into 35 mm glass bottom dishes, incubated at 37 °C for 24 h until completely being attached, then incubated with 2.5 × 10−6m TPE, Rho, and TRc at 37 °C for 30 min, respectively. Cells were then rinsed with phosphate‐buffered saline (PBS) twice and incubated with LysoTracker Deep Red (1000 times dilution in PBS) for lysosomes staining. After 10 min incubation, cells were rinsed with PBS twice and imaged using a CLSM  with excitation at 405 nm for TPE, 543 nm for Rho, both 405 and 543 nm for TRc, and 630 nm for LysoTracker Deep Red.The authors declare no conflict of interest.SupplementaryClick here for additional data file.