Transgene Was Silenced in Hybrids between Transgenic Herbicide-Resistant Crops and Their Wild Relatives Utilizing Alien Chromosomes.

The commercialization of transgenic herbicide-resistant (HR) crops may cause gene flow risk. If a transgene in progenies of transgenic crops and wild relatives is silencing, these progenies should be killed by the target herbicide, thus, the gene flow risk could be decreased. We obtained the progenies of backcross generations between wild Brassca juncea (AABB, 2n = 36) and glufosinate-resistant transgenic Brassica napus (AACC, 2n = 38, PAT gene located on the C-chromosome). They carried the HR gene but did not express it normally, i.e., gene silencing occurred. Meanwhile, six to nine methylation sites were found on the promoter of PAT in transgene-silencing progenies, while no methylation sites occurred on that in transgene-expressing progenies. In addition, transgene expressing and silencing backcross progenies showed similar fitness with wild Brassica juncea. In conclusion, we elaborate on the occurrence of transgene-silencing event in backcross progenies between transgenic crop utilizing alien chromosomes and their wild relatives, and the DNA methylation of the transgene promoter was an important factor leading to gene silencing. The insertion site of the transgene could be considered a strategy to reduce the ecological risk of transgenic crops, and applied to cultivate lower gene flow HR crops in the future.

The genomic landscape of cholangiocarcinoma reveals the disruption of post-transcriptional modifiers.

Molecular variation between geographical populations and subtypes indicate potential genomic heterogeneity and novel genomic features within CCA. Here, we analyze exome-sequencing data of 87 perihilar cholangiocarcinoma (pCCA) and 261 intrahepatic cholangiocarcinoma (iCCA) cases from 3 Asian centers (including 43 pCCAs and 24 iCCAs from our center). iCCA tumours demonstrate a higher tumor mutation burden and copy number alteration burden (CNAB) than pCCA tumours, and high CNAB indicates a poorer pCCA prognosis. We identify 12 significantly mutated genes and 5 focal CNA regions, and demonstrate common mutations in post-transcriptional modification-related potential driver genes METTL14 and RBM10 in pCCA tumours. Finally we demonstrate the tumour-suppressive role of METTL14, a major RNA N6-adenosine methyltransferase (m6A), and illustrate that its loss-of-function mutation R298H may act through m6A modification on potential driver gene MACF1. Our results may be valuable for better understanding of how post-transcriptional modification can affect CCA development, and highlight both similarities and differences between pCCA and iCCA.

Gene Flow Risks From Transgenic Herbicide-Tolerant Crops to Their Wild Relatives Can Be Mitigated by Utilizing Alien Chromosomes.

Integration of a transgene into chromosomes of the C-genomes of oilseed rape (AACC, 2n = 38) may affect their gene flow to wild relatives, particularly Brassica juncea (AABB, 2n = 36). However, no empiric evidence exists in favor of the C-genome as a safer candidate for transformation. In the presence of herbicide selections, the first- to fourth-generation progenies of a B. juncea × glyphosate-tolerant oilseed rape cross [EPSPS gene insertion in the A-genome (Roundup Ready, event RT73)] showed more fitness than a B. juncea × glufosinate-tolerant oilseed rape cross [PAT gene insertion in the C-genome (Liberty Link, event HCN28)]. Karyotyping and fluorescence in situ hybridization-bacterial artificial chromosome (BAC-FISH) analyses showed that crossed progenies from the cultivars with transgenes located on either A- or C- chromosome were mixoploids, and their genomes converged over four generations to 2n = 36 (AABB) and 2n = 37 (AABB + C), respectively. Chromosome pairing of pollen mother cells was more irregular in the progenies from cultivar whose transgene located on C- than on A-chromosome, and the latter lost their C-genome-specific markers faster. Thus, transgene insertion into the different genomes of B. napus affects introgression under herbicide selection. This suggests that gene flow from transgenic crops to wild relatives could be mitigated by breeding transgenic allopolyploid crops, where the transgene is inserted into an alien chromosome.

