The Pol II preinitiation complex (PIC) influences Mediator binding but not promoter-enhancer looping.

Knowledge of how Mediator and TFIID cross-talk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genome-wide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II preinitiation complex (PIC) at promoters, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by promoter capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect on Mediator integrity but led to a consistent twofold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but had no effect on the other PIC components. PCHi-C revealed no consistent effect of MED4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

Robotic pancreaticoduodenectomy for pancreatic ductal adenocarcinoma: Analysis of surgical outcomes and long-term prognosis in a high-volume center.

BACKGROUND: Robotic pancreaticoduodenectomy (RPD) has been reported to be safe and feasible for patients with pancreatic ductal adenocarcinoma (PDAC) of the pancreatic head. This study aimed to analyze the surgical outcomes and risk factors for poor long-term prognosis of these patients. METHODS: Data from patients who underwent RPD for PDAC of pancreatic head were retrospectively analyzed. Multivariate Cox regression analysis was used to seek the independent prognostic factors for overall survival (OS), and an online nomogram calculator was developed based on the independent prognostic factors. RESULTS: Of the 273 patients who met the inclusion criteria, the median operative time was 280.0 minutes, the estimated blood loss was 100.0 mL, the median OS was 23.6 months, and the median recurrence-free survival (RFS) was 14.4 months. Multivariate analysis showed that preoperative carbohydrate antigen 19-9 (CA19-9) [hazard ratio (HR) = 2.607, 95% confidence interval (CI): 1.560-4.354, P < 0.001], lymph node metastasis (HR = 1.429, 95% CI: 1.005-2.034, P = 0.047), tumor moderately (HR = 3.190, 95% CI: 1.813-5.614, P < 0.001) or poorly differentiated (HR = 5.114, 95% CI: 2.839-9.212, P < 0.001), and Clavien-Dindo grade ≥ III (HR = 1.657, 95% CI: 1.079-2.546, P = 0.021) were independent prognostic factors for OS. The concordance index (C-index) of the nomogram constructed based on the above four independent prognostic factors was 0.685 (95% CI: 0.640-0.729), which was significantly higher than that of the AJCC staging (8th edition): 0.541 (95% CI: 0.493-0.589) (P < 0.001). CONCLUSIONS: This large-scale study indicated that RPD was feasible for PDAC of pancreatic head. Preoperative CA19-9, lymph node metastasis, tumor poorly differentiated, and Clavien-Dindo grade ≥ III were independent prognostic factors for OS. The online nomogram calculator could predict the OS of these patients in a simple and convenient manner.

Step-by-step and orderly lowering of the height of inferior vena cava tumor thrombus is the key to robot-assisted thrombectomy for Mayo III/IV tumor thrombus.

BACKGROUND: The surgical management of Mayo III/IV tumor thrombi is difficult and risky, and robotic surgery is even more difficult. The purpose of this study was to introduce the step-by-step and orderly lowering of the height of inferior vena cava tumor thrombus, which was the core technique of robot operation for Mayo III/IV tumor thrombus. METHOD: A total of 18 patients were included in this study. The average tumor thrombus height was 2.4 cm above the level of the second porta hepatis (SPH), and 9 patients were prepared for cardiopulmonary bypass (CPB) before surgery. During the operation, the height of the tumor thrombus was lowered orderly for 2-3 times, and the blood flow blocking method was changed sequentially. The CPB was required when tumor thrombus in the atrium; After the height of the thrombus was lowered to the atrium entrance, CPB was stopped and the blood flow was blocked in the upper- and retro-hepatic inferior vena cava (IVC); After the tumor thrombus continued to descend to the lower part of the SPH, liver blood flow could be restored, and then, the blood flow was simply blocked in the retro-hepatic IVC to complete the removal of the thrombus and the repair or resection of the IVC. Finally, the diseased kidney and renal vein were removed. RESULTS: All operations were successfully completed, and 2 cases were transferred to laparotomy. Seven cases received CPB, while the other 11 did not. 15 patients underwent two times of the lowering of the tumor thrombus, 2 patients underwent one time and 1 patient underwent three times. The mean liver/IVC dissociation and vascular suspension time was 22.0 min. All patients had less than Clavien-Dindo grade III complications, no serious complications occurred during operation, and no patient died within 90 days. CONCLUSIONS: The step-by-step and orderly decline of tumor thrombus height is the key to the success of robot Mayo III / IV tumor thrombus surgery. This method can shorten FPH and CPB time and improve the success rate of surgery.

