EUS­guided antegrade stenting for malignant jaundice following Roux­en­Y reconstruction: the treatment of multiple biliary strictures.

Multiple malignant biliary strictures are rare, and the application of multiple stents can achieve better drainage. EUS-guided biliary drainage (EUS-BD) can be offered as an alternative technique when ERCP unsuccessful. We applied Endoscopic ultrasonic guided antegrade stenting technique to treat a case of multiple biliary strictures following Roux-en-Y reconstruction.

Bending the distal tip of a guidewire aids in cannulation of an occluded uncovered biliary metal stent.

Biliary metal stent implantation is an effective treatment for malignant obstructive jaundice. But it's well known that stents put in for a long time can become occluded and cause jaundice and cholangitis. At this time, endoscopic intervention is usually required to replace the stent or re-insert the stent. Re-cannulation for metal stent occlusion is challenging because the guide wire may pass through the side holes of the uncovered metal stents, resulting in prolonged surgical time and exposure to radiation. Here we present a small tip that may help endoscopists complete the re-cannulation of an uncovered metal stent in a very short time.

Endoscopic management of stent displacement during EUS-guided pancreatic pseudocyst drainage.

The therapeutic effect of EUS-Guided Pancreatic Pseudocyst Drainage (EUS-PPD) is widely recognized, and intraoperative stent displacement is a rare but potentially serious condition. We report a case of the cyst stent displace into the cyst cavity during EUS-PPD, we successfully reduced the stent in time under the guidance of EUS and fluoroscopy in the final.

Endoscopic ultrasound combined with enteroscopy for bilioenterostomy stenosis after pancreatoduodenectomy. The first attempt of a two-person operation.

Bilioenterostomy stenosis is very challenging, and effective endoscopic treatment can prevent patients from undergoing surgery. We present a case of a patient with extremely narrow bilioenterostomy treated with simultaneous endoscopic ultrasonography and enteroscopy. It provides a new and feasible idea of endoscopic therapy for the treatment of such patients.

Cystic duct stones in postcholecystectomy Mirizzi syndrome - A novel endoscopic treatment.

Mirizzi syndrome is a rare type of cholelithiasis, and the main treatment is still surgery. The development of endoscopic technology has made surgeons more active in the management of rare diseases of the biliary tract and pancreas. Here we report that our center applied the new endoscopic method to treat a Mirizzi patient with residual cystic neck duct stones after laparoscopic cholecystectomy.

Reprogramming of m6A epitranscriptome is crucial for shaping of transcriptome and proteome in response to hypoxia.

Hypoxia causes a series of responses supporting cells to survive in harsh environments. Substantial post-transcriptional and translational regulation during hypoxia has been observed. However, detailed regulatory mechanism in response to hypoxia is still far from complete. RNA m6A modification has been proven to govern the life cycle of RNAs. Here, we reported that total m6A level of mRNAs was decreased during hypoxia, which might be mediated by the induction of m6A eraser, ALKBH5. Meanwhile, expression levels of most YTH family members of m6A readers were systematically down-regulated. Transcriptome-wide analysis of m6A revealed a drastic reprogramming of m6A epitranscriptome during cellular hypoxia. Integration of m6A epitranscriptome with either RNA-seq based transcriptome analysis or mass spectrometry (LC-MS/MS) based proteome analysis of cells upon hypoxic stress revealed that reprogramming of m6A epitranscriptome reshaped the transcriptome and proteome, thereby supporting efficient generation of energy for adaption to hypoxia. Moreover, ATP production was blocked when silencing an m6A eraser, ALKBH5, under hypoxic condition, demonstrating that m6A pathway is an important regulator during hypoxic response. Collectively, our studies indicate that crosstalk between m6A and HIF1 pathway is essential for cellular response to hypoxia, providing insights into the underlying molecular mechanisms during hypoxia.

DNA Damage-Regulated Autophagy Modulator 1 (DRAM1) Mediates Autophagy and Apoptosis of Intestinal Epithelial Cells in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: DNA damage-regulated autophagy modulator 1 (DRAM1) is required for induction of autophagy and apoptosis. However, the influence of DRAM1 on the pathogenesis of inflammatory bowel disease (IBD) has not been explored. METHODS: DRAM1 expression was examined in the intestinal mucosa of patients with IBD and colons of colitis mice. We used a recombinant adeno-associated virus carrying small hairpain DRAM1 to knock down the DRAM1 gene to treat colitis in the mice. The effect of DRAM1 on autophagy and apoptosis of intestinal epithelial cells was explored. DRAM1-mediated interaction with the c-Jun N-terminal kinase (JNK) pathway was also examined. RESULTS: DRAM1 expression in the intestinal mucosa of the IBD patients was higher than that in the control participates. DRAM1 expression in the inflammatory cells in patients with Crohn's disease (CD) was lower than that in patients with ulcerative colitis (UC). Additionally, DRAM1 expression was correlated with the Simple Endoscopic Score for CD and the Mayo endoscopic score for UC. Serum levels of DRAM1 in the IBD group were substantially higher than those in the normal group. The knockdown of DRAM1 could alleviate colitis symptoms in mice. In in vitro experiments, knocking down DRAM1 could reduce autophagy and apoptosis levels. Mechanistically, DRAM1 may participate in the regulation of these two processes by positively regulating JNK activation. CONCLUSIONS: During intestinal inflammation, the upregulation of DRAM1 may promote the activation of JNK and further aggravate intestinal epithelium damage.

