In this study, a small-diameter hybrid vascular graft was prepared by co-electrospinning PCL and PDS fibers. The incorporation of PDS improves mechanical properties, hydrophilicity of the hybrid grafts compared to PCL grafts. The in vitro/vivo degradation assay showed that PDS fibers completely degraded within 12 weeks, which resulted in the increased pore size of PCL/PDS grafts. The healing characteristics of the hybrid grafts were evaluated by implantation in rat abdominal aorta replacement model for 1 and 3 months. Color Doppler ultrasound demonstrated PCL/PDS grafts had good patency, and did not show aneurysmal dilatation. Immunofluorescence staining showed the coverage of endothelial cells (ECs) was significantly enhanced in PCL/PDS grafts due to the improved surface hydrophilicity. The degradation of PDS fibers provided extra space, which facilitated vascular smooth muscle regeneration within PCL/PDS grafts. These results suggest that the hybrid PCL/PDS graft may be a promising candidate for the small-diameter vascular grafts.Electrospun polycaprolactone (PCL) vascular grafts showed good mechanical properties and patency. However, the slow degradation of PCL limited vascular regeneration in the graft. Polydioxanone (PDS) is a biodegradable polymer with high mechanical strength and moderate degradation rate  Bypass surgery has been one of the most effective treatments for CVD, and helped patients live longer. The artificial blood vessel have shown great potential applications in bypass surgery, and provided a good alternative to the autologous vein. Although three kinds of commercialized vascular grafts, expanded polytetrafluoroethylene (ePTFE), poly (Dacron) and polyurethane (PU), have been used in vascular surgery almost 50 years as large diameter prostheses, intimal hyperplasia and thrombosis limit the application of these polymer in smaller diameter grafts (<6mm)2. Thus, it is very important to find an appropriate biodegradable material for fabrication of smaller diameter grafts.Cardiovascular disease (CVD) is the NO.1 killer in the world, and it is responsible for >17.3 million deaths every year, a number that represents 31% of all global deaths3. Many studies have shown that electrospun PCL vascular grafts have remarkable patency because of its optimal hemocompatibility and mechanical properties7. But the number of cells and capillaries in the electrospun PCL vascular graft walls decreased on the long term (12 and 18 months), meanwhile, calcification was observed, due to the slow degradation, lower compliance and dense fibrous structure4. Our group fabricated an electrospun PCL vascular graft with thick fibers and large pores in order to enhance cell infiltration, vascularization and efficient regeneration of functional tunica media6. Although the grafts have much better effects in vascular regeneration and remodeling compared to thin fiber PCL grafts, the slow degradation of PCL is still a problem.PCL is a Food and Drug Administration (FDA)-approved biodegradable polymer with wide applications in tissue engineering8. The graft regenerated into compliant neoarteries resembled native arteries in the components and structures of tissue within 3 months. Rapid degradation can reduce inflammatory responses caused by long-lasting materials, in addition, and also provide more space for cell infiltration and extracellular matrix (ECM) secretion. However, the safety issue should be taken into account when the PGS grafts are used in large animals or human due to the rapid degradation of PGS which does not match the slow tissue regeneration process in large animals or human. To solve this problem, researchers combined fast-degrading and slow-degrading materials to fabricate vascular grafts with an appropriate degradation rate.A fast-degrading vascular graft was made of elastic poly (glycerol sebacate) (PGS), and wrapped with a dense electrospun PCL sheath which enhanced graft strength and prevented bleedinget al. developed a dual-polymer composite scaffold containing PCL (a slow-degrading polyester) and poly(ethylene-oxide) (PEO) (a water-soluble polymer) via co-electrospun from two separate spinnerets9. The fast solution of PEO provided space for cell infiltration. But the excessive solution of PEO resulted in an overall loss in structural integrity of scaffold. In order to maintain scaffold integrity with an appropriate degradation rate, this group fabricated a multicomponent nanofibrous scaffold by a tri-jet electrospinning device. These fibers included PEO, PCL, and poly(lactide-co-glycolide) (PLGA) (medium-degrading) with different degradable rates10. After the PEO fibers fast dissolved, the PCL and PLGA fibers would serve to maintain scaffold integrity initially, and PLGA fibers gradually degraded to augment pore size and porosity. The presence of the three types of fibers provided time-dependent characteristics in the composite scaffold, and could also be used to further refine the mechanical properties.Baker ®, and approved by FDA11. PDS lost about 50% of original strength at four weeks in vivo, and completely absorbed after 240 days without obvious tissue reaction12. The degradation products of PDS had been proven low-toxicity, and caused a lower inflammatory response rates than Vicryl (PLGA) and Dexon (poly(glycolic acid))13. Compared to polyglycolide (PGA) and Dacron synthetic grafts, the grafts made of PDS have been shown to be less thrombogenic14. However, when tested in situ in a rat model alone, the insufficient cellular infiltration and fast degradation of PDO grafts led to aneurismal formations15.PDS has been widely used as absorbable wound closure suture in clinical practice under the trade name PDSin vivo/vitro and tissue regeneration and remolding of the hybrid PCL/PDS grafts in rat abdominal aorta model.Inspired from above previous reports, we fabricated a hybrid grafts consisting of fast-degrading PDS fibers and slow-degrading PCL fibers by co-electrospinning. We anticipated that fast degradation of PDS fibers can enlarge the pore spaces to promote cell infiltration and tissue regeneration, meanwhile, the slow-degrading PCL fibers maintained the structural integrity of scaffold and provided enough mechanical strength to prevent aneurysm. Here we describe the degradation behavior of the hybrid PCL/PDS scaffolds 17. Figure 6. In this study, the optimized PCL fibers were selected to prepared PCL/PDS vascular grafts. The relative ratio of two components is a key factor for this study. So we planned to fabricate a series of vascular grafts over a wide range of ratios  by adjusting electrospun PDS parameters  of 131.28 ± 0.65° for electrospun PCL. After the incorporation of hydrophilic PDS component (11.54 ± 0.60°), the hydrophicity was improved, that is, the mean WCA of PCL/PDS decreased to 78.06 ± 2.20° Fig. . This wa21. Compared to PCL grafts, the PCL/PDS grafts had a significant increase in Tensile strength and Young’s modulus , which indicated that the larger pores generated by PDS degradation can enhance cells migration to some extent.Longitudinal sections were stained with H&E to identify the neointimal formation Fig. . One mon23. Therefore, rapid endothelialization is a prerequisite for artificial vascular grafts.The lumen of native blood vessels is lined with a layer of ECs, the endothelium, which provides an anti-coagulating layer to prevent thrombosisThe lumen of the explanted grafts was observed by SEM to analyze the endothelium coverage at three different positions, the near suture site, the midportion and the site between them Fig. . At 1 moEn face staining of the lumen surface of grafts showed the coverage of CD31+ cells on the PCL/PDS grafts was faster than that on PCL grafts at 1 month  higher than that of the PCL grafts (43.02 ± 6.06%) at 1 month  was slightly higher than that of PCL (81.77 ± 6.76%)  was significantly (p < 0.05) higher than that of PCL (51.42 ± 7.66%) . Similarly, the area of MYH+ SMCs within PCL/PDS grafts was about 2 times larger than that in the PCL grafts . At 1 month, the explanted PCL and PCL/PDS grafts are comparable in terms of fiber density. However, at 3 months, the fiber density of PCL/PDS grafts was lower than that of PCL grafts  and Von Kossa staining were performed to detect the collagen, elastin fibers, glycosaminoglycan and calcification formation, respectively. The neointima of the two types of grafts both showed ECM deposition, which was similar to the SMCs layer of native abdominal aorta Fig. . Howeverfts Fig. .Figure 6in vitro/vivo, which resulted in the increased pore size of PCL/PDS grafts. The hybrid grafts exhibited good blood compatibility and patency in rat abdominal aorta model, similar to PCL grafts. The PCL/PDS grafts could resist aneurysm formation despite degradation of PDS fibers. Endothelialization and contractile phenotype SMCs coverage was significantly better in the PCL/PDS grafts. The degradation of PDS fibers provided extra space for improving SCMs regeneration within hybrid graft walls. In summary, our hybrid PCL/PDS grafts may be a promising off-the-shelf vascular graft candidate and are worthy of further evaluation with long term.In this study, a hybrid vascular graft composed of PCL and PDS was prepared via electrospinning with a side-by-side dual spinneret method. The incorporation of PDS improved mechanical properties, hydrophicity of the hybrid grafts compared to PCL grafts. The PDS fibers completely degraded within 12 weeks Mn = 80,000) were purchased from Sigma . PDS pellets were purchased from Tianjin Dongnanhesheng Medical Technologies Co., Ltd . 1, 1, 1, 3, 3, 3-fluoro-2-propanol (HFIP) was purchased from Aladdin . Methanol, chloroform and alcohol were obtained from Tianjin Chemical Reagent Company . 