A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy.

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Precancerous nature of intestinal metaplasia with increased chance of conversion and accelerated DNA methylation.

OBJECTIVE: The presence of intestinal metaplasia (IM) is a risk factor for gastric cancer. However, it is still controversial whether IM itself is precancerous or paracancerous. Here, we aimed to explore the precancerous nature of IM by analysing epigenetic alterations. DESIGN: Genome-wide DNA methylation analysis was conducted by EPIC BeadArray using IM crypts isolated by Alcian blue staining. Chromatin immunoprecipitation sequencing for H3K27ac and single-cell assay for transposase-accessible chromatin by sequencing were conducted using IM mucosa. NOS2 was induced using Tet-on gene expression system in normal cells. RESULTS: IM crypts had a methylation profile unique from non-IM crypts, showing extensive DNA hypermethylation in promoter CpG islands, including those of tumour-suppressor genes. Also, the IM-specific methylation profile, namely epigenetic footprint, was present in a fraction of gastric cancers with a higher frequency than expected, and suggested to be associated with good overall survival. IM organoids had remarkably high NOS2 expression, and NOS2 induction in normal cells led to accelerated induction of aberrant DNA methylation, namely epigenetic instability, by increasing DNA methyltransferase activity. IM mucosa showed dynamic enhancer reprogramming, including the regions involved in higher NOS2 expression. NOS2 had open chromatin in IM cells but not in gastric cells, and IM cells had frequent closed chromatin of tumour-suppressor genes, indicating their methylation-silencing. NOS2 expression in IM-derived organoids was upregulated by interleukin-17A, a cytokine secreted by extracellular bacterial infection. CONCLUSIONS: IM cells were considered to have a precancerous nature potentially with an increased chance of converting into cancer cells, and an accelerated DNA methylation induction due to abnormal NOS2 expression.

Research on Classification Algorithm of Silicon Single-Crystal Growth Temperature Gradient Trend Based on Multi-Level Feature Fusion.

In the process of silicon single-crystal preparation, the timely identification and adjustment of abnormal conditions are crucial. Failure to promptly detect and resolve issues may result in a substandard silicon crystal product quality or even crystal pulling failure. Therefore, the early identification of abnormal furnace conditions is essential for ensuring the preparation of perfect silicon single crystals. Additionally, since the thermal field is the fundamental driving force for stable crystal growth and the primary assurance of crystal quality, this paper proposes a silicon single-crystal growth temperature gradient trend classification algorithm based on multi-level feature fusion. The aim is to accurately identify temperature gradient changes during silicon crystal growth, in order to promptly react to early growth failures and ensure the stable growth of high-quality silicon single crystals to meet industrial production requirements. The algorithm first divides the temperature gradient trend into reasonable categories based on expert knowledge and qualitative analysis methods. Then, it fuses the original features of actual production data, shallow features extracted based on statistical information, and deep features extracted through deep learning. During the fusion process, the algorithm considers the impact of different features on the target variable and calculates mutual information based on the difference between information entropy and conditional entropy, ultimately using mutual information for feature weighting. Subsequently, the fused multi-level feature vectors and their corresponding trend labels are input into a Deep Belief Network (DBN) model to capture process dynamics and classify trend changes. Finally, the experimental results demonstrate that the proposed algorithm can effectively predict the changing trend of thermal field temperature gradients. The introduction of this algorithm will help improve the accuracy of fault trend prediction in silicon single-crystal preparation, thereby minimizing product quality issues and production interruptions caused by abnormal conditions.

HSD17B4 methylation enhances glucose dependence of BT-474 breast cancer cells and increases lapatinib sensitivity.