A novel online calculator to predict recurrence risk in patients with distal cholangiocarcinoma after radical pancreaticoduodenectomy.

BACKGROUND: Patients with distal cholangiocarcinoma (DCC) are prone to relapse even after radical pancreaticoduodenectomy. In this study, we sought to create an online nomogram calculator to accurately predict the recurrence risk of DCC. METHODS: A total of 184 patients were included. Multivariate Cox regression analysis was used to identify independent prognosis factors for recurrence-free survival and overall survival. A nomogram was constructed according to the prognostic factors in the training cohort and then tested in the validation cohort. RESULTS: Multivariate Cox analysis showed preoperative carbohydrate antigen 19-9 (p < 0.001), maximum tumor size (p = 0.076), perineural invasion (p = 0.044), and N stage (p = 0.076) were independent prognostic factors for DCC relapse. We then constructed a nomogram with these four factors. The consistency index (C-index) of the nomogram in the training and validation cohorts were 0.703 and 0.665, respectively. Time-dependent receiver operating characteristic and decision curve analyses revealed that the nomogram provided higher diagnostic power and net benefit compared with other staging systems. CONCLUSION: In this study, we developed an online nomogram calculator that can accurately predict the recurrence risk of DCC and identify patients with a high risk of recurrence in a simple and convenient manner.

Comparison of robotic and laparoscopic liver resection in ordinary cases of left lateral sectionectomy.

BACKGROUND: Laparoscopy was considered the standard method of left lateral sectionectomy. The robotic approach showed advantages in complex cases of left lateral sectionectomy. However, the impact of the robotic system on ordinary cases is still unknown. METHODS: Retrospective review of consecutive robotic left lateral sectionectomy (R-LLS) and laparoscopic left lateral sectionectomy (L-LLS) from January 2015 to December 2019. Univariate and multivariate logistic regression was used to determine the effects of surgical method and surgical complexity on postoperative length of stay, surgical and overall cost. RESULTS: 258 consecutive patients who underwent minimally invasive left lateral sectionectomy were analyzed. L-LLS had comparable outcomes and decreased surgery (USD 2416.3 vs 4624.5; p < 0.001) and overall costs (USD 8004.5 vs 11897.1; p < 0.001) compared with R-LLS in the ordinary-case group, whereas R-LLS was associated with shorter postoperative LOS (5.0 vs 3.5 days; p = 0.004) in the complex-case group. On multivariable analysis, R-LLS was predictive of shorter postoperative LOS [odds ratio (OR) 0.388, 95% confidence interval (CI) 0.198-0.760, p = 0.006], whereas R-LLS was predictive of higher surgery (OR 65.640, 95% CI 17.406-247.535, p < 0.001) and overall costs (OR 102.233, 95% CI 22.241-469.931, p < 0.001). CONCLUSION: Results of this study showed no clinical benefit to the R-LLS compared with L-LLS in ordinary cases. R-LLS had potential advantages in selected complex cases.

Short- and long-term outcomes after robotic and open liver resection for elderly patients with hepatocellular carcinoma: a propensity score-matched study.