3, 3′-dioctade-cyloxacarbocyanine perchlorate (DiO) and 1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorate (DiI) were products of Molecular Probes . Triton X-100 was purchased from Alfa Aesar . Sprague Dawley (SD) rats  were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences . All animal studies were performed under the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals, and the overall project protocols were approved by the Animal Ethics Committee of Nankai University.PCL pellets (The hybrid PCL/PDS vascular grafts were prepared by co-electrospinning. A 25% w/v solution of PCL was prepared in a 5:1 (V/V) mixture of chloroform and methanol by stirring overnight. PDS was dissolved in the HFIP with stirring at room temperature for 6 h to obtain 20% w/v solution. Two 10-mL syringes were filled with PCL or PDS solution and connected to a 21 G blunt-ended needle that served as the charged spinneret. The apparatus consists of a syringe pump , a high-voltage generator  and a rotating steel mandrel (2.0 mm in diameter) as collector. The flow rate of PCL was set at 8 mL/h. The flow rate of PDS was set at 4 mL/h, 6 mL/h, 10 mL/h and 16.67 mL/h, respectively, to fabricate hybrid PCL/PDS grafts with different ratios. The voltages between the needle tip and the rotating mandrel were set as 11 kV for PCL and 15 kV for PDS. The distance between the needle tip and collector were 25 cm for PCL and 15 cm for PDS. The pure PCL grafts and pure PDS grafts were regarded as control and prepared by the same method as described above. The obtained electrospun grafts were vacuum-dried over 48 h at room temperature before further treatment.et al.30. At least six pores per image, five images per sample and three samples per group were included to obtain the calculation.The cross-section and lumen surface of grafts were mounted on an aluminum foil and sputter coated with gold and palladium. Scanning electron microscope  at an accelerating voltage of 15 kV was used to observe the morphology of grafts structure. Based on the SEM images, fiber diameter was analyzed using Image-Pro Plus software (IPP). The pore diameter was calculated according to the method described by Dong To visualize the distribution of the PCL and PDS fibers, two different fluorescent dyes were incorporated into each fibers of the graft. DiI  was added to PCL solution and DiO  was added to PDS solution. After co-electrospinning, the PCL/PDS grafts were visualized using a laser scanning confocal microscope .The weighted hybrid PCL/PDS grafts (1.1 cm in length) were soaked in 10 ml of chloroform in a 15 ml centrifugal tube, and kept in a shaker at room temperature for 12 h. The chloroform was changed every 4 h to completely leach the PCL. After leaching, the grafts were vacuum-dried over 48 h at room temperature. Then the weight of grafts was measured. The ratio of PCL and PDS in the hybrid graft was determined as follows: the ratio of PCL and PDS = (weight of graft before leaching−weight of graft after leaching)/weight of graft after leaching.Mechanical properties of the grafts in transverse directions were measured on a tensile-testing machine with a load capacity of 100 N . Grafts with 0.3 cm in length were fixed on two steel rings which were clamped by machine chuck, and then pulled radially at a rate of 10 mm/min until rupture. Tensile strength and ultimate elongation at break were measured. Young’s modulus was obtained by measuring the slope of the stress-strain curve in the elastic region. These mechanical tests were performed in triplicate according to the ANSI guidelines.Twenty SD rats were anesthetized with intraperitoneal injection of chloral hydrate (330 mg/kg body weight). Heparin (100 unit/kg) was used for anticoagulation by tail vein injection before surgery. A midline laparotomy incision was made and the abdominal aorta was isolated, clamped, and transected. The PCL, PCL/PDS and PDS grafts (1.1 cm in length) were sewed in an end-to-end fashion with 8 interrupted stitches using 9-0 monofilament nylon sutures . The wound was closed with 3-0 monofilament nylon sutures. No anticoagulation drug was administered after surgery. The grouping of animals was based on the type of scaffolds and duration of observation for 1 and 3 months.At the predetermined time points (1 and 3 months), the patency and blood velocity of the grafts was visualized by high-resolution ultrasound  after the rats were anesthetized with isoflurane.En face immunofluorescence staining.At each time point, the rats were sacrificed by injection of over-dose urethane. The grafts were explanted and perfused with saline before they were harvested. The explants were cut into two parts from the middle. One part was snap-frozen in optimal cutting temperature (OCT) Compound (Tissue Tek) for frozen cross-section. The other part was longitudinally cut into three pieces. One piece was observed by stereomicroscope, and then snap-frozen in OCT for longitudinal section. Other two pieces were prepared for SEM and The samples embedded in OCT were cut into 6 μm in thickness. Subsequently, the sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, Safranin O, Verhoeffe-Van Gieson (VVG) and Von Kossa. Images were observed under upright microscope (Leica DM3000).For immunofluorescent staining, the frozen sections were fixed in acetone at −20 °C for 10 min, air-dried, and rinsed once with 0.01 mM PBS. Then slides were incubated in 5% normal goat serum  for 45 min at 4 °C. To stain intracellular antigens, 0.1% Triton-PBS was used to permeate the membrane before incubation with serum. The sections were incubated with the following primary antibodies overnight at 4 °C: Mouse anti- CD31 , mouse anti-alpha smooth muscle actin , mouse anti-smooth muscle myosin heavy chain I . After rinsing with PBS for five times, Alexa Fluor 488 goat anti-mouse IgG  was applied for 2 h at room temperature. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) containing mounting solution . The sections without incubation with primary antibody were used as negative controls. Slides were observed under a fluorescence microscope , and the images were acquired with a digital camera .To quantify the cell density of graft walls, the cross-sections of grafts were stained with DAPI. Ten high-magnification DAPI images per sample, five samples per group at 1month and four samples per group at 3 months were included to quantify the cell density.To prepare samples for SEM, the explanted grafts were fixed with 2.5% glutaraldehyde for 12 h, and dehydrated in ascending series of ethanol. They were affixed onto aluminum stubs with carbon tape, sputter-coated with gold, and observed by SEM.The explanted grafts were fixed with 2% paraformaldehyde for 30 min at room temperature. Then the samples were washed with PBS, blocked with 2% bovine serum albumin for 2 h at 4 °C, and incubated with primary antibody against endothelial cells marker CD31 overnight at 4 °C, and then incubated with Alexa-Fluor 488 labeled secondary antibodies. To prepare stained whole mounts for imaging, segments were mounted on glass slides, flattened under a coverslip. The luminal surface of the explanted grafts was imaged under a confocal laser scanning microscope.p < 0.05 and #p < 0.001. All data were shown as mean ± standard error of the mean (SEM). Other Parts are available in the Supporting Information.GraphPad Prism Software Version 5.0  was used for statistical analysis. Single comparisons were made with a paired Student’s t-test. Multiple comparisons were carried out using one-way ANOVA and Tukey’s post-hoc analysis. The minimum significance level was set at *Supporting Information


Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal carcinoma in China. This study was to develop a staging model to predict outcomes of patients with ESCC.Using Cox regression analysis, principal component analysis (PCA), partitioning clustering, Kaplan–Meier analysis, receiver operating characteristic (ROC) curve analysis, and classification and regression tree (CART) analysis, we mined the Gene Expression Omnibus database to determine the expression profiles of genes in 179 patients with ESCC from GSE63624 and GSE63622 dataset.P < 0.05). Accordingly, we constructed a molecular disaggregated model comprising one lncRNA and two PCGs, which we designated as the LSB staging model using CART analysis in the GSE63624 dataset. This LSB staging model classified the GSE63622 dataset of patients into three different groups, and its effectiveness was validated by analysis of another cohort of 105 patients.Univariate cox regression analysis of the GSE63624 dataset revealed that 2404 protein-coding genes (PCGs) and 635 long non-coding RNAs (lncRNAs) were associated with the survival of patients with ESCC. PCA categorized these PCGs and lncRNAs into three principal components (PCs), which were used to cluster the patients into three groups. ROC analysis demonstrated that the predictive ability of PCG-lncRNA PCs when applied to new patients was better than that of the tumor-node-metastasis staging (area under ROC curve [AUC]: 0.69 vs. 0.65, The LSB staging model has clinical significance for the prognosis prediction of patients with ESCC and may serve as a three-gene staging microarray. Esophageal cancer ranks as the world’s sixth deadliest cancer and has two major histological types: adenocarcinoma and squamous cell carcinoma , 2. In CWith the development of high-throughput sequencing technology, such as microarray analysis , 12, theA new molecular staging using the G-factor, which is based on the expression of p53 and matrix metalloproteinase-7 (MMP-7), can supplement applying the TNM system to classify gastric cancer , and a pHOTAIR) is associated with breast cancer metastasis [GAS5) and LINC00538 (Yiya) are promising prognostic biomarkers for liver metastases in patients with early-stage colorectal cancer [Similar to PCGs, certain dysregulated long non-coding RNAs (lncRNAs) act as oncogenes . For exatastasis . Growth l cancer . An 8-lnl cancer .Here we used two microarray datasets and relevant clinical information from the GEO dataset to explore the association between PCGs and lncRNAs and the survival of patients with ESCC. For this purpose, we constructed a PCG-lncRNA transcriptome staging model to predict the prognosis of patients with ESCC.https://www.ncbi.nlm.nih.gov/geo/). We excluded cases without clinical survival information. Two ESCC microarray datasets (GSE53624 and GSE53622) generated using the Agilent-038314 CBC Homo sapiens lncRNA + mRNA microarray V2.0 (http://www.genomics.agilent.com/) were selected. We divide GSE53624 into the training and test dataset randomly. The GSE53624 test dataset and GSE53622 was used for internal validation. The main clinicopathological characteristics of patients are summarized in Table PCG and lncRNA expression data and clinical data of corresponding patients with ESCC were obtained from the publicly available GEO database (Homo sapiens lncRNA + mRNA microarray V2.0 were downloaded from the Agilent website (https://www.agilent.com/). PCG and lncRNA expression data from the Agilent-based expression profile of ESCC cohorts (GSE53624 and GSE53622) was obtained by re-annotating microarray probes according to the sequences of the probe sets and the annotations of PCG and lncRNA records in GENCODE . We used BLASTn (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST/) to align the probe sequences to those of noncoding and coding transcript sequences from GENCODE.The GPL18109 probe set sequences for the Agilent-038314 CBC The alignments were filtered as follows: (i) only probes perfectly matched to a transcript were retained, resulting in two sets of probes targeting protein-coding and -non-coding transcripts, respectively; (ii) probes targeting noncoding transcripts that perfectly matched cDNA coding sequences were removed; (iii) all transcripts corresponding to the retained probes were mapped to the genome and annotated as PGCs or lncRNAs.Besides above 179 ESCC patients from GEO database, we collected 105 ESCC patients as the experimental set from the Chaoshan District of Guangdong Province, which has a high prevalence of ESCC . The exp−ΔΔCt method. Quantitative RT-PCR (qRT-PCR) was performed in triplicate and repeated at least three times, as described previously [Total RNA was extracted using TRIzol  and purified using a PureLink RNA Mini Kit  according to the manufacturer’s protocol. The purity and concentration of RNA were determined according to the ratio of absorbance of 260 nm/280 nm light using a NanoDrop ND-2000 spectrophotometer . The cDNA synthesis was performed by reverse transcription using random hexamer primers . Real-time PCR was performed using a SYBR Premix Ex Taq kit . Briefly, reverse transcription was performed at 37 °C for 15 min and at 85 °C for 5 s. Real-time PCR was performed using an ABI 7500 real-time PCR system (Life Technologies) as follows: 95 °C, 30 s; 95 °C, 5 s; 60 °C, 34 s (30 cycles). Relative quantification of mRNA expression was calculated using the 2eviously . All metwww.r-project.org), including packages named pROC, rpart, and survival downloaded from Bio-conductor (http://www.bioconductor.org/). Univariate Cox regression analyses were used to identify common PCGs and lncRNAs that associated with OS. P < 0.05 indicates a statistically significant difference. All insignificant PCGs and lncRNAs with P >0.05 were excluded. We performed principal component analysis (PCA) [All analyses were performed using the R program (is (PCA) –38 to reWe used the R NbClust package , and it provides 30 indices for determining the number of clusters. The output of the software proposes the best clustering scheme from the results obtained by varying combinations of the clusters, distance measures, and clustering methods. These operations identify the optimal number of clusters by calculating several cluster indices. The clusters were validated using the Calinski and Harabasz index , 40.Kaplan–Meier survival analysis was performed to test the equality of survival distributions of different groups for each ESCC cohort, and statistical significance was assessed using the two-sided log-rank test. Further, time-dependent receiver operating characteristic (ROC) curves were used to compare the sensitivity and specificity of the survival prediction, and the area under the curve (AUC) value was calculated from the ROC curve. Multiclass ROC curves were used to evaluate the effectiveness of multi classification predictions, which were calculated using the R packages called pROC , 42. ClaTo begin the CART analysis, patients identified from the GSE53624 dataset were randomly split into the training and testing groups using the function “sample” of the R program . CART waP < 0.05 were considered significant [The associations between coexpressed lncRNA and PGCs in the molecular staging model were computed using Pearson correlation coefficients visualized with Cytoscape. We next performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the coexpressed protein-coding genes to predict biological functions using the ClueGo plugin of Cytoscape (version 3.2.3) , which inificant .Homo sapiens lncRNA + mRNA microarray V2.0 identified 17,434 PCGs and 6253 lncRNAs. We retained probes that were uniquely mapped to the genomic coordinates of PCGs or lncRNAs derived from GENCODE. Multiple probes (or probe sets) mapping to the same PCGs or lncRNAs were integrated using the arithmetic mean of the values of multiple probes (or probe sets) and were used to generate new PCG and lncRNA expression profile values from the GSE53624 and GSE53622 datasets. Further, we used fold-change values  on a log2 scale in the next analysis.According to the dataset screening criteria described in Methods, 179 samples (119 from the GSE53624 dataset and 60 from the GSE53622 dataset) were selected. All these ESCC samples contained tumor tissues and adjacent normal tissues. Probe reannotation of the Agilent-038314 CBC n = 119) was selected to explore the association of OS with PCGs or lncRNAs. We conducted univariate Cox proportional hazards regression analysis of the PCG and lncRNA expression profiling data, with OS as the dependent variable, and identified 2404 PCGs and 635 lncRNAs that significantly associated with OS (P < 0.05)   Fig. a. The 5-P < 0.05)  Fig. c.n = 59) and a testing set (n = 60) for internal validation. Next, we selected the first 100 genes with the highest absolute loading, e.g., the 100 genes with the highest positive or negative correlation corresponding to each principal component. The clinical attributes of patients, such as age, sex, tobacco use, alcohol use, tumor location, tumor grade, T stage, N stage, and TNM stage, were used as variables to perform the CART routines.Kaplan–Meier and ROC analyses showed that the PCG-lncRNA grouping improved the classification of patients, indicating its value as a novel, efficient staging plan. We next pursued identifying markers to classify the three groups as follows. We randomly divided the GSE53624 dataset into a training set (P < 0.05) in the training set.Subsequently, CART analysis of the training set generated the final tree composed by LINC01800, semaphorin 3A (SEMA3A), and brain-expressed X-linked 2 (BEX2) identified from the survival-related PCGs and lncRNAs. Moreover, there were no clinical attributes of patients with lowest error rate remaining in the classification tree produced using RPART  and entire GSE53624 dataset (n = 119), and the AUC values were 0.79 and 0.83 (P < 0.05), indicating that this model efficiently stratified patients into different prognostic groups . Kaplan–Meier survival curves for patients with LSB I, II, and III ESCC, which were classified according to the respective cutoff values of the three molecular markers, are shown in Fig. P = 0.01). The 5-year OS rates were of was 63.4% in patients with LSB I ESCC, 39.6% in patients with LSB II ESCC, and 21.2% in patients with LSB III ESCC. The percentages of patients with LSB I, LSB II, and LSB III ESCC were 33.3%, 38.3% and 28.4%, respectively, similar to those in the training group . Moreover, the AUC of the LSB staging model was 0.68, greater than 0.66 for the TNM staging (P < 0.05)  . These findings suggest that SEMA3A, BEX2, and LINC01800 may be involved in tumorigenesis through interacting with those coexpressed PCGs that influence biological processes such as angiogenesis, cell migration, cell differentiation, and cell adhesion  . Overexp−ΔΔCt values to group patients, in contrast to the same cutoff values of the three genes in microarray. Thus, the data were insufficient to confirm cutoff values of the three genes in RT-PCR. Nevertheless, the robustness of our LSB staging model using independent and experimental datasets indicates that this model has potential clinical significance for translation to the clinic as a three-gene microarray, likely to serve as a powerful prognostic staging model for ESCC.Certain limitations to this study, other than the limited robustness of the data for ESCC need to be acknowledged. First, only a fraction of human lncRNAs  and PCGs  were included in the analysis. Therefore, the prognostic lncRNA and PCGs identified here may not represent all candidates. Second, experimental studies on these three genes may provide important information that will enhance our understanding of their functional roles. Third, the expression values of microarrays differ from those of RT-PCR , 53. TheIn conclusion, we show here that the LSB staging model can accurately predict the survival of patients with ESCC. Moreover, the method used to construct the LSB staging model suggests a general strategy and effective methodology that will facilitate research aimed at identifying new clinical staging markers for other types of cancer.