PURPOSE: HER2-positive breast cancer has a high chance of achieving pathological complete response when HSD17B4, responsible for peroxisomal ß-oxidation of very long-chain fatty acids (VLCFA) and estradiol, is methylation-silenced. Here, we aimed to identify the underlying molecular mechanism. METHODS: Using a HER2-positive breast cancer cell line, BT-474, control and knock-out (KO) clones were obtained. Metabolic characteristics were analyzed using a Seahorse Flux analyzer. RESULTS: HSD17B4 KO suppressed cellular proliferation, and enhanced sensitivity to lapatinib approximately tenfold. The KO led to accumulation of VLCFA and a decrease of polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid. HSD17B4 KO increased Akt phosphorylation, possibly via decreased DHA, and genes involved in oxidative phosphorylation (OxPhos) and electron transport chain (ETC) were upregulated. Increased mitochondrial ATP production in the KO cells was confirmed by extracellular flux analyzer. Increased OxPhos led to severe dependence of the KO cells on pyruvate from glycolysis. Suppression of glycolysis by lapatinib led to severe delayed suppression of OxPhos in KO cells. CONCLUSION: HSD17B4 KO in BT-474 cells caused a decrease of PUFAs, increased Akt phosphorylation, enhanced glucose dependence of OxPhos, and increased sensitivity to inhibition of HER2, upstream of Akt. This mechanism may be applicable to other HER2-positive glucose-dependent breast cancer cells with HSD17B4 silencing.

Nickel-Catalyzed Three-Component Tandem Radical Cyclization 1,5-Difunctionalization of 1,3-Enynes and Alkyl Bromide.

A nickel-catalyzed three-component tandem radical cyclization reaction of aryl bromides with 1,3-enynes and aryl boric acids to construct γ-lactam-substituted allene derivatives has been described. This protocol provides lactam alkyl radicals through the free radical cyclization process, which can be effectively used to participate in the subsequent multicomponent coupling reaction so that 1,3-enynes could directly convert into corresponding poly-substituted allene compounds. In addition, this efficient method enjoys a broad substrate scope and provides a series of 1,5-difunctionalized allenes in a one-pot reaction.

The methylation level of a single cancer risk marker gene reflects methylation burden in gastric mucosa.

BACKGROUND: Gastric cancer risk can be accurately predicted by measuring the methylation level of a single marker gene in gastric mucosa. However, the mechanism is still uncertain. We hypothesized that the methylation level measured reflects methylation alterations in the entire genome (methylation burden), induced by Helicobacter pylori (H. pylori) infection, and thus cancer risk. METHODS: Gastric mucosa of 15 healthy volunteers without H. pylori infection (G1), 98 people with atrophic gastritis (G2), and 133 patients with gastric cancer (G3) after H. pylori eradication were collected. Methylation burden of an individual was obtained by microarray analysis as an inverse of the correlation coefficient between the methylation levels of 265,552 genomic regions in the person's gastric mucosa and those in an entirely healthy mucosa. RESULTS: The methylation burden significantly increased in the order of G1 (n = 4), G2 (n = 18), and G3 (n = 19) and was well correlated with the methylation level of a single marker gene (r = 0.91 for miR124a-3). The average methylation levels of nine driver genes tended to increase according to the risk levels (P = 0.08 between G2 vs G3) and was also correlated with the methylation level of a single marker gene (r = 0.94). Analysis of more samples (14 G1, 97 G2, and 131 G3 samples) yielded significant increases of the average methylation levels between risk groups. CONCLUSIONS: The methylation level of a single marker gene reflects the methylation burden, which includes driver gene methylation, and thus accurately predicts cancer risk.

Prediction of tissue origin of adenocarcinomas in the esophagogastric junction by DNA methylation.

BACKGROUND: Prediction of tissue origin of esophagogastric junction (EGJ) adenocarcinomas can be important for therapeutic decision, but no molecular marker is available. Here, we aimed to develop such a marker taking advantage of tissue-specific profiles of DNA methylation. METHODS: DNA methylation profiles of gastric adenocarcinomas (GACs) were obtained by an Infinium HumanMethylation450 BeadChip array, and those of esophageal adenocarcinoma (EACs) were obtained from the TCGA database. DNA from formalin-fixed paraffin-embedded (FFPE) samples was analyzed by bisulfite pyrosequencing. RESULTS: In the screening set, 51 of 145,841 CpG sites in CpG islands were methylated at significantly higher levels in 30 GACs compared to those in 30 EACs. Among them, SLC46A3 and cg09177106 were unmethylated in all the 30 EACs. Predictive powers of these two markers were successfully confirmed in an independent validation set (18 GACs and 18 EACs) (SLC46A3, sensitivity = 77.8%, specificity = 100%; cg09177106, sensitivity = 83.3%, specificity = 94.4%), and could be applied to FFPE samples (37 GACs and 18 EACs) (SLC46A3, P = 0.0001; cg09177106, P = 0.0028). On the other hand, EAC-specific markers informative in the FFPE samples could not be isolated. Using these GAC-specific markers, nine of 46 (19.6%) TCGA EGJ adenocarcinomas were predicted to be GACs. CONCLUSIONS: Two GAC-specific markers, SLC46A3 and cg09177106, had a high specificity for identifying the tissue origin of EGJ adenocarcinoma.

Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease.

End-stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of 2 major histopathological types: clear-cell RCCs (ESRD-ccRCCs) and acquired cystic disease (ACD)-associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. Here, we investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD-ccRCCs and 7 ACD-associated RCCs to identify their molecular alterations and cellular origins. Targeted sequencing of 409 cancer-related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD-ccRCCs harbored frequent VHL mutations, while ACD-associated RCCs did not. CNA analysis showed that ESRD-ccRCCs had a frequent loss of chromosome 3p while ACD-associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD-ccRCCs had methylation profiles similar to those of sporadic ccRCCs, while ACD-associated RCCs had profiles similar to those of papillary RCCs. Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD-ccRCCs and ACD-associated RCCs had high expression of proximal tubule cell marker genes, while chromophobe RCCs had high expression of distal tubule cell/collecting duct cell marker genes. In conclusion, ESRD-ccRCCs and ACD-associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these 2 histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron.

Progress on Range of Motion After Total Knee Replacement by Sensor-Based System.

For total knee replacement (TKR) patients, rehabilitation after the surgery is key toregaining mobility. This study proposes a sensor-based system for effectively monitoringrehabilitation progress after TKR. The system comprises a hardware module consisting of thetriaxial accelerometer and gyroscope, a microcontroller, and a Bluetooth module, and a softwareapp for monitoring the motion of the knee joint. Three indices, namely the number of swings, themaximum knee flexion angle, and the duration of practice each time, were used as metrics tomeasure the knee rehabilitation progress. The proposed sensor device has advantages such asusability without spatiotemporal constraints and accuracy in monitoring the rehabilitation progress.The performance of the proposed system was compared with the measured range of motion of theCybex isokinetic dynamometer (or Cybex) professional rehabilitation equipment, and the resultsrevealed that the average absolute errors of the measured angles were between 1.65° and 3.27° forthe TKR subjects, depending on the swing speed. Experimental results verified that the proposedsystem is effective and comparable with the professional equipment.

Biosynthesis of polyketides by two type III polyketide synthases from Aloe barbadensis.

Various bioactive polyketides have been found in Aloe barbadensis. However, the polyketide synthases (PKSs), which participate in biosynthesis of polyketides in A. barbadensis remain unknown. In this study, two type III PKSs (AbPKS1 and AbPKS2) were identified from A. barbadensis. AbPKS1 and AbPKS2 were able to utilize malonyl-CoA to yield heptaketides (TW93a and aloesone) and octaketides (SEK4 and SEK4b), respectively. AbPKS1 also exhibited catalytic promiscuity in recognizing CoA thioesters of aromatics to produce unusual polyketides. What Is more, a whole cell biocatalysis system with the capability of producing 26.4 mg/L of SEK4/SEK4b and 2.1 mg/L of aloesone was successfully established.

Photophysical Identification of Three Kinds of Low-Energy Green Band Defects in Wide-Bandgap Polyfluorenes.

Blue-light-emitting semiconductors based on polyfluorenes often exhibit an undesired green emission band. In this report, three well-defined oligofluorenes corresponding to three types of "defects" attributed to aggregation, keto formation, and chain entanglement, respectively, are systemically investigated to unveil the origins of the green emission band in fluorene-based materials. First, the optical properties of defect molecules in different states are studied. The defect associated with aggregation is absent in dilute solutions and in films doped at 0.01 wt % with poly(methyl methacrylate). Second, the dependence of the emission spectra on the solvent was monitored to compare the effects of the "keto-" and "chain-entanglement defect" molecules. The green emission of keto defects exhibited a strong dependence on solvent polarity, whereas this cannot be observed in case of chain-entanglement defect. Third, energy transfer between poly[4-(octyloxy)-9,9-diphenylfluoren-2,7-diyl]- co-[5-(octyloxy)-9,9-diphenyl-fluoren-2,7-diyl] and the keto or chain-entanglement defect molecules is illustrated. Compared to those of the chain-entanglement defect, the spectra of the keto defect molecule (1:10-3) show signs of defect emission at lower proportions. These investigations not only provide insight into the photophysics of oligofluorenes but also supply a new strategy to explore defects in semiconductor polymers, which will aid in the development of effective approaches to obtain stable, pure blue organic light-emitting diodes based on polyfluorenes.