An exonuclease protection and CRISPR/Cas12a integrated biosensor for the turn-on detection of transcription factors in cancer cells.

Transcription factors (TFs) are critical proteins that regulate the expression of genes, and the abnormal change of TFs levels is directly related to physical dysfunctions. Herein, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensor for the measurement of TFs level with the assistance of exonuclease protection assay. A dsDNA (activator) with the ability to activate Cas12a was engineered to contain TFs binding domain, and the binding between TFs and the activator can protect the dsDNA from being digested by exonuclease III (Exo III). The reserved activator then triggered a CRISPR/Cas12a reporting reaction to produce fluorescent signal for detection. In the detection of nuclear factor-kappa B (NF-κB) p50 subunit, the limit of detection of 0.2 pM and limit of quantification of 0.6 pM were obtained respectively, and the performance of this biosensor has been challenged by cell nucleoprotein extracts. Additionally, this method can be applied in the screening and evaluation of TFs inhibitors, calculating the IC50 of oridonin. Integrating merits including high sensitivity, low cost, and good portability, this method may enrich the arsenal for TFs-related applications.

Centipede KCNQ Inhibitor SsTx Also Targets KV1.3.

It was recently discovered that Ssm Spooky Toxin (SsTx) with 53 residues serves as a key killer factor in red-headed centipede's venom arsenal, due to its potent blockage of the widely expressed KCNQ channels to simultaneously and efficiently disrupt cardiovascular, respiratory, muscular, and nervous systems, suggesting that SsTx is a basic compound for centipedes' defense and predation. Here, we show that SsTx also inhibits KV1.3 channel, which would amplify the broad-spectrum disruptive effect of blocking KV7 channels. Interestingly, residue R12 in SsTx extends into the selectivity filter to block KV7.4, however, residue K11 in SsTx replaces this ploy when toxin binds on KV1.3. Both SsTx and its mutant SsTx_R12A inhibit cytokines production in T cells without affecting the level of KV1.3 expression. The results further suggest that SsTx is a key molecule for defense and predation in the centipedes' venoms and it evolves efficient strategy to disturb multiple physiological targets.

Tip60 Suppresses Cholangiocarcinoma Proliferation and Metastasis via PI3k-AKT.

BACKGROUND/AIMS: Aberrant expression of Tip60 is associated with progression in many cancers. However, the role of Tip60 in cancer progression remains contradictory. The aim of this study was to investigate the clinical significance, biological functions and underlying mechanisms of Tip60 deregulation in cholangiocarcinoma (CCA) for the first time. METHODS: Quantitative real-time PCR (QRT-PCR), western blotting and immunohistochemistry staining (IHC) were carried out to measure Tip60 expression in CCA tissues and cell lines. Kaplan-Meier analysis and the log-rank test were used for survival analysis. In vitro, cell proliferation was evaluated by flow cytometry and CCK-8, colony formation, and EDU assays. Migration/ invasion was evaluated by trans-well assays. Phosphokinase array was used to confirm the dominant signal regulated by Tip60. Tumor growth and metastasis were demonstrated in vivo using a mouse model. RESULTS: Tip60 was notably downregulated in CCA tissues, which was associated with greater tumor size, venous invasion, and TNM stage. Down-regulation of Tip60 was associated with tumor progression and poorer survival in CCA patients. In vitro and in vivo studies demonstrated that Tip60 suppressed growth and metastasis throughout the progression of CCA. We further identified the PI3K/AKT pathway as a dominant signal of Tip60 and suggested that Tip60 regulated CCA cell proliferation and metastasis via PT3K-AKT pathway. Pearson analysis revealed that PTEN was positively correlated with the Tip60 level in CCA tissues. CONCLUSION: Tip60, as a tumor suppressor in CCA via the PI3K/AKT pathway, might be a promising therapeutic target or prognostic marker for CCA.