Eukaryotic elongation factor 2 kinase promotes angiogenesis in hepatocellular carcinoma via PI3K/Akt and STAT3.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Angiogenesis is crucial for tumor formation, development and metastasis in HCC. Previous studies indicated that high expression levels of elongation factor 2 kinase (eEF2K), a protein kinase that negatively regulates the elongation stage of translation, were associated with poor prognosis of HCC. Here, we show that pharmacological inhibition or knockdown of eEF2K in highly metastatic liver cancer cells inhibits their colony forming and migratory capacities, as well as reducing their invasiveness. Importantly, knocking down eEF2K by lentiviral directed shRNA prevented tumor growth and angiogenesis of HCC in mice. Silencing of eEF2K in endothelial cells (HUVECs) led to a reduction in vascularization, evidenced by a decrease in capillary-like structures in the matrigel. Notably, knocking down eEF2K reduced the expression of angiogenesis-related growth factors in liver cancer cells and the expression of growth factor receptors on HUVECs, and thus restricted signaling crosstalk that promotes angiogenesis between HCC cells and endothelial cells. We also showed that silencing of eEF2K effectively reduced protein levels of SP1/KLF5 transcription factors and hence decreased the levels of bound SP1/KLF5 to the VEGF promoter, resulted in a decrease in VEGF mRNA expression. Knocking down eEF2K also led to a striking decrease in the phosphorylation of PI3K/Akt and STAT3, indicating inactivation of these tumorigenic pathways. Taken together, our data suggest that eEF2K contributes to angiogenesis and tumor progression in HCC via SP1/KLF5-mediated VEGF expression, as well as the subsequent stimulation of PI3K/Akt and STAT3 signaling.

AKT2 regulates development and metabolic homeostasis via AMPK-depedent pathway in skeletal muscle.

Skeletal muscle is responsible for the majority of glucose disposal in the body. Insulin resistance in the skeletal muscle accounts for 85-90% of the impairment of total glucose disposal in patients with type 2 diabetes (T2D). However, the mechanism remains controversial. The present study aims to investigate whether AKT2 deficiency causes deficits in skeletal muscle development and metabolism, we analyzed the expression of molecules related to skeletal muscle development, glucose uptake and metabolism in mice of 3- and 8-months old. We found that AMP-activated protein kinase (AMPK) phosphorylation and myocyte enhancer factor 2 (MEF2) A (MEF2A) expression were down-regulated in AKT2 knockout (KO) mice, which can be inverted by AMPK activation. We also observed reduced mitochondrial DNA (mtDNA) abundance and reduced expression of genes involved in mitochondrial biogenesis in the skeletal muscle of AKT2 KO mice, which was prevented by AMPK activation. Moreover, AKT2 KO mice exhibited impaired AMPK signaling in response to insulin stimulation compared with WT mice. Our study establishes a new and important function of AKT2 in regulating skeletal muscle development and glucose metabolism via AMPK-dependent signaling.

Two-Dimensional Layered Ultrathin Carbon/TiO2 Nanosheet Composites for Superior Pseudocapacitive Lithium Storage.

Intercalation of carbon nanosheets into two-dimensional (2D) inorganic materials could enhance their properties in terms of mechanics and electrochemistry, but sandwiching these two kinds of materials in an alternating sequence is a great challenge in synthesis. Herein, we report a novel strategy to construct TiO2 nanosheets into 2D pillar-layer architectures by employing benzidine molecular assembly as pillars. Then, 2D carbon/TiO2 nanosheet composite with a periodic interlayer distance of 1.1 nm was obtained following a polymerization and carbonization process. This method not only alleviates the strain arising from the torsion of binding during carbonization but also hinders the structural collapse of TiO2 due to the intercalation of the carbon layer by rational control of annealing conditions. The composite material possesses a large carbon/TiO2 interface, providing abundant active sites for ultrafast pseudocapacitive charge storage, thus displaying a superior high-rate performance with a specific capacity of 67.8 mAh g-1 at a current density of 12.8 A g-1 based on the total electrode and excellent cyclability with 87.4% capacity retention after 3000 cycles.

Comparative Transcriptome Analysis Reveals Candidate Genes Involved in Isoquinoline Alkaloid Biosynthesis in Stephania tetrandra.

The roots of Stephania tetrandra are used as a traditional Chinese medicine. Isoquinoline alkaloids are considered to be the most important and effective components in this herb, but little is known about the molecular mechanism underlying their biosynthesis. In this context, this study aimed to reveal candidate genes related to isoquinoline alkaloid biosynthesis in S. tetrandra. Determination of tetrandrine and fangchinoline in the roots and leaves of S. tetrandra by HPLC showed that the roots had much higher contents of the two isoquinoline alkaloids than the leaves. Thus, a comparative transcriptome analysis of the two tissues was performed to uncover candidate genes involved in isoquinoline alkaloid biosynthesis. A total of 71 674 unigenes was obtained and 31 994 of these were assigned putative functions based on BLAST searches against 6 annotation databases. Among the 79 isoquinoline alkaloid-related unigenes, 51 were differentially expressed, with 42 and 9 genes upregulated and downregulated, respectively, when the roots were compared with the leaves. The upregulated differentially expressed genes were consistent with isoquinoline alkaloid accumulation in roots and thus were deemed key candidate genes for isoquinoline alkaloid biosynthesis in the roots. Moreover, the expression profiles of 10 isoquinoline alkaloid-related differentially expressed genes between roots and leaves were validated by quantitative real-time polymerase chain reaction, which indicated that our transcriptome and gene expression profiles were reliable. This study not only provides a valuable genomic resource for S. tetrandra but also proposes candidate genes involved in isoquinoline alkaloid biosynthesis and transcription factors related to the regulation of isoquinoline alkaloid biosynthesis. The results lay a foundation for further studies on isoquinoline alkaloid biosynthesis in this medicinal plant.

CIRDES: an efficient genome-wide method for in vivo RNA-RNA interactome analysis.

Complex RNA-RNA interactions underlie fundamental biological processes. However, a large number of RNA-RNA interactions remain unknown. Most existing methods used to map RNA-RNA interactions are based on proximity ligation, but these strategies also capture a huge amount of intramolecular RNA secondary structures, making it almost impossible to detect most RNA-RNA interactions. To overcome this limitation, we developed an efficient, genome-wide method, Capture Interacting RNA and Deep Sequencing (CIRDES) for in vivo capturing of the RNA interactome. We designed multiple 20-nt CIRDES probes tiling the whole RNA sequence of interest. This strategy obtained high selectivity and low background noise proved by qRT-PCR data. CIRDES enriched target RNA and its interacting RNAs from cells crosslinked by formaldehyde in high efficiency. After hybridization and purification, the captured RNAs were converted to the cDNA library after a highly efficient ligation to a 3' end infrared-dye-conjugated RNA adapter based on adapter ligation library construction. Using CIRDES, we detected highly abundant known interacting RNA, as well as a large number of novel targets of U6 snRNA. The enrichment of U4 snRNA, which interacts with U6, confirmed the robustness of the identification of the RNA-RNA interaction by CIRDES. These results suggest that the CIRDES is an efficient strategy for genome-wide RNA-RNA interactome analysis.

Identification of Pyroptosis Gene Signature Related Molecular Pattern, Clinical Implication, and Tumor Immunity in Hepatocellular Carcinoma`.

BACKGROUND: Pyroptosis is a novel form of programmed cell death in cancers, which regulates tumor cell invasion, proliferation, and metastasis, thereby affecting the prognosis of cancer patients. However, the role of Pyroptosis-Related Genes (PGs) in Hepatocellular Carcinoma (HCC) remains unclear. METHODS: Somatic mutation, copy number variation, and expression of 41 PGs were assessed in HCC and normal liver from the TCGA dataset. The Least Absolute Shrinkage and Selection Operator (LASSO) was used to construct the prognostic model. K-M curves, ROC curves, nomograph, and univariate and multivariate Cox regression were conducted to evaluate the predictive value of PGs. Immune infiltration was analyzed by CIBERSOFT and ssGSEA algorithm. The expression of prognostic PGs was validated by qPCR. RESULTS: Significant mutation and copy number variation of PGs were found in HCC. These genes were involved in an inflammatory response. In addition, 9 out of 41 PGs were differentially expressed in HCC and found to correlate significantly with patient survival. Then, these signature genes were selected to build a prognosis model and were utilized to stratify HCC patients into high and low PGs-score groups. It showed that the high-PGs group had a worse prognosis. Univariate and multivariate Cox regression verified that PGs-score was an independent risk factor for HCC. By ROC curves and nomogram, we showed that PGs-score effectively predicted the 1-, 3-, and 5-year survival of HCC patients and correlated with AFP level and disease stage. Immune infiltration analysis further showed that tumor immunity correlated with the PGs-score, and the expression of immune checkpoint molecule was significantly enhanced in the high PGs group. The PGs-score was also validated in the external validation cohort (ICGC). Finally, the expression of 9 signature genes was validated in normal liver and HCC cell lines. CONCLUSION: This study elucidated the aberrant regulation of PGs in HCC, and those pyroptosisrelated genes may be applied as a prognostic factor of HCC.

EUS-guided drainage for pancreatic duct stone combined with main pancreatic duct stenosis after pancreaticoduodenectomy: a case report.

.

Genome-Wide Identification and Analysis of TCP Gene Family among Three Dendrobium Species.

Dendrobium orchids, which are among the most well-known species of orchids, are appreciated for their aesthetic appeal across the globe. Furthermore, due to their strict living conditions, they have accumulated high levels of active ingredients, resulting not only in their medicinal value but also in their strong ability to respond to harsh environments. The TCP gene family plays an important role in plant growth and development, and signal transduction. However, these genes have not been systematically investigated in Dendrobium species. In this study, we detected a total of 24, 23, and 14 candidate TCP members in the genome sequences of D. officinale, D. nobile, and D. chrysotoxum, respectively. These genes were classified into three clades on the basis of a phylogenetic analysis. The TCP gene numbers among Dendrobium species were still highly variable due to the independent loss of genes in the CIN clade. However, only three gene duplication events were detected, with only one tandem duplication event (DcTCP9/DcTCP10) in D. chrysotoxum and two pairs of paralogous DoTCP gene duplication events (DoTCP1/DoTCP23 and DoTCP16/DoTCP24) in D. officinale. A total of 25 cis-acting elements of TCPs related to hormone/stress and light responses were detected. Among them, the proportions of hormone response, light response, and stress response elements in D. officinale (100/421, 127/421, and 171/421) were similar to those in D. nobile (83/352, 87/352, and 161/352). Using qRT-PCR to determine their expression patterns under MeJA treatment, four DoTCPs (DoTCP2, DoTCP4, DoTCP6, and DoTCP14) were significantly upregulated under MeJA treatment, which indicates that TCP genes may play important roles in responding to stress. Under ABA treatment, seven DoTCPs (DoTCP3, DoTCP7, DoTCP9, DoTCP11, DoTCP14, DoTCP15, and DoTCP21) were significantly upregulated, indicating that TCP genes may also play an important role in hormone response. Therefore, these results can provide useful information for studying the evolution and function of TCP genes in Dendrobium species.

Identification of a hypoxia-related gene prognostic signature in colorectal cancer based on bulk and single-cell RNA-seq.

Colorectal cancer (CRC) is the most common and fatal tumor in the gastrointestinal system. Its incidence and mortality rate have increased in recent years. Hypoxia, a persistent physiological tumor feature, plays a vital role in CRC tumorigenesis, metastasis, and tumor microenvironment (TME). Therefore, we constructed a hypoxia-related gene (HRG) nomogram to predict overall survival (OS) and explored the role of HRGs in the CRC TME. The Cancer Genome Atlas (TCGA) dataset was used as the training set, and two Gene Expression Omnibus datasets (GSE39582 and GSE103479) were used as the testing sets. HRGs were identified using the Gene Set Enrichment Analysis (GSEA) database. An HRG prognostic model was constructed in the training set using the least absolute shrinkage and selection operator regression algorithm and validated in the testing sets. Then, we analyzed tumor-infiltrating cells (TICs) using the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm. Furthermore, single-cell next-generation RNA sequencing (RNA-seq) was used to investigate HRG expression in different TICs in the GSE139555 dataset. Finally, reverse transcription polymerase chain reactions (RT-PCR) were used to validate HRG mRNA expression in ten pairs of CRC normal and cancer tissue samples. A six HRG prognostic signature was constructed, with a superior OS prediction ability in CRC patients (area under the receiver operating characteristic curve (AUC) at one year: 0.693, AUC at three years: 0.712, and AUC at five years: 0.780). GSEA enrichment analysis identified six pathways enriched in the high-risk group. The TIC analysis indicated that the high-risk group had lower T-cell expression and higher neutrophil expression than the low-risk group. Furthermore, immune-related genes had an inseparable relationship with the HRG prognostic signature. Based on single-cell RNA-seq data, we found elevated hexokinase 1 (HK1) and glucose-6-phosphate isomerase (GPI) gene expression in natural killer (NK) and CD8+ T cells. RT-PCR in ten CRC normal-tumor tissue pairs showed that expression of the signature's six HRGs varied differently in cancerous and paracancerous tissues. The constructed HRG signature successfully predicted the OS of whole-stage CRC patients. In addition, we showed that the signature's six HRGs were closely associated with the TME in CRC, where hypoxia inhibits the antitumor function of T cells.