Sperm-inherited organelle clearance in C. elegans relies on LC3-dependent autophagosome targeting to the pericentrosomal area.

Macroautophagic degradation of sperm-inherited organelles prevents paternal mitochondrial DNA transmission in C. elegans. The recruitment of autophagy markers around sperm mitochondria has also been observed in mouse and fly embryos but their role in degradation is debated. Both worm Atg8 ubiquitin-like proteins, LGG-1/GABARAP and LGG-2/LC3, are recruited around sperm organelles after fertilization. Whereas LGG-1 depletion affects autophagosome function, stabilizes the substrates and is lethal, we demonstrate that LGG-2 is dispensable for autophagosome formation but participates in their microtubule-dependent transport toward the pericentrosomal area prior to acidification. In the absence of LGG-2, autophagosomes and their substrates remain clustered at the cell cortex, away from the centrosomes and their associated lysosomes. Thus, the clearance of sperm organelles is delayed and their segregation between blastomeres prevented. This allowed us to reveal a role of the RAB-5/RAB-7 GTPases in autophagosome formation. In conclusion, the major contribution of LGG-2 in sperm-inherited organelle clearance resides in its capacity to mediate the retrograde transport of autophagosomes rather than their fusion with acidic compartments: a potential key function of LC3 in controlling the fate of sperm mitochondria in other species.

Tanshinol alleviates impaired bone formation by inhibiting adipogenesis via KLF15/PPARγ2 signaling in GIO rats.

Glucocorticoid (GC)-induced osteoporosis (GIO) is characterized by impaired bone formation, which can be alleviated by tanshinol, an aqueous polyphenol isolated from Salvia miltiorrhiza Bunge. In this study we investigated the molecular mechanisms underlying GC-induced modulation of osteogenesis as well as the possibility of using tanshinol to interfere with GIO. Female SD rats aged 4 months were orally administered distilled water (Con), prednisone (GC, 5 mg·kg-1·d-1), GC plus tanshinol (Tan, 16 mg·kg-1·d-1) or GC plus resveratrol (Res, 5 mg·kg-1·d-1) for 14 weeks. After the rats were sacrificed, samples of bone tissues were collected. The changes in bone formation were assessed using Micro-CT, histomorphometry, and biomechanical assays. Expression of Kruppel-like factor 15 (KLF15), peroxisome proliferator-activated receptor γ 2 (PPARγ 2) and other signaling proteins in skeletal tissue was measured with Western blotting and quantitative RT-PCR. GC treatment markedly increased the expression of KLF15, PPARγ2, C/EBPα and aP2, which were related to adipogenesis, upregulated FoxO3a pathway proteins (FoxO3a and Gadd45a), and suppressed the canonical Wnt signaling (ß-catenin and Axin2), which was required for osteogenesis. Thus, GC significantly decreased bone mass and bone quality. Co-treatment with Tan or Res effectively counteracted GC-impaired bone formation, suppressed GC-induced adipogenesis, and restored abnormal expression of the signaling molecules in GIO rats. We conclude that tanshinol counteracts GC-decreased bone formation by inhibiting marrow adiposity via the KLF15/PPARγ2/FoxO3a/Wnt pathway.

Detection of trapped charges in the blend films of polystyrene/SFDBAO electrets by electrostatic and Kelvin probe force microscopy.

The charge trapping properties of the blend of polystyrene (PS) and a sterically hindered organic semiconductor SFDBAO (spiro[fluorene-9,7-dibenzo[c,h]acridin-5-one]) are investigated by electrostatic and Kelvin probe force microscopy (EFM and KPFM). EFM signals of trapped charge spots injected with controllable tip biases, which are recorded with different dissipation times t, the percent of SFDBAO in blends, and the scanning tip bias, have been measured. By the quantitative analysis, the excellent trapped charge density of PS/SFDBAO blend films for the holes (∼×10(-5) C m(-2)) is much higher than that of the SFDBAO film (∼×10(-6) C m(-2)) and the PS film (∼×10(-7) C m(-2)). However, the trapped charge density of electrons (∼×10(-7) C m(-2)) has the same order magnitude for SFDBAO, PS and the blend films. The results indicate that the blend of PS and SFDBAO enhances the high-density storage and retention abilities of the holes to a larger extent, but the endurance improvement of the electrons is not that obvious. By the KPFM measurement, we further verify the different diffusion rates of the trapped holes and electrons in the PS/SFDBAO blend films, and discuss the possible physical mechanism. The qualitative and quantitative determination of charge trapping properties in this work can be very useful for the characterization of PS/SFDBAO based charge trapping memory devices.