BACKGROUND: Robotic liver resection (RLR) has increasingly been accepted as it has overcome some of the limitations of open liver resection (OLR), while the outcomes following RLR in elderly patients with hepatocellular carcinoma (HCC) are still uncertain. This study aimed to evaluate the short and long-term outcomes of RLR vs. OLR in elderly HCC patients. METHODS: Perioperative data of elderly patients (≥ 65 years) with HCC who underwent RLR or OLR between January 2010 and December 2020 were retrospectively analyzed. A 1:2 propensity score-matched (PSM) analysis was performed to minimize the differences between RLR and OLR groups. Univariable and multivariable Cox regression analyses were used to identify independent prognosis factors for overall survival (OS) and recurrence-free survival (RFS) of these patients. RESULTS: Of the 427 elderly HCC patients included in this study, 113 underwent RLR and 314 underwent OLR. After the 1:2 PSM, there were 100 and 178 patients in the RLR and the OLR groups, respectively. The RLR group had a less estimated blood loss (EBL), a shorter postoperative length of stay (LOS), and a lower complications rate (all P < 0.05), compared with the OLR group before and after PSM. Univariable and multivariable analyses showed that advanced age and surgical approaches were not independent risk factors for long-term prognosis. The two groups of elderly patients who were performed RLR or OLR had similar OS (median OS 52.8 vs. 57.6 months) and RFS (median RFS 20.4 vs. 24.6 months) rates after PSM. CONCLUSIONS: RLR was comparable to OLR in feasibility and safety. For elderly patients with HCC, RLR resulted in similar oncologic and survival outcomes as OLR.

Robotic versus open pancreaticoduodenectomy for distal cholangiocarcinoma: a multicenter propensity score-matched study.

BACKGROUND: Pancreatoduodenectomy is the only potentially curative treatment for distal cholangiocarcinoma (DCC). In this study, we sought to compare the perioperative and oncological outcomes of robotic pancreaticoduodenectomy (RPD) and open pancreaticoduodenectomy (OPD) based on a multicenter propensity score-matched study. METHODS: Consecutive patients with DCC who underwent RPD or OPD from five centers in China between January 2014 and June 2019 were included. A 1:1 propensity score matching (PSM) was performed. Univariable and multivariable Cox regression analyses were used to identify independent prognosis factors for overall survival (OS) and recurrence-free survival (RFS) of these patients. RESULTS: A total of 217 patients and 228 patients underwent RPD and OPD, respectively. After PSM, 180 patients in each group were enrolled. There were no significant differences in operative time, lymph node harvest, intraoperative transfusion, vascular resection, R0 resection, postoperative major morbidity, reoperation, 90-day mortality, and long-term survival between the two groups before and after PSM. Whereas, compared with the OPD group, the RPD group had significantly lower estimated blood loss (150.0 ml vs. 250.0 ml; P < 0.001), and a shorter postoperative length of stay (LOS) (12.0 days vs. 15.0 days; P < 0.001). Multivariable analysis showed carbohydrate antigen 19-9 (CA19-9), R0 resection, N stage, perineural invasion, and tumor differentiation significantly associated with OS and RFS of these patients. CONCLUSIONS: RPD was comparable to OPD in feasibility and safety. For patients with DCC, RPD resulted in similar oncologic and survival outcomes as OPD.

Comparison of robotic and open pancreaticoduodenectomy for primary nonampullary duodenal adenocarcinoma: a retrospective cohort study.

PURPOSE: Robotic surgery has been increasingly applied in pancreatic surgery and showed many advantages over conventional open surgery. The robotic pancreaticoduodenectomy (RPD) is a surgical option for primary nonampullary duodenal adenocarcinoma (PNDA). However, whether RPD is superior to open pancreaticoduodenectomy (OPD) for PNDA has not been reported. The comparative study was designed to analyze the short- and long-term outcomes of RPD versus OPD on patients with PNDA. METHODS: Demographics, perioperative, and survival outcomes among patients who underwent RPD (n = 49) versus OPD (n = 43) for PNDAs between January 2013 and March 2018 were collected and analyzed RESULTS: Demographic characteristics were comparable between the RPD group and the OPD group. The RPD group demonstrated a decreased estimated blood loss (100 vs. 200 ml, p < 0.001), time to oral intake (4.0 vs. 4.0 days, p = 0.04), and postoperative hospital stay (12.9 vs. 15.0 days, p = 0.01) compared with the OPD group. However, no differences were observed between the two groups in terms of operative time and the rates of major complications, grade B and C POPF, PPH, grade B and C DGE, biliary fistular, reoperation, and 90-day readmission. No patient died within 90 days. There were no significant differences in tumor size, differentiation, TNM stage, number of harvested lymph nodes, and the rates of nerve invasion, lymph node invasion, R0 resection, and the median overall survival between the two groups (p > 0.05) CONCLUSIONS: RPD is a safe, feasible, and effective treatment for PNDA compared with OPD and can be used as an alternative for surgeons in the treatment of PNDA. Further multicenter randomized controlled trials are needed to evaluate the effectiveness of RPD in patients with PNDA.