Triticum aestivum), one of the world’s most important crops, is facing unprecedented challenges due to global warming. To evaluate the gene resources for heat adaptation in hexaploid wheat, small heat shock proteins (sHSPs), the key plant heat protection genes, were comprehensively analysed in wheat and related species. We found that the sHSPs of hexaploid wheat were massively expanded in A and B subgenomes with intrachromosomal duplications during polyploidization. These expanded sHSPs were under similar purifying selection and kept the expressional patterns with the original copies. Generally, a strong purifying selection acted on the α-crystallin domain (ACD) and theoretically constrain conserved function. Meanwhile, weaker purifying selection and strong positive selection acted on the N-terminal region, which conferred sHSP flexibility, allowing adjustments to a wider range of substrates in response to genomic and environmental changes. Notably, in CI, CV, ER, MI and MII subfamilies, gene duplications, expression variations and functional divergence occurred before wheat polyploidization. Our results indicate the massive expansion of active sHSPs in hexaploid wheat may also provide more raw materials for evolving functional novelties and generating genetic diversity to face future global climate changes, and highlight the expansion of stress response genes with wheat polyploidization.Wheat ( Small heat shock proteins (sHSPs), one type of HSPs, are typically induced by heat, and function in protein folding and preventing or reversing substrate aggregation4. SHSPs contain a conserved α-crystallin domain (ACD), preceded by a highly divergent N-terminal region and followed by a C-terminal extension. The ACD comprises seven or eight anti-parallel β-strands which form a β-sandwich. In their native state, sHSPs form oligomers (200–800 kDa) that operate as a functional unit6. The ACD and C-terminal extension are the major regions responsible for oligomer formation, while the flexible N-terminal region is responsible for substrate recognition and oligomer stability11. Extensive studies have demonstrated that manipulating the sHSPs of plants remarkably altered their thermotolerance16, highlighting their role in heat adaptation. The composition and expression of sHSPs has recently been studied in several plants, including Arabidopsis thaliana, rice, algae, Populus trichocarpa, pepper, sugarcane and soybean, and the identified sHSPs have been categorized into sixteen subfamilies, including eleven cytoplasmic/nuclear localized (CI-CXI) and five organelle localized subfamilies24. However, the evolutionary mechanisms of sHSPs in plants, especially in plant polyploidization process, remain largely unknown.Diverse heat shock proteins (HSPs), one key component of cellular protein quality control network, are very important for plant heat stress adaptation27. Bread wheat, also known as allohexaploid wheat (AABBDD), originated from two hybridization events among the genera Triticum and Aegilops29. The first hybridization, between Triticum urartu (AA) and a close relative Aegilops speltoides (BB), occurred <0.8 million years ago (Ma) and gave rise to the allotetraploid emmer wheat . The second hybridization, between emmer wheat and Aegilops tauschii (DD), which arose from the hybridization of the AA and BB lineages ~5.5 Ma, occurred <0.4 Ma and gave rise to the allohexaploid wheat30. Owing to the high degree of autonomy in the subgenomes and the shorter evolutionary history31, bread wheat and its related progenitors provide a good system for studying the polyploidization process30.Productivity of bread wheat (Triticum aestivum L.) cropping systems is at risk due to increasing temperatures as a result of global warming32. The autopolyploid which resulted from whole genome duplication or unreduced gametes fusion within a single species, and the allopolyploidy which resulted from interspecific hybridisation, generate extra gene copies34. These duplicated genes then underwent subfunctionalization, neofunctionalization, specialization, pseudogenization or concerted evolution, leading to greater genetic diversity36, which is the key factor in adaptation to new habitats and distribution to larger geographical areas than those covered by their progenitors34. The genome size of hexaploid wheat approximates to the sum of three diploid progenitors with 2–10% DNA loss with polyploidization, suggesting that most genes are redundant compared with their progenitors and some genes may be lost28. As the key heat protection genes, twenty-seven candidate sHSPs have been identified in hexaploid wheat, with all found to localise to the mitochondria (26) or nucleus (1)37. This differed from the literature on the cellular localization of other identified sHSPs, which has shown that most sHSPs are actually located in the cytoplasm22. Further elucidation and clarification of sHSP candidates in wheat is therefore required.Recent studies have suggested that all flowering plants have undergone at least two ancient polyploidy events during their evolutionary historyWith the accumulation of genome data on hexaploid wheat and its relatives, systematic analyses of sHSP evolution and function in Triticeae have become practicable. Here, we demonstrate the evolutionary pattern and mechanism of sHSPs in Triticeae and provide evidence for massive intrachromosomal gene duplications, selection pressure variations and the expression pattern diversity during wheat polyploidization. Our results provide fundamental evidence that will be valuable for further wheat sHSP applications as well as for its evolutionary pattern variations with polyploidization.et al.30. Diploid progenitors were represented by T. urartu (AAuu) and Triticum monococcum (AAmm) as the wheat subgenome A donors, Ae. Tauschii (DD) as the wheat subgenome D donor, and Ae. sharonensis (SshSsh) and Ae. speltoides (SS) as the wheat subgenome B donors. Tetraploid progenitors were represented by T. turgidum  and T. turgidum  as the reference AABB genomes41.To characterize the copy number variation of sHSPs during wheat polyploidization, we performed comparative analyses on hexaploid wheat and its relatives (diploid and tetraploid). The evolutionary relationships among Triticeae were constructed Fig. , with reA. thaliana and rice22, 404 sHSP candidates were identified in Triticeae, including 321 sHSP genes and 83 Acd genes (which shared homology with the ACD domain of sHSPs but were divergent from sHSPs), respectively  of a tetraploid (AABB) and the DD progenitors, or the sum  of its three diploid progenitors  originated recently (around 0.8 Ma)30. Our result shows that the genome size of current tetraploid is close to the total of its two diploid progenitors, along with 13.1% (AAuu and SshSsh to AABBTdi)–31.2% (AAuu and SS to AABBTdc) gene loss  of nonsynonymous (dN) to synonymous (dS) substitution rates (ω = dN/dS) with codon-based models provided in the PAML programs46. The ω values estimated by the one-ratio model were 0.05 and 0.1 for the ACD domain and the full length proteins, respectively, indicating that strong purifying selection acted on this protein family to remove nonsynonymous mutations and to maintain their important biological functions  and the null model A were applied  and MI  subfamilies. The N-terminal region plays a major role in substrate recognition and binding11. Thus, the weaker purifying selection and the evident positive selection in the N-terminal region may confer greater flexibility in sHSPs, allowing the binding of more substrates.To evaluate the selection pressures that acted on each region of the sHSPs in Triticeae, we aligned the sHSP sequences of each subfamily to Ta16.9, the model used for sHSP studiesdue Fig. . The res34. To test the evolutionary patterns of sHSPs estimated by the above sequence analysis in this study, an integration transcriptional analysis was performed on published RNA-seq datasets from 28 hexaploid wheat samples, covering five tissues, three developmental stages and two types of abiotic stress  of hexaploid wheat are highly active and massive pseudogenization did not occurred after polyploidization , a similar proportion as seen with A. thaliana and rice sHSPs (87–90%)22. This finding indicates that expanded sHSPs in the current wheat genome maintain their conserved function in heat protection  adjusted ion Fig. . Consistion Fig. . The expion Fig. , and simion Fig. , indication Fig. , highligt-test p > 0.05, p = 0.1354 for CI and p = 0.1348 for CII, respectively), consistenting with the unaltered purifying seletions acted on them.The transcriptional profiles and tissue preference of sHSPs displayed strikingly consistency with the evolutionary analysis based on sequences variations, further supporting the purifying selection and differential evolution. In addition, the duplicated sHSPs of CI and CII subfamilies kept similar transcripton patterns with original sHSPs –31.2% (AAuu and SS to AABBTdc) gene loss 56. First, the keywords “alpha crystallin protein” and “small heat shock protein” were searched against each genome annotation to predict candidate sHSP genes. Then, we used the Hmmsearch program in HMMER57 to search for the family-specific HMM profiles of sHSPs (PF00011) downloaded from the Pfam database (http://pfam.xfam.org/)58, using each of the predicted proteomes as queries. These two results were merged to remove redundancy and examined for the ACD in the InterPro (http://www.ebi.ac.uk/interpro/) and PROSITE (http://prosite.expasy.org/) databases. The sequences in which ACD domain were detected in InterPro or PROSITE, were aligned using the M-Coffee program, which combines the output of popular alignment programs59. A phylogenetic tree was constructed based on the ACD using the neighbour-joining method and a bootstrap test of 1000 iterations, and the tree was presented by iTOL60. Triticeae sHSPs were grouped into different subfamilies by phylogenetic analysis using sHSPs from A. thaliana and rice as markers. Wheat sHSPs were mapped to the chromosome based on the annotation information and presented by Circos61.The bread wheat genome sequences and annotation information were downloaded from the EnsemblPlants database (ftp://ftp.ensemblgenomes.org/pub/plants/release-26/fasta/triticum_aestivum/dna/). The genomes and gene predictions of the wheat diploid and tetraploid relatives, 46 was used to estimate the ω values for Triticeae relatives and sHSP subfamilies. One-ratio model (model 0) which assumes a constant ω ratio along all branches and two-ratio model (model 2), which allows different ω ratios between foreground and background