[Research progress of plant BAHD acyltransferase family].

Acylation conducted by acyltransferase is a ubiquitous process in structure modification of secondary metabolites. It plays an important role in the structural diversity of natural products and contributes significantly to their improved stabilities, increased solubilities, and enhanced bioavailabilities. BAHD acyltransferase family is a typical kind of acyltransferase original from plants, which involved in the biosynthesis of various bioactive acylated natural products. In order to provide references for future investigations of BAHD acyltransferase family, research progresses on basic properties, three-dimensional structures, catalytic mechanisms, enzymatic functional identifications and phylogenetic analyses of BAHD family from plants is summarized in this paper.

Revealing the interactions between pentagon-octagon-pentagon defect graphene and organic donor/acceptor molecules: a theoretical study.

Defect engineering and the non-covalent interaction strategy allow for dramatically tuning the optoelectronic features of graphene. Herein, we theoretically investigated the intrinsic mechanism of non-covalent interactions between pentagon-octagon-pentagon (5-8-5) defect graphene (DG) and absorbed molecules, tetrathiafulvalene (TTF), perfluoronaphthalene (FNa), tetracyanoquinodimethane (TCNQ) and 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4TCNQ), through geometry, distance, interaction energy, Mulliken charge distribution, terahertz frequency vibration, visualization of the interactions, charge density difference, electronic transition behaviour, band structure and density of state. All the calculations were performed using density functional theory including a dispersion correction (DFT-D). The calculated results indicate that the cyano- (CN) group (electron withdraw group) in TCNQ and F4TCNQ, rather than the F group, gain the electron from DG effectively and exhibit much stronger interactions via wavefunction overlap with DG, leading to a short non-covalent interaction distance, a large interaction energy and a red-shift of out-of-plane terahertz frequency vibration, changing the bands near the Fermi level and enhancing the infrared (IR) light absorption significantly. The enhancement of such IR absorbance offering a broader absorption (from 300 to 1200 nm) will benefit light harvesting in potential applications of solar energy conversion.

Development of a nomogram for predicting liver transplantation prognosis in hepatocellular carcinoma.

BACKGROUND: At present, liver transplantation (LT) is one of the best treatments for hepatocellular carcinoma (HCC). Accurately predicting the survival status after LT can significantly improve the survival rate after LT, and ensure the best way to make rational use of liver organs. AIM: To develop a model for predicting prognosis after LT in patients with HCC. METHODS: Clinical data and follow-up information of 160 patients with HCC who underwent LT were collected and evaluated. The expression levels of alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin, Golgi protein 73, cytokeratin-18 epitopes M30 and M65 were measured using a fully automated chemiluminescence analyzer. The best cutoff value of biomarkers was determined using the Youden index. Cox regression analysis was used to identify the independent risk factors. A forest model was constructed using the random forest method. We evaluated the accuracy of the nomogram using the area under the curve, using the calibration curve to assess consistency. A decision curve analysis (DCA) was used to evaluate the clinical utility of the nomograms. RESULTS: The total tumor diameter (TTD), vascular invasion (VI), AFP, and cytokeratin-18 epitopes M30 (CK18-M30) were identified as important risk factors for outcome after LT. The nomogram had a higher predictive accuracy than the Milan, University of California, San Francisco, and Hangzhou criteria. The calibration curve analyses indicated a good fit. The survival and recurrence-free survival (RFS) of high-risk groups were significantly lower than those of low- and middle-risk groups (P < 0.001). The DCA shows that the model has better clinical practicability. CONCLUSION: The study developed a predictive nomogram based on TTD, VI, AFP, and CK18-M30 that could accurately predict overall survival and RFS after LT. It can screen for patients with better postoperative prognosis, and improve long-term survival for LT patients.

Marked intestinal trans-differentiation by autoimmune gastritis along with ectopic pancreatic and pulmonary trans-differentiation.

BACKGROUND: Autoimmune gastritis (AIG) is a prevalent chronic inflammatory disease with oncogenic potential that causes destruction of parietal cells and severe mucosal atrophy. We aimed to explore the distinctive gene expression profiles, activated signaling pathways, and their underlying mechanisms. METHODS: A comprehensive gene expression analysis was conducted using biopsy specimens from AIG, Helicobacter pylori-associated gastritis (HPG), and non-inflammatory normal stomachs. Gastric cancer cell lines were cultured under acidic (pH 6.5) conditions to evaluate changes in gene expression. RESULTS: Gastric mucosa with AIG had a unique gene expression profile compared with that with HPG and normal mucosa, such as extensively low expression of ATP4A and high expression of GAST and PAPPA2, which are involved in neuroendocrine tumorigenesis. Additionally, the mucosa with AIG and HPG showed the downregulation of stomach-specific genes and upregulation of small intestine-specific genes; however, intestinal trans-differentiation was much more prominent in AIG samples, likely in a CDX-dependent manner. Furthermore, AIG induced ectopic expression of pancreatic digestion-related genes, PNLIP, CEL, CTRB1, and CTRC; and a master regulator gene of the lung, NKX2-1/TTF1 with alveolar fluid secretion-related genes, SFTPB and SFTPC. Mechanistically, acidic conditions led to the downregulation of master regulator and stemness control genes of small intestine, suggesting that increased environmental pH may cause abnormal intestinal differentiation in the stomach. CONCLUSIONS: AIG induces diverse trans-differentiation in the gastric mucosa, characterized by the transactivation of genes specific to the small intestine, pancreas, and lung. Increased environmental pH owing to AIG may cause abnormal differentiation of the gastric mucosa.

DNA Methylation Analysis.

DNA methylation of promoter CpG islands silences their downstream genes, and enhancer methylation can be associated with decreased or increased gene expression. DNA methylation alterations in normal and diseased cells provide rich information, such as tissue origin, disease risk, patient response, and prognosis. DNA methylation status is detected by bisulfite conversion, which converts unmethylated cytosines into uracils but methylated cytosines very inefficiently. A genome-wide DNA methylation analysis is conducted by a BeadChip microarray or next-generation sequencing (NGS) of bisulfite-treated DNA. A region-specific DNA methylation analysis can be conducted by various methods, such as methylation-specific PCR (MSP), quantitative MSP, and bisulfite sequencing. This chapter provides protocols for bisulfite-mediated conversion, a BeadChip array-based method (Infinium), quantitative MSP, and bisulfite sequencing.

Partial endothelial-to-mesenchymal transition mediated by HIF-induced CD45 in neointima formation upon carotid artery ligation.

AIMS: Endothelial-to-mesenchymal transition (EndMT) is a fundamental process in vascular remodelling. However, the precise regulatory mechanism of vascular remodelling during neointima formation and the source of neointima cells are not entirely understood. METHODS AND RESULTS: To investigate the origin of neointima cells and their relevance to vascular wall remodelling, we used an endothelial cell (EC)-specific lineage tracing system [VE-Cadherin (Cdh5)-BAC-CreERT2 mice] and carotid artery ligation model and showed evidence that resident ECs transdifferentiate into neointima cells with the expression of CD45. During the early stages of neointima formation, ECs transiently expressed CD45, a haematopoietic marker, accompanied by a host of EndMT markers, and CD31 and αSMA were prominently expressed in developing neointima. In vitro, CD45-positive EndMT was induced by stabilization of HIF1α with cobalt chloride or with a VHL inhibitor in human primary ECs, which mimicked the hypoxic condition of the ligated artery, and promoted the formation of an integrin α11-shank-associated RH domain-interacting protein (SHARPIN) complex. Notably, a CD45 phosphatase inhibitor disrupted this integrin α11-SHARPIN complex, thereby destabilizing cell-cell junctions. Deletion of Hif1α in ECs suppressed expression of CD45 and EndMT markers and ameliorated neointima formation. CONCLUSION: These results suggest that the HIF-induced CD45 expression is normally required for the retention of an EC fate and cell-cell junctions, CD45-positive EndMT (termed as 'partial EndMT') contributes to neointima formation and vascular wall remodelling.