Short- and long-term outcomes of middle hepatic vein-oriented hepatectomy for advanced perihilar cholangiocarcinoma.

BACKGROUND: This study aimed to compare clinical outcomes of the middle hepatic vein (MHV)-oriented versus conventional hemihepatectomy for perihilar cholangiocarcinoma (PHC). METHODS: From 2008 to 2017, medical records of patients undergoing hemihepatectomy with caudate lobectomy for advanced PHC were reviewed retrospectively. MHV-oriented hepatectomy was defined as full exposure of the MHV on the dissection plane. Predictors of morbidity and survival were identified. RESULTS: A total of 125 patients were enrolled. MHV-oriented and conventional hepatectomies were performed in 44 and 81 patients, respectively. The curative resection rate, blood loss, transfusion, and survival were comparable between two groups; however, severe morbidity rate was significantly lower in the MHV-oriented group (9.1% vs 38.3%, P < 0.001). MHV-oriented approach was an independent predictor of severe morbidity, as were the age, bilirubin level, and blood transfusion. Severe morbidity was associated with significantly decreased overall survival and recurrence-free survival (RFS) (median 29.0 vs 46.9 months, P = 0.011 and 20.3 vs 31.1 months, P = 0.003, respectively). Multivariate analysis revealed that severe morbidity independently predicted shorter RFS (P = 0.025). CONCLUSIONS: MHV-oriented approach for advanced PHC is safe and associated with a significant decrease in severe morbidity. Severe morbidity adversely affects survival after surgery; therefore, optimal preoperative preparation and MHV-oriented hepatectomy with meticulous dissection remain of critical importance.

Suppression of miR-16 promotes tumor growth and metastasis through reversely regulating YAP1 in human cholangiocarcinoma.

BACKGROUND & AIMS: Aberrant expression of microRNAs is associated with many cancers progression. Many studies have shown that miR-16 is down-regulated in many cancers. However, its role in cholangiocarcinoma (CCA) is unknown. METHODS: Quantitative real-time PCR (qRT-PCR) was developed to measure miR-16 expression in CCA tissues and cell lines. CCK-8, colony formation and transwell assays were used to reveal the role of miR-16 in CCA cell proliferation and malignant transformation in vitro. The loss-and-gain function was further validated by subcutaneous xenotransplantation and tail vein injection xenotransplantation model in vivo. Dual-luciferase reporter assay was performed to validate the relationship of miR-16 with YAP1. RESULTS: MiR-16 was notably downregulated in CCA tissues, which was associated with tumor size, metastasis, and TNM stage. Both in vitro and in vivo studies demonstrated that miR-16 could suppress proliferation, invasion and metastasis throughout the progression of CCA. We further identified YAP1 as a direct target gene of miR-16 and found that miR-16 could regulate CCA cell growth and invasion in a YAP1-dependent manner. In addition, YAP1 was markedly upregulated in CCA tissues, which was reversely correlated with miR-16 level in tissue samples. Besides, Down-regulation of miR-16 was remarkably associated with tumor progression and poor survival in CCA patients through a Kaplan-Meier survival analysis. CONCLUSIONS: miR-16, as a novel tumor suppressor in CCA through directly targeting YAP1, might be a promising therapeutic target or prognosis biomarker for CCA.

Radiologic evaluation of vasculobiliary anatomy in the umbilical fissure.