lineages, were used to detect different selective pressures between the duplicated clades. The site-specific discrete model (model 3), which assumes two classes of sites with different ω ratios, and the clade model (model D), which allows selective pressure at one class of sites (foreground clade) to be different from the rest of the phylogeny, were used to detect functional divergence after gene duplication. The branch-site model A (model A), which assumes one class of sites in the foreground lineage ω > 1 and null model A, with fixed ω = 1, were compared to detect positive selection in specific lineages. In PAML analysis, the protein sequence alignments were converted into corresponding codon alignments using the PAL2NAL program62. The Selecton program63 was used to detect the selective pressure acting on each amino acid residue and the SPEER-SERVER64 was used to identify the type I and type II residues that contribute to functional divergence between duplicated clades.The codeml program from the PAML4.7 package31, and the RNA-seq data from different cell types of grain at three development stages were downloaded from NCBI (Accession: PRJEB5135)29. RNA-seq data from wheat seedlings under heat and drought treatments were downloaded from NCBI (PRJNA257316)65. The quality of the public RNA-seq data was checked with the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The high-quality paired-end RNA-seq reads from each library were aligned to the wheat reference genome  using hisats v2.0.066 at “very-sensitive” preset parameters in the “end-to-end” mode. The intron length was set to 20 to 5000 nucleotides during alignment. Alignments between reads and wheat reference genome sequences were input into stringTie v1.1.167 for the normalization and estimation of the gene expression level. Gene expression levels were reported as fragments per kilobase of exon model per million mapped reads (FPKM). A gene was regarded as expressed in a sample if the FPKM was greater than zero. The wheat genome annotation information used in these analyses was obtained from the EnsemblPlants database (ftp://ftp.ensemblgenomes.org/pub/plants/release-26/gff3/triticum_aestivum). The expression data were presented by HemI68 and iTOL60.We performed expression profiling analysis of wheat sHSPs using high-throughput RNA-seq data. The RNA-seq data from five tissues  at three development stages were obtained from URGI (https://urgi.versailles.inra.fr/files/RNASeqWheat/)Chinese Spring, which had been subjected to heat stress, using a RNA extraction kit with TRIzol reagent . A FastQuant RT kit  was used to synthesize cDNA from the treated RNA with an oligo-dT primer in a 50-μl reaction. Real-time quantitative PCR primers were designed in Primer Premier 5.0 software. The CDS sequence of bread wheat is downloaded from the Ensemble database (ftp://ftp.ensemblgenomes.org/pub/release-26/plants/fasta/triticum_aestivum/cds/). The size of the PCR product was set to 100–200 bp and the length of the primer was 15–25 bp. A pre-PCR was performed using wheat cDNA as a template, and primer produced a single specific band was further used for real-time PCR. For PCR, 0.1–0.25 μM cDNA were amplified with specific primers for each of eight different gene models, with the 1 × SYBR Green Master Mix Kit  in a final volume of 12.5 μL.Total RNA was isolated from the leaves of 7-day seedlings of the hexaploid wheat variety Supplementary Figures and Tables


Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine. Previous studies have indicated that SSEA-4 and PODXL are two promising markers to isolate MSCs with stronger multipotency or clonogenicity910Chemical conversion of fibroblasts into other functional cell types has drawn substantial attention recently131415highPODXLhigh MSC-like cells , three iMSCs from these fibroblasts, and BMMSCs. We also retrieved the expression information of one BMMSC from the publicly available Gene Expression Omnibus (GEO) database. Principal component analysis (PCA) of stemness genes revealed that iMSCs derived from dermal fibroblasts were similar to each other, while the parental fibroblasts and BMMSCs were quite different . Of notehighPODXLhigh cells, but not SSEA-4lowPODXLlow cells, were able to efficiently differentiate into osteoblasts and adipocytes  consistently induced the conversion with an efficiency ranging from 26.4–64.0% in ten experiments (mean = 38%) . These iipocytes .highPODXLhigh iMSCs express traditional MSC markers by flow cytometry. The marker expression profiles of iMSCs were nearly identical to BMMSCs   , fulfill HLA-DR) , while a HLA-DR) . Both of HLA-DR) . In summ HLA-DR)  and surf HLA-DR)  both sugTo investigate if iMSCs are multipotent, we examined their ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Alkaline phosphatase (ALP) activity and Alizarin Red S staining (ARS) were examined to determine osteogenesis ability. ALP activity is required for bone formation in early osteogenesis; and ARS reveals the extent of calcium deposition, which is required for bone matrix formation in late osteogenesis. After osteogenic induction, iMSCs induced from fibroblasts CRL2097 exhibited ALP activity at day 10 and ARS at day 21 to an extent comparable to that of primary BMMSCs. In contrast, primary fibroblasts could not differentiate into osteoblasts . To detein vivo, an acute lung injury (ALI) mouse model was performed  are required for generating iMSCs. By adding one cytokine at a time, we found that all three growth factors, TGF-β, bFGF, and LIF, were dispensable for iMSC generation , since sTo avoid (1) insertional mutagenesis by virus infection or plasmid transfection, (2) the tedious processes of virus preparation, and (3) repeated transfection/transduction, the use of chemical/growth factors to convert fibroblasts has drawn substantial attention recently. To date, chemical/growth factor conversion has been performed in fibroblasts to generate iPSCs (40–60 days)1628293031In this study, we established the first method to chemically induce MSCs from fibroblasts in six days, which might be one of the fastest chemical conversion protocols of fibroblasts to date. Our data reveal that the cocktail containing six chemical inhibitors  with or without and three growth factors  can efficiently generate functional iMSCs from human primary dermal fibroblasts within 6 days .For the small molecules in the chemical cocktail that is capable of reprogramming human fibroblasts into iMSCs, all have been suggested to have the possibility to promote stemness by repressing differentiation or promoting stem cell expansion. For example, SB202190 is a specific inhibitor of p38 signaling. The p38 pathway is critical for inducing chondrogenesis, osteoblastogenesis, and neuronal differentiation of MSCs353440413443The six chemicals and three growth factors  we used also happened to be included as a part of the components in the naïve human stem cell medium (NHSM)45et al. reported that ectopic expression of OCT4 with GSK inhibitor, CHIR99021, could convert the human hematopoietic stem cells into iMSCset al. and Piccinato et al. also indicated that self-renewal and differentiation ability of MSCs could be upregulated under hypoxia condition with high expression of OCT4 and other factors22Although there is a lack of defined master regulator(s) in MSCs, several studies have indicated that pluripotency markers of ESCs, such as OCT4 and NANOG, might play critical roles in maintaining the potency and proliferative ability of MSCs. Meng, Like BMMSCs, iMSCs can differentiate into osteoblasts, adipocytes, and chondrocytes . Moreovein vivo function of our iMSCs748494951Due to the known immunosuppressive ability of MSCs, we applied the well-established endotoxin-induced acute lung injury model as in previous studies to test highPODXLhigh)   and had DXLhigh) . The iMSDXLhigh) . They alDXLhigh) . TherefoThe morphology of iMSCs was similar to BMMSCs and fibroblasts without senescence after the chemical induction. To obtain sufficient amounts of iMSCs for differentiation and mouse anti-sepsis assay, we expanded the induced cells in normal MSC culture medium for 6–8 passages. These iMSCs still expressed functional markers SSEA-4 and PODXL and maintained the morphology and proliferation ability at passage 8, suggesting that they are relatively stable and suitable for clinical applications.www.clinicaltrials.gov). Thus, chemically induced MSCs hold great promise for the treatment of acute lung injury (ALI)www.clinicaltrials.gov). Since the components of the conversion medium are well-defined and serum-free, our chemical reprogramming method allows standardization and reproducibility with great ease. The method also does not involve any processes that may lead to insertional mutagenesis, therefore we envision it to profoundly benefit the study of stem cell biology and regenerative medicine.To date, MSCs have been approved to treat GvHD in Canada and New Zealand, and degenerative arthritis and anal fistula in KoreaAll methods were performed in accordance with the relevant guidelines and regulations.All culture media (unless otherwise specified) were purchased from Invitrogen . All chemicals in the cocktails were purchased from LC Laboratories , except Go6983, which was obtained from TOCRIS  . All rec2. All primary cells were cultured within 10 passages after isolation. The informed consent forms were obtained from all the subjects by ATCC and Lonza.All experiments with primary human cells were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Human Subject Research Ethics, Academia Sinica  (ID: AS-IRB03–105122). Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC  and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) . Primary adult abdominal skin fibroblasts derived from two adult females (DF440547 and DF443480), who are 42 and 56 years old, respectively . These cells were cultured in DMEM-HG with 10% FBS and started in culture from passage 1. Human primary bone marrow mesenchymal stem cells (BMMSCs) were purchased from LONZA and were cultured with Dulbecco’s Modified Eagle Media-low glucose (DMEM-LG) medium containing 10% FBS. All cells were cultured at 37 °C under 5% CO2. To obtain sufficient amounts of iMSCs for differentiation assay and mouse anti-sepsis assay, the cells were expanded to 6–8 passages.For iMSC generation, primary fibroblasts maintained for less than 10 passages were cultured in DMEM-HG for 2 days. The culture medium was then replaced with the chemical cocktail medium (6C+3GF)  or diffehigh and PODXLhigh cells were isolated using a cell sorter . The data was further analyzed by FACSDiva software (BD Biosciences).For cell surface marker analysis, BMMSCs, iMSCs, and fibroblasts were incubated with FITC-conjugated anti-human SSEA-4  and PE-conjugated anti-human PODXL  antibodies. The cells were then analyzed by FACSCanto . SSEA-4In this assay, the decrease of seeding cell density increases the incidence of wells with no colonies. Sorted cells were seeded into 96-well plates containing 8 cell-dose groups: 0, 5, 10, 20, 40, 80, 160, 320 sorted cells per well. After incubation in optimal condition for 14 days, the wells with no colony were counted for each condition. The seeding cell number in which 37% of the wells are negative for colony formation is defined as the cell concentration of one CFU-F20® Human Genome U133A 2.0 chips following the manufacturer’s protocol . The array results were analyzed using the Gene Spring GX 12.6 software . The microarray profiling of BMMSC_2 was described previously in the Pubmed GEO database (GSM1533333), and all other array data were uploaded to the Pubmed GEO database (GSE72693).RNA was purified using an RNeasy kit . Samples were prepared for array hybridization using the human Affymetrix 3’ IVT Express kit with GeneChip4 cells/cm2) were cultured with osteogenic induction medium . The media were replaced twice per week during differentiation.Human primary BMMSCs and iMSCs were cultured in DMEM-LG medium with 10% FBS. Fibroblasts were cultured in DMEM-HG with 10% FBS. To induce differentiation, cells  staining was performed using alkaline phosphatase kits in accordance with the manufacturer’s instruction (Sigma-Aldrich). For the quantification of ALP activity, the cells were washed twice with PBS and incubated with the ALP substrate p-nitrophenyl phosphate (pNPP) at room temperature for 5–20 minutes. The absorbance at an optical density (O.D.) at 405 nm was measured.2O before being air dried. For quantification, the cells were incubated with 1 mL of cetylpyridinium chloride buffer for 1 h to extract ARS, and the O.D. at 550 nm was then recorded.After 21 days of osteogenic differentiation, cells were fixed with ice-cold 70% ethanol at −20 °C for 1 hour and then washed with PBS. The cells were then stained with 40 mM Alizarin Red S (ARS) (pH 4.2) for 10 minutes and subsequently washed five times with ddHFor adipogenic induction, cells were cultured in adipogenic induction medium , which was replaced twice per week during the 21-day differentiation period.Cells were fixed with 4% PFA for 1 hour, washed with 60% isopropanol, and then air dried. The lipid vesicles were stained with Oil Red O staining medium (30 ml of 0.5% oil red solution in 2-propanol diluted with 20 ml of water) for 10 minutes and then washed with distilled water. For quantification, Oil Red O was extracted with isopropanol, and the absorbance at 530 nm was measured.5 cells) in separate 15-mL tubes were centrifuged at 500 g for 10 minutes. The pelleted cells were then incubated with chondrogenic induction medium . The cells formed a spherical aggregate after overnight incubation. The cells were continuously induced for 21 days, and paraffin sections were taken to analyze the samples. After deparaffinization, the slides were stained with Hematoxylin-eosin or an Alcian blue solution.For chondrogenic differentiation, BMMSCs, iMSCs, and fibroblasts  for 30–45 minutes. The slides were then washed with water for 2 minutes, dehydrated with xylene, and mounted with mounting solution .E. coli O55:B5 (Sigma-Aldrich) or 100 μL PBS. At four hours after LPS treatment, mice were anesthetized again and then randomly divided into four groups: (1) PBS, (2) human fibroblasts (106 cells in 100 μL PBS), (3) iMSCs (106 cells in 100 μL PBS), and (4) human BMMSCs (106 cells in 100 μL PBS). The survival of mice was followed for 48 h. The survival rate of each group was observed every 6 hours. For the histology analysis and lung injury analysis, the samples were collected before or at 48 hours post injection. The lung sections were fixed and stained. The whole lung area was scanned by Aperio ScanScope XT machine . Then, Aperio ImageScope program (Leica) was used to analyze the total lung section under 4× zoom by adding 100 μm × 100 μm grid. Images of each lung section were further quantified by counting lung section area and inflammatory area. The injury area, which has alveolar and interstitial inflammation (immune cell number >5 in a 100 μm × 100 μm grid), was counted. The ratio of injury area in lung section is calculated as (injury area)/(lung section area). Injury score was determined by the following criteria: 0, no injury; 1, 25% injury; 2, 50% injury; 3, 75% injury; 4, 100% injury.All animal experimental procedures and methods were performed in accordance with the relevant guidelines and regulations. All animal protocols were approved by Academia Sinica Institutional Animal Care and Utilization Committee (Protocol ID: 15–07–840). BALB/c female mice  were first anesthetized with Tiletamine/Zolazepam (25 mg/kg) and xylazine (10 mg/kg) via an intraperitoneal route. Acute lung injury (ALI) was then induced by the intratracheal (i.t.) instillation of 40 mg/kg lipopolysaccharides (LPS) purified from To quantify the pro-inflammatory cytokine secretion levels, the antibody-coated beads in a high sensitivity MILLIPLEX kit  was used in accordance with the manufacturer’s protocol. To measure the amounts of selected cytokines , BALF from LPS-induced acute lung injury mice injected with PBS, fibroblasts, iMSCs, and BMMSCs respectively were collected. Then different interleukins antibody-coated beads were incubated with 100 μg total BALF protein for 16 hours and further analyzed by Luminex 200 (Merck)t-test, where p-value < 0.05 was considered a significant difference.All statistical data are presented as the mean ± standard deviation (S.D.) of at least three biological replicates. Statistically significant differences were assessed by Student’s unpaired two-tailed Accession codes: The microarray data have been deposited in GEO database under the accession code GSE72693.How to cite this article: Lai, P.-L. et al. Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts. Sci. Rep.7, 44534; doi: 10.1038/srep44534 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in obese children. Early diagnosis and treatment are essential for curing or slowing down the disease progression. The aim of the study was to assess the prevalence of NAFLD in this population and to identify anthropometrical and metabolic risk factors for NAFLD prediction and its development. The study included 108 obese children. Anthropometric measurements, NAFLD diagnosis (based on ALT level and/or liver ultrasound), and metabolic syndrome (MS) components were assessed in all patients. Patients were divided into groups with and without NAFLD.p = 0.089]. NAFLD patients had significantly greater waist circumference, WHR, and WHtR and significantly higher total cholesterol, triglyceride, and fasting insulin concentrations as well as higher glucose and insulin concentrations in 120 minutes of OGTT and higher HOMA-IR levels compared to group of patients without NAFLD. In NAFLD patients, MS was significantly more likely to be diagnosed than in group without NAFLD , but among the MS components only hypertriglyceridemia was significantly more frequently diagnosed in the group with NAFLD (p = 0.002). Among analysed parameters the best independent risk factor for NAFLD was fasting insulin concentration with the cut-off point = 18,9 uIU/ml (AUC = 0.829). NAFLD was diagnosed in 49 (45%) patients with similar prevalence in boys  and girls [22 (44.9%),  NAFLD is a very common disease in obese children. NAFLD predictive risk factors include increased waist circumference, elevated WHR and WHtR, and elevated total cholesterol, triglycerides, and fasting insulin as well as elevated glucose and insulin concentration in the OGTT and HOMA-IR index. NAFLD increases the risk of potential cardiovascular complications expressed by diagnosis of metabolic syndrome. The best independent predictive risk factor for diagnosing NAFLD in obese children is fasting insulin > 18.9 uIU/ml. Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in children . Due to NAFLD not only in adults but also in children is associated with severe metabolic disorders as hypertension, dyslipidaemia, increased risk of type 2 diabetes, metabolic syndrome, and cardiovascular diseases , 13. ForThe aim of the study was to assess the prevalence of NAFLD, as well as identifying additional predictive anthropometrical and metabolic risk factors for NAFLD development in the obese children.The study was prospective and included 108 children aged 6 to 18, hospitalized in our department between years 2012 and 2014, whose BMI exceeded 97 pc for sex and age, after informed consent. Children with acute infectious disease, chronic hepatitis of known etiology, using hepatotoxic drugs, or consuming alcohol were excluded from the study. A general medical examination, anthropometric measurements, laboratory tests, blood pressure measurements, and abdomen ultrasound imaging were performed in all subjects.All anthropometric measurements were made in the morning, with fasting with empty bladder in the upright position. Subjects were barefoot and lightly dressed. Height and weight were measured to the nearest 0,1 cm and 0,1 kg, respectively, by using calibrated scale and Harpenden stadiometer. Waist and hip circumference were measured to the nearest 0,5 cm using standard technique with nonelastic tape, at the end of normal expiration. Waist circumference (WC) was measured at a point midway between the lower border of the ribs and the iliac crest, and hip circumference was measured at the widest part of the hip. BMI, waist-hip ratio (WHR), and waist-to-height ratio (WHtR) were calculated. Standard deviations scores for height and BMI were calculated using the LMS method based on Polish reference values .μA) current was applied is painless and lasts for several seconds. Of the many variables, the following results were analysed: total body water in kg and % (TBW), fat mass in kg and fat%, fat-free mass in kg (FFM), muscle mass in kg, visceral fat in %, and basal metabolic rate in kJ (BMR). Moreover the following indicators were calculated: visceral fat to total body fat , basal metabolic rate per kilogram body weight (BMR/kg), and standard deviations scores for fat percentage (fat% Z-score = 2  ∗ (fat%  − 50 pc fat%)/(98 pc fat%  − 50 pc fat%)) based on children reference values [Body composition was evaluated by bioelectrical impedance using the Tanita MC 980 MA device-multifrequency segmental analyser. Estimates of body composition were obtained from the equipped software. Patient stand barefoot on the marked electrodes and in straightened hands held the handles equipped with electrodes. The measurement of the tissue impedance through which the six-frequency  and low intensity , gamma glutamyltransferase (GGT), total cholesterol (Tch), high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG), and bilirubin, were performed in hospital laboratory by using standard methods. Oral glucose tolerance test  was performed in all patients. Glucose and insulin concentration were measured during fasting and in 120 minutes of the test. In this study, elevated ALT levels were identified for ALT > 35 U/l in children aged 3–11 years and ALT > 40 U/l in children aged ≥ 11 years. Hypertriglyceridemia was defined as fasting triglycerides ≥ 150 mg/dl, and low HDL cholesterol as fasting HDL < 40 mg/dl in children aged < 16 years and <40 mg/dl for boys or <50 mg/dl for girls aged > 16 years . ImpaireBlood pressure (BP) was measured three times daily by the Korotkoff method using sphygmomanometer, on the right arm after 5–10 minutes of rest in the sitting position. The results were referred to Polish children reference values . PatientInsulin resistance was measured by homeostasis model assessment of insulin resistance (HOMA-IR) described by Matthews et al. , definedWe used the definition of the International Diabetes Federation (IDF) for the metabolic syndrome in children and adolescents . The firNAFLD was diagnosed based on ultrasound examination and/or elevated ALT. U, ANOVA Kruskal-Wallis, Spearman rank correlation tests were used to compare continuous variables. Chi-square test was used for qualitative variables. The receiver operating characteristics (ROC) analysis was used to verify the characteristics of the independent variables and to assess an appropriate cut-off. Statistical significance was set at p value less than 0.05.The statistical software (Statistica v. 12.5 and Microsoft Excel) was used in data analysis. Descriptive data were expressed as median, mean values, and standard deviations (SD) for continuous variables. Patients were divided into two groups: with and without NAFLD, which were then compared for anthropometric features, biochemical parameters, and metabolic syndrome components. Mann–Whitneyp = 0.074). The age distribution of the examined group is shown in n = 50) and females (n = 58) had similar BMI and height Z-scores while males had significantly higher mean WC and WHR. Among important anthropometrical data for obesity diagnosis, parameters of body composition males had significantly higher fat% and fat% Z-score; however no difference in visceral fat/fat% was observed.The study included 108 obese children (50 boys) aged 6 years and 2 months to 17 years and 10 months. Mean age was 14.24 ± 2.73 SD. Mean age of girls was 14.75 ± 2.79 SD and of boys 13.9 ± 2.64 SD. Girls were about one year older than boys, but the difference was not statistically significant  and (II) non-NAFLD patients with normal liver and normal ALT concentration .NAFLD was diagnosed in patients with hyperechogenic liver on ultrasound and/or elevated ALT concentration. Ultrasonographic features of fatty liver were diagnosed in 34 patients (16 girls and 18 boys) and elevated ALT in 30 patients (8 girls and 22 boys). In 15 patients (2 girls and 13 boys) both were reported. Based on proposed criteria, two groups were identified: (I) NAFLD patients (p = 0.089).NAFLD was diagnosed more often in boys than in girls; however the difference was insignificant , total body water (TBW), and basal metabolic rate (BMR); however BMR per kilogram remained similar in both groups. The comparison of the two groups in terms of basic anthropometric and body composition parameters is presented in NAFLD patients did not differ significantly from patients without NAFLD in age, height, body weight, BMI, or BMI Of the biochemical parameters, the serum concentration of total cholesterol, triglycerides, fasting insulin, glucose, and insulin in 120 minutes of OGTT and HOMA-IR were significantly higher in patients with NAFLD.p = 0.00 for HOMA-IR > 90 percentile; 85% versus 15%, p = 0.00 for HOMA-IR > 97 percentile). A detailed summary of the biochemical parameters is presented in NAFLD was significantly more often diagnosed in patients with HOMA-IR exceeding reference values  than in The ROC analysis was performed on variables that differentiated NAFLD patients from non-NAFLD ones. Based on the area under receiver operating curves (AUC) we established a threshold of each variable that showed the highest accuracy for identifying children at high risk of NAFLD. p = 0.04), but of the MS components only hypertriglyceridemia was significantly more often diagnosed in NAFLD patients. The frequency of the MS and its components in the study population is presented in In the study population 34 (31.48%) patients were diagnosed with metabolic syndrome (MS). Of the metabolic syndrome components abdominal obesity and hypertriglyceridemia were most often recognised. MS was diagnosed significantly more frequently in NAFLD group than in patients without fatty liver  and specificity (84–100%) of ultrasonography make this examination suitable for screening . AlanineIn this study we diagnosed NAFLD in 45% of patients. Such a high prevalence is caused by both group selection  and diagnostic criteria. Fatty liver on ultrasound was reported in 34 (31.48%) patients, and elevated ALT levels were reported in 30 (27.78%) patients.The authors of meta-analysis from 2015 , which ip = 0.31). However, in that study prepubertal population was analysed, which was significantly younger than in other studies. In our study prevalence of NAFLD was similar in males and females, which could have come from the sex distribution in age groups .Males have higher prevalence of NAFLD than females, which is confirmed by most studies , 24, 30,Z-score. There was also no difference in the most important parameters of the body composition . Children with NAFLD had only significantly higher fat-free mass, total body water content, and BMR (but not BMR/kg); however, these parameters, due to their weight, age, or gender dependence, have little diagnostic value.In our study NAFLD patients were similar to non-NAFLD ones in age and basic anthropometric parameters such as height, weight and even BMI, and BMI The only anthropometric parameters discriminating NAFLD patients from non-NAFLD, in our study, are waist circumference (WC) and the indicators associated with WC, WHR and WHtR. These are important, practical pieces of information indicating that BMI, even referred to reference values, is not sufficient for obese patients, while WC, which is still not a routine medical examination, may be an effective tool for detecting patients at risk of metabolic disorders, including NAFLD. Many studies have shown that waist circumference may be used in central obesity screening, cardiovascular risk assessment in children , and NAFp < 0.0001) but, in contrast to our results, NAFLD patients had significantly higher BMI-Z score  [Similar results were obtained by Italian and Taiwanese researchers who did not observe differences in BMI and body composition between the groups with and without NAFLD, but waist circumference was significantly higher in NAFLD group , 34. Den 0.0001) .Considering the complexity of links between NAFLD, insulin resistance (IR), and type 2 diabetes, it is extremely difficult to find out whether NAFLD is the cause or the effect of insulin resistance. For many years, NAFLD has been treated as the hepatic consequence of IR, but recent studies suggest that hepatic steatosis may precede type 2 diabetes and metabolic syndrome and may even be a risk factor for their development , 15.μIU/ml with 75% sensitivity and 87.3% specificity diagnosed NAFLD. Studies in obese patients [μIU/ml ± 9.4, while in Pacifico et al. it was 10.6 μIU/ml in obese children without steatosis and 15.6 μIU/ml in obese children with NAFLD [μIU/ml ± 6.0 in obese children without NAFLD and 24.8 μIU/ml + 14.6 in children with NAFLD [μIU/ml ± 6.76 in children without steatosis versus 28.48 μIU/ml ± 21.53 in children with NAFLD).In our survey in diagnostics of carbohydrate metabolism fasting glucose remained useless, which was confirmed in other studies , 36. Sigpatients , 36, 37 patients  mean fasNAFLD patients had significantly higher HOMA-IR values compared to non-NAFLD ones. Similar observations have been made by Denzer et al.  and D'Adp = 0.00 for HOMA-IR > 90 pc; 85% versus 15%, p = 0.00 for HOMA-IR > 97 pc), and HOMA-IR > 4.089 is a good indicator of NAFLD .It is proven that insulin resistance increases in obese patients; therefore separate standards are developed for the population of obese children . Authorsp < 0.05), but no correlation was found between the number of metabolic syndrome criteria and the prevalence NAFLD (p = 0.052), which may be caused by the fact that only fasting glucose but not the insulin resistance is considered in the IDF criteria for MS.Metabolic syndrome (MS) in adult patients increases the risk of cardiovascular disease. In the pediatric population such conclusions are not clear. Due to both lack of long-term follow-up of pediatric patients and different criteria for MS, some authors suggest that the prevalence of individual components of metabolic syndrome in the pediatric population would be more relevant for assessment of the cardiovascular risk than the diagnosis of metabolic syndrome , 39. In NAFLD is a very common disease in obese children. NAFLD risk factors include increased waist circumference, elevated WHR and WHtR, and elevated total cholesterol, triglycerides, and fasting insulin as well as glucose and insulin concentration in 120 min of OGTT and HOMA-IR index. NAFLD increases the risk of potential cardiovascular complications expressed by diagnosis of metabolic syndrome. The best independent risk factor for diagnosing NAFLD in obese children is fasting insulin concentration > 18.9 uIU/ml.In our study, we focused on the diagnosis of obese children in the context of metabolic disorders, especially related to obesity and fatty liver disease. The prevalence of NAFLD and other metabolic disorders in the study population indicates the need to improve diagnostics in obese children already at primary health care level. Simple diagnostic methods such as waist circumference measurement and fasting plasma insulin concentration may contribute to the early identification and prediction of patients at risk of NAFLD and other metabolic complications. Appropriate therapy and lifestyle change in these patients and their families will help to prevent the negative effects of obesity in the future.


Mycobacterium tuberculosis infection was receiving oral isoniazid 300 mg daily. She was admitted to the hospital for epigastric and right sided flank pain of one-week duration. Laboratory results and imaging confirmed hepatitis. After ruling out all other possible causes, she was diagnosed with isoniazid-induced acute hepatitis . After discharge, the patient was readmitted and suffered from severe coagulopathy, metabolic acidosis, acute kidney injury, hepatic encephalopathy, and cardiorespiratory arrest necessitating two rounds of cardiopulmonary resuscitation. Despite maximal hemodynamic support, the patient did not survive. A review of the literature, from several European countries and the United States of America, revealed a low incidence of mortality due to isoniazid-induced hepatotoxicity when used as a single agent for latent Mycobacterium tuberculosis infection. As for the management, the first step consists of withdrawing isoniazid and rechallenge is usually discouraged. Few treatment modalities have been proposed; however there is no robust evidence to support any of them. Routine monitoring for hepatotoxicity in patients receiving isoniazid is warranted to prevent morbidity and mortality.This paper describes a case of an acute and fatal isoniazid-induced hepatotoxicity and provides a review of the literature. A 65-year-old female diagnosed with latent Mycobacterium tuberculosis organisms. Due to its significant potential in reducing morbidity, the use of INH in treating latent Mycobacterium tuberculosis infection (LTBI) is recommended as a first-line option since the 1965 American Thoracic Society guidelines especially in high risk individuals [Isoniazid (INH) is an antituberculosis agent that is commonly used for the treatment and prophylaxis of tuberculosis (TB). It inhibits bacterial cell wall synthesis, thus killingSince its introduction in 1952, several cases of INH-induced hepatotoxicity were reported . Drug-inMultiple risk factors may contribute to INH-related hepatotoxicity. Some of them have conflicting evidence such as racial difference –15 and fReports of severe and fatal hepatitis associated with INH emerged after the 1970 TB outbreak in Capitol Hill in Washington, DC, among workers who were receiving it for prophylaxis. Nineteen patients developed signs and symptoms of liver damage; and two of them died .Accordingly, the guidelines published in 1971 were updated to include pretreatment screening and monitoring to reduce the risk of such complications . In 1979The most recent American Thoracic Society guidelines (2003) recommends INH 300 mg monotherapy daily for 9 months for human immunodeficiency virus (HIV) negative and positive individuals. Routine baseline and follow-up laboratory monitoring is only recommended for those who are HIV positive, have chronic liver disease, and are regular alcohol consumers or pregnant . A recenHere, we present a probable case, as determined by the Naranjo adverse drug reaction probability scale score , of INH-We present the case of a 65-year-old  Sri Lankan black woman, known to have LTBI treated with INH and dyslipidemia. She was diagnosed with LTBI based on a positive purified protein derivative test. The patient's main indication for LTBI treatment was recent arrival (within 5 years) from a high prevalence country. She presented to the emergency department with epigastric and right sided flank pain of one-week duration. The history goes back to one week prior to admission when the patient started to experience pain along with one episode of vomiting and dark (tea-colored) urine without any other urinary symptoms. She reported a severe pain of 8/10, relieved by sitting and lying forward and worsened by lying backwards. The patient denied any nausea, diarrhea, constipation, or fever prior to the presentation.Her past medical history includes LTBI  and dyslipidemia without any known liver disease. Her medications are Rimifon® (INH) 150 mg two tablets by mouth once daily and Panadol® (Acetaminophen) 500 mg two tablets by mouth as needed for headache. The patient was previously on Liponorm® (Atorvastatin) 10 mg one tablet by mouth once daily and Neurobion®  both of which were discontinued when INH was initiated (6 months prior to the presentation). The patient works as a housekeeper; she denied any known drug or food allergies, the use of recreational drugs, tobacco smoke, or alcohol.Her vital signs were as follows: blood pressure of 120/78 mmHg, heart rate of 83 beats per minute, respiratory rate of 15 breaths per minute, and a body temperature of 36.2°C. Her review of systems was normal except for icteric sclera, pale conjunctiva, and epigastric tenderness. Pending laboratory results, she was given Perfalgan® (acetaminophen) 1 g IV STAT for pain and normal saline 0.9% 1 L every 24 hours. The patient's laboratory data upon admission are shown in The patient was admitted to the internal medicine service for further investigation. INH was discontinued and medication orders were the following: Profenid® (ketoprofen) 100 mg IV every 8 hours as needed for pain, Nexium® (Esomeprazole) 40 mg IV once daily, and Buscopan® (Scopolamine Butylbromide) 1 ampoule of 20 mg IV every 8 hours as needed for abdominal pain. Based on negative infectious serology, hepatitis A, hepatitis B, hepatitis C, cytomegalovirus (CMV), and Epstein-Barr virus were all ruled out. The patient had a positive CMV IgG Ab (160 AU/mL) suggesting a previous infection. Hepatic autoimmune disease was ruled out as well based on negative immunology markers. An abdominal ultrasound showed a normal liver size of 11 cm, perihepatic fluid effusion, and pericholecystic edema that are most probably related to hepatitis rather than cholecystitis. CT scan of the abdomen and pelvis showed a diffusely thickened gallbladder wall with neither the evidence of obstructive process nor portal vein thrombosis. After ruling out all the possible causes of acute hepatitis in our patient, the final diagnosis was made as INH-induced acute hepatitis.Throughout the hospital stay, there was a slight reduction (around 15% from baseline) in liver function tests (LFTs); however INR and bilirubin remained elevated. The patient was clinically improving and her laboratory values were slightly better. Upon physician's order, the patient was discharged on Nexium 40 mg one tablet by mouth daily for one week. The patient did not resume the isoniazid treatment upon discharge after the initial hospital admission.γ-GTP 95 U/L, ALP 283 U/L, ALT (SGPT) 873 U/L, AST (SGOT) 1582 U/L, amylase 164 U/L, lipase 65 U/L, Cr 0.66 mg/dL, and INR 2.28. Bilirubin was also detected in urine. Accordingly, the patient was admitted to the internal medicine floor for fulminant hepatic failure due to INH.Six days later, the patient was readmitted to the hospital for abdominal distention and increased upper right quadrant pain that radiated to the flanks and increases with food intake and exertion. She also reported decreased appetite, fatigue, and dyspnea with exertion. Upon readmission, she presented with jaundice, icteric sclera, dark urine, and prominent ascites confirmed by wave fluid test. In the ER, she received Nexium 40 mg IV, Profenid 100 mg IV STAT, and NS 0.9% 500 mL every 24 hours which was increased to 1 L every 24 hours when her blood pressure dropped to 85/55 mmHg. Laboratory values were the following: total bilirubin 27.8 mg/dL, In an attempt to normalize INR, multiple administrations of Konakion® (Phytomenadione) were given. Profenid was discontinued due to the increased risk of bleeding and Buscopan 20 mg IV every 8 hours as needed for abdominal pain was ordered. Furthermore, Primperan® (Metoclopramide) 10 mg IV STAT, one vial of Albumin (20%) IV STAT, and morphine sulfate 2 mg SC STAT twice for pain were given. The patient had several episodes of hypotension responsive to fluid resuscitation with NS. The ultrasound showed diffused and clear moderate ascites as well as marked edematous thickening of the gallbladder wall with clear content related to intra-abdominal fluid effusion. The patient developed hypoglycemia (32–37 mg/dL) and oxygen saturation dropped to 86%. Due to deterioration, reduced level of consciousness, and poor response to oxygen, she was intubated and transferred to the intensive care unit. Escherichia coli and sputum cultures showed α-Streptococcus and Neisseria species. The patient had cardiorespiratory failure necessitating cardiopulmonary resuscitation (CPR). Spontaneous circulation was achieved after 15 minutes of CPR. Unfortunately, this was followed by a second cardiac arrest and despite maximal hemodynamic support, the patient passed away.On physical exam she was unresponsive, her pupils were middilated, and nonreactive, her lungs had few scattered rhonchi, and she had abdominal distention and ascites. She had metabolic acidosis (pH 6.8) and multiple doses of sodium bicarbonate were administered. The patient was consistently hypoglycemic and hypotensive despite fluid resuscitation and vasopressor administration. Serum creatinine increased from 0.66 mg/dL to 2.34 mg/dL in two days confirming acute kidney injury. She was also suffering from severe coagulopathy and hepatic encephalopathy. Additionally, blood cultures revealed a sensitiveR = ALT/ULN ÷ ALP/ULN) to differentiate between three different types of drug-induced liver injury (DILI): acute hepatocellular (R ≥ 5), mixed (R 2–5), or cholestatic (R ≤ 2) [R score was 10 confirming the acute hepatocellular injury as expected. Other reports show latency period of approximately one week to three months; in most cases, however, the INH-induced hepatotoxicity can occur up to one year later or more [The American College of Gastroenterology (ACG) clinical guidelines recommend the use of a specific formula ( (R ≤ 2) . The mos (R ≤ 2) . The for (R ≤ 2) –36. In c (R ≤ 2) . Finally (R ≤ 2) , 33. The or more . An acut or more .We presented the case of a 65-year-old female with acute INH-induced hepatic failure that progressed dramatically in a short period of time leading to death. As mentioned earlier, hepatic involvement is a common side effect of INH. The patient's Naranjo adverse drug reaction probability score was 7, indicating a probable association . SimilarA recent case report presents a 53-year-old Japanese male receiving INH for LTBI . SimilarThe Center for Disease Control and Prevention published a report that quantified the frequency of severe adverse effects in patient receiving isoniazid for LTBI treatment during 2004–2008 . Severe According to The Diagnosis and Management of Idiosyncratic Drug-Induced Liver Injury published by the ACG, it is strongly discouraged to rechallenge a patient with a drug that was considered to be a causative agent for hepatotoxicity especially when the injury is severe (LFTs > 5x ULN). However, exceptions do occur in life-threatening situations when no other alternative is available . As for A 4-month regimen of rifampin can be considered for persons who cannot tolerate INH, who have been exposed to INH-resistant tuberculosis, or who require a shortened duration of therapy for better completion rates . It shouWe reported a case of acute, fulminant, and fatal hepatotoxicity due to INH in a 65-year-old female with LTBI. Although hepatotoxicity is a well-known side effect of INH, mortality rates remain low as reported in the literature. It is important for clinicians and pharmacists to appropriately follow up patients, counsel them on the signs and symptoms of hepatotoxicity, and encourage them to report it.