Methylation patterns partition pancreatic cancer into distinct prognostic subtypes.

In the initiation and progression of pancreatic cancer, DNA methylation plays a critical role. The present study attempts to explore specific prognosis subtypes based on DNA methylation data and develop an epigenetic signature to predict the overall survival (OS) of patients with pancreatic cancer.147 samples were included in the training cohort, whereas the validation cohort included 226 samples. The 298 OS-related methylation sites in the training cohort were selected for consensus clustering, and the authors identified three subtypes with a significant difference in prognosis. Cluster1 was associated with poor OS, low-grade disease and high lymph node involvement. In addition, we identified 33 specific methylation sites in Cluster1. Subsequently, we developed a robust qualitative signature consisting of 14 methylation sites to individually predict OS in the training cohort, and the predictive accuracy of this model was confirmed in the validation cohort. Functional enrichment analysis showed that the selected genes in the model were mainly enriched in known cancer-related pathways. Patients were divided into high- and low-risk groups by the model, and a significant difference in OS was observed between these groups. Classification based on the modeling of a specific DNA methylation site can reveal the heterogeneity of pancreatic cancer and provide guidance for clinicians in predicting the prognosis of pancreatic cancer and providing personalized treatment.

Robotic versus open total pancreatectomy: a retrospective cohort study.

PURPOSE: Robotic total pancreatectomy (RTP), although considered safe and feasible, has rarely been reported. This study aimed to evaluate whether RTP has advantages over open TP (OTP). METHODS: Demographics and perioperative outcomes among patients who underwent RTP (n=14) versus OTP (n=15) between May 2015 and September 2020 were retrospectively analyzed. RESULTS: RTP reduced the operative time (307.2 vs. 382.0 min, p=0.01) and estimated blood loss (EBL) (200 vs. 700 ml, p=0.002) compared to those of OTP. The patients in the RTP group got out of their beds and stood, received their first liquid, and took oral diets earlier (2.0 vs. 3.0 days, p=0.002; 2.0 vs. 4.0 days, p=0.009; 3.0 vs. 5.0 days, p=0.006) and experienced a shorter postoperative hospital stay (PHS) (9.0 vs. 12.0 days, p=0.03). There were no significant differences in the rates of spleen preservation, splenic vessel preservation, bile leakage, delayed gastric emptying, morbidity, or the number of lymph nodes harvest between the two groups. CONCLUSION: This study demonstrates that RTP is safe and feasible in selected patients with different indications in experienced robotic center. RTP was associated with a shorter operative time, lower EBL, and shorter PHS than OTP.

The standardized technique in robotic radical antegrade modular pancreatosplenectomy using the flip-up approach.

PURPOSE: Radical antegrade modular pancreatosplenectomy (RAMPS) was first introduced in 2003. It has been accepted as an alternative technique for pancreatic cancer of the body and tail. However, robotic RAMPS is not yet popular because of its technical difficulty and lack of standardized technique. This study describes in detail the standard steps of robotic RAMPS using the flip-up approach with the benefit of a robotic view when treating pancreatic cancer of the body and tail. METHOD: We took advantage of our single-center experience to provide a step-by-step technique of robotic RAMPS procedure using the da Vinci Si system. RESULTS: We divided the procedure into 11 key steps. The surgical steps are optimized to achieve margin-negative curative resection and sufficient regional lymphadenectomy. The artery-first approach is usually used to determine tumor resectability early before performing an irreversible operative step. We also determine the borders of surgical resection and divide the splenic artery after dividing the pancreatic neck and the splenic vein, which facilitates a complete lymphadenectomy around the celiac axis with a bottom-up view. CONCLUSION: Robotic RAMPS using the flip-up approach is safe and feasible in performing curative resection for well-selected pancreatic cancer of the body and tail. A randomized controlled trial comparing open and robotic RAMPS is needed in the future.