BACKGROUND: Preoperative evaluation of vasculobiliary anatomy in the umbilical fissure (U-point) is pivotal for perihilar cholangiocarcinoma (PCCA) applied to right-sided hepatectomy. The purpose of our study was to review the vasculobiliary anatomy in the U-point using three-dimensional (3D) reconstruction technique, to investigate the diagnostic ability of 2D scans to evaluate anatomic variations, and to discuss its surgical implications. METHODS: A retrospective study of 159 patients with Bismuth type I, II, and IIIa PCCA, who received surgery at our institution from November 2012 to September 2016, was conducted. Anatomic structures were assessed using multidetector computed tomography (MDCT) by one hepatobiliary surgeon, whereas 3D images were reconstructed by an independent radiologist. Normal confluence pattern of left biliary system was defined as the left medial segmental bile duct (B4) joining the common trunk of segment II (B2) and segment III (B3) ducts, whereas aberrant confluence patterns were classified into 3 types: type I, triple confluence of B2, B3, and B4; type II, B2 draining into the common trunk of B3 and B4; type III, other patterns. Surgical anatomy of B4 was classified into the central, peripheral, and combined type according to its relation to the hepatic confluence. The lengths from the bile duct branch of Spiegel's lobe (B1l) to the orifice of B4 and the junction of B2 and B3 were measured on 3D images. The anatomy of left hepatic artery (LHA) was classified according to different origins and the spatial relationship related to the U-point. RESULTS: 3D reconstruction revealed that normal confluence pattern of left biliary system was observed in 71.1% (113/159) of all patients, and variant patterns were type I in 11.9% (19/159), type II in 12.6% (20/159), and type III in 4.4% (7/159). The length from B1l to the junction of B2 and B3 was 12.1 ± 3.1 mm in type I variation, which was significantly shorter than that in normal configuration (30.0 ± 6.8 mm, P < 0.001) but significantly longer than that in type II variation (9.6 ± 3.4 mm, P = 0.019). Surgical anatomy of B4: the peripheral type was most commonly seen (74.2%, 118/159), followed by central type (15.7%, 25/159) and combined type (10.1%, 16/159). The distance between the B1l and B4 was 8.4 ± 2.4 mm in central and combined type, which was significantly shorter than that in peripheral type (14.5 ± 4.1 mm, P < 0.001). A replaced or accessory LHA from the left gastric artery was present in 6 (3.8%) and 9 (5.7%) patients, respectively. LHA running along the left caudal position of U-point was present in 143 cases (89.9%), along the right cranial position of U-point in nine cases (5.7 %), and combined position in seven cases (4.4%). Interobserver agreement of two imaging modalities was almost perfect in biliary confluence pattern (kappa = 0.90; 95% confidence interval: 0.79-1.00), substantial in surgical anatomy of B4 (kappa = 0.74; 95% confidence interval: 0.62-0.86), and perfect in LHA (kappa = 1.00). CONCLUSIONS: Thoroughly understanding the imaging characters of surgical anatomy in the U-point may be benefit for preoperative evaluation of PCCA by successive review of 2D images alone, whereas 3D reconstruction technique allows detailed hepatic anatomy and individualized surgical planning for advanced cases.

Clinical Implications of Biliary Confluence Pattern for Bismuth-Corlette Type IV Hilar Cholangiocarcinoma Applied to Hemihepatectomy.

BACKGROUND: Since biliary variations are commonly seen, our aims are to clarify these insidious variations and discuss their surgicopathologic implications for Bismuth-Corlette (BC) type IV hilar cholangiocarcinoma (HC) applied to hemihepatectomy. METHODS: Three-dimensional images of patients with distal bile duct obstruction (n = 97) and advanced HC (n = 79) were reconstructed and analyzed retrospectively. Normal biliary confluence pattern was defined as the peripheral segment IV duct (B4) joining the common trunk of segment II (B2) and segment III (B3) ducts to form the left hepatic duct (LHD) that then joined the right hepatic duct (RHD). The lengths from left and right secondary biliary ramifications to the right side of the umbilical portion of the left portal vein (Rl-L) and the cranio-ventral side of the right portal vein (Rr-R) were measured, respectively, and compared with the resectable bile duct length in HCs. Surgicopathologic findings were compared between different BC types. RESULTS: The resectable bile duct length in right hemihepatectomy for eradication of type IV tumors was significantly longer than the Rl-L length in normal biliary configuration (17.4 ± 1.8 and 10.3 ± 3.4 mm, respectively, p < 0.001), and type III variation (B2 joining the common trunk of B3 and B4) was the predominant configuration (53.8%). The resectable length in left hemihepatectomy for eradication of type IV tumors was comparable with the Rr-R length in RHD absent cases (15.2 ± 2.5 and 16.4 ± 2.6 mm, respectively, p = 0.177) but significantly longer than that in normal configuration (p < 0.001). The estimated length was 8.5 ± 2.0 mm in unresectable cases. There was no significant difference between type III and IV tumors, except for the rate of nodal metastasis (29.7 and 76.0%, respectively, p < 0.001). CONCLUSION: Hemihepatectomy might be selected for curative-intent resection of BC type IV tumors considering the advantageous biliary variations, whereas anatomical trisegmentectomy is recommended for the resectable bile duct length less than 10 mm. Biliary variations might result in excessive classification of BC type IV but require validation on further study.

A giant and insidious subphrenic biloma formation due to gallbladder perforation mimicking biliary cystic tumor: A case report.

Gallbladder perforation (GBP) represents a rare, but potentially life-threatening, complication of acute cholecystitis. GBP is subdivided into three categories whereas the development of biloma is extremely rare. The present case study reports on a 40-year-old man with a 10-year history of calculus cholecystitis, who was referred to The First Affiliated Hospital of Nanjing Medical University (Nanjing, China) for the surgical treatment of an emerging massive hepatic entity with insidious symptoms and normal laboratory tests. A preoperative imaging study demonstrated the collection with internal septations and mural nodules, but no visible communication with the biliary system. Given the suspected biliary cystic tumor, a laparotomy was performed and the lumen was scattered with papillae. An intraoperative frozen section examination illustrated a simple hepatic cyst. Biochemical analysis of the collection and histopathology of the gallbladder and capsule substantiated the diagnosis of biloma formation due to GBP. The purpose of the present case report was to demonstrate how a pinhole-sized perforation with extravasation of unconcentrated bile from the gallbladder may result in insidious clinical presentation and an undetected leak site. According to the clinicopathological characteristics and composition, formation of biloma should be classified as type IV GBP. To differentiate bilomas with intracystic septations and mural nodules from BCTs is difficult via a preoperative examination, and the definitive diagnosis should be based on a histological examination. Laparotomy with frozen section examination may be the optimal approach in such a case.

miR-940 Suppresses Tumor Cell Invasion and Migration via Regulation of CXCR2 in Hepatocellular Carcinoma.

Aim. To investigate the expression of miR-940 in the hepatocellular carcinoma (HCC) and its impact on function and biological mechanism in the HCC cells. Methods. Quantitative RT-PCR analysis was used to quantify miR-940 expression in 46 cases of tissues and cells. Transfection of HCC cell lines was performed by miR-940 mimics; the abilities of invasion and migration were assessed through Transwell array. Western blot represents the alteration in expression of CXCR2 by miR-940 mimics. Results. miR-940 expression was decreased significantly in the HCC tissues and the relevant cell lines. miR-940 upregulation suppressed the invasion and migration of HCC cells in vitro. Furthermore, the CXCR2 was downregulated to suppress invasion and migration after miR-940 mimics. Moreover, decreased miR-940 expression was negatively correlated with Edmondson grade (P = 0.008), tumor microsatellite or multiple tumors (P = 0.04), vascular invasion (P = 0.035), and recurrence and metastasis (P = 0.038). Kaplan-Meier analysis demonstrated that decreased miR-940 expression contributed to poor overall survival (P < 0.05). Conclusions. Our findings present that miR-940 acts as a pivotal adaptor of CXCR2 and its transcription downregulated CXCR2 expression to decrease HCC invasion and migration in vitro. Our study suggests that miR-940 may be a novel poor prognostic biomarker for HCC.