Robotic isolated partial and complete hepatic caudate lobectomy: A single institution experience.

BACKGROUND: Current reports on robotic hepatic caudate lobectomy are limited to Spiegel lobectomy. This study aimed to compare the safety and feasibility of robotic isolated partial and complete hepatic caudate lobectomy. METHODS: Clinical data of 32 patients who underwent robotic resection of the hepatic caudate lobe in our department from May 2016 to January 2020 were retrospectively analyzed. The patients were divided into three groups according to the lobectomy location: left dorsal segment lobectomy (Spiegel lobectomy), right dorsal segment lobectomy (caudate process or paracaval portion lobectomy), and complete caudate lobectomy. General information and perioperative results of the three groups were compared and analyzed. RESULTS: Among the 32 patients, none had conversion to laparotomy, three received intraoperative blood transfusion (9.38%), and none had complications of Clavien-Dindo grade III or higher or died in the perioperative period. Among them, 17 patients (53.13%) underwent Spiegel lobectomy, 7 (21.88%) underwent caudate process or paracaval portion lobectomy, and 8 (25.00%) underwent complete caudate lobectomy. The operative time and blood loss in the left dorsal segment lobectomy group were significantly better than those in the right dorsal segment lobectomy and complete caudate lobectomy groups (operative time: P = 0.010 and P = 0.005; blood loss: P = 0.005 and P = 0.017, respectively). The postoperative hospital stay in the left dorsal segment lobectomy group was significantly shorter than that in the complete caudate lobectomy group (P = 0.003); however, there was no difference in the postoperative hospital stay between the left dorsal segment lobectomy group and right dorsal segment lobectomy group (P = 0.240). CONCLUSIONS: Robotic isolated partial and complete caudate lobectomy is safe and feasible. Spiegel lobectomy is relatively straightforward and suitable for beginners.

Robotic vs laparoscopic hemihepatectomy: A comparative study from a single center.

BACKGROUND: Robotic surgery is increasingly being used in hepatectomy. Previous studies comparing the robotic and laparoscopic minor hepatectomy have been documented, but comparative studies on robotic and laparoscopic hemihepatectomy (LH) involving a large patient cohort are rare. The objective of this study was to compare perioperative outcomes between robotic and LH. METHODS: Data on the demographics, clinicopathologic characteristics, and perioperative outcomes of consecutive patients who underwent robotic or LH in a single center between November 2011 and July 2017 were analyzed. RESULTS: A total of 92 patients underwent robotic and 48 LH. Multiple linear regression analysis showed no significant difference in perioperative outcomes including operative time, postoperative hospital stay, postoperative complications, and mortality between the groups. Compared to the laparoscopic cohort, the robotic cohort had a significantly less estimated blood loss (120.24 mL; 95% confidence interval, 53.72-186.76) and a significantly lower conversation rate (1.09% vs 10.42%; P = .034). Stratified and interaction analyses demonstrated that disease type had an interaction effect on the association between the operative approach and the estimated blood loss. CONCLUSIONS: Robotic hemihepatectomy was safe and feasible in selected patients. It had similar perioperative outcomes as LH and was better than LH regarding estimated blood loss and open conversion.

MicroRNA-4656 is a prognostic factor and tumor suppressor in human pancreatic cancer through a downstream target of TrkA.

BACKGROUND: In the present study, we investigated the expression profile and functional mechanism of microRNA-4656 in human pancreatic cancer (PC). METHODS: MiR-4656 expression in PC tumors was examined using a quantitative reverse transcriptase-polymerase chain reaction in 134 patients. Associations between tumorous miR-4656 expression and clinicopathological parameters of patients, as well as overall survival, were analyzed. MiR-4656 expression was also examined in PC in vitro cell lines. In Capan-1 and AsPC-1 PC cells, lentivirus-induced miR-4656 overexpression or downregulation was applied to investigate its functional regulations on PC in vitro proliferation and invasion, as well as in vivo transplant growth. The association of miR-4656 and its downstream target, the tropomyosin receptor kinase A (TrkA) gene, was investigated in both cell line and clinical pancreatic tumors. In miR-4656-overerxpressed PC cells, TrkA was overexpressed with the aim of investigating its role in miR-4656-induced functional regulation in PC. RESULTS: MiR-4656 was downregulated in PC. Low tumorous miR-4656 expression was associated with a poor prognosis and overall survival of patients. MiR-4656 was also found to be downregulated in PC cell lines. MiR-4656 overexpression in Capan-1 and AsPC-1 cells significantly inhibited cancer proliferation and invasion in vitro, as well as explant growth in vivo, whereas miR-4656 downregulation had no effect on cancer development. The TrkA gene was directly bound by miR-4656, and reversely expressed in PC tumors as miR-4656. TrkA overexpression reversed the inhibitory effect of miR-4656-overexpression on PC proliferation and invasion. CONCLUSIONS: MiR-4656 is expressed to a low extent and is a potential biomarker in PC. Overexpressing miR-4656 has tumor suppressive effects on PC development both in vitro and in vivo, likely through its downstream target of the TrkA gene.

Robotic versus laparoscopic distal pancreatectomy: A propensity score-matched study.

BACKGROUND: Robotic distal pancreatectomy (RDP) is considered a safe and feasible alternative to laparoscopic distal pancreatectomy (LDP). However, previous studies have some limitations including small sample size and selection bias. This study aimed to evaluate whether the robotic approach has advantages over laparoscopic surgery in distal pancreatectomy. METHODS: Demographics and perioperative outcomes among patients undergoing RDP (n = 102) and LDP (n = 102) between January 2011 and December 2015 were reviewed. A 1:1 propensity score matched analysis was performed between both groups. RESULTS: Both groups displayed no significant differences in perioperative outcomes including operative time, blood loss, transfusion rate, and rates of overall morbidities and pancreatic fistula. Robotic approach reduced the rate of conversion to laparotomy (2.9% vs 9.8%, P = 0.045), especially in patients with large tumors (0% vs 22.2%, P = 0.042). RDP improved spleen (SP) and splenic vessels preservation (SVP) rates in patients with moderate tumors (60.0% vs 35.5%, P = 0.047; 37.1% vs 12.9%, P = 0.025), especially in patients without malignancy (95.5% vs 52.4%, P = 0.001; 59.1% vs 19.0%, P = 0.007). RDP also reduced postoperative hospital stay (PHS) significantly (7.67% vs 8.58, P = 0.032). CONCLUSIONS: RDP is associated with less rate of conversion to laparotomy, shorter PHS, and improved SP and SVP rates in selected patients than LDP.

The surgical outcomes of robot-assisted laparoscopic pancreaticoduodenectomy versus laparoscopic pancreaticoduodenectomy for periampullary neoplasms: a comparative study of a single center.

BACKGROUND: Pancreaticoduodenectomy (PD) is a difficult and complex operation. The introduction of robotics has opened up new angles in pancreatic surgery. This study aims to assess the surgical outcomes of robot-assisted laparoscopic pancreaticoduodenectomy relative to its laparoscopic counterpart. METHODS: A retrospective study was designed to compare the surgical outcomes of 27 robot-assisted laparoscopic pancreaticoduodenectomy (RPD) and 25 laparoscopic pancreaticoduodenectomy (LPD). Perioperative data, including operating time, complication, morbidity and mortality, estimated blood loss, and postoperative length of stay, were analyzed. RESULTS: The robotic group exhibited significantly shorter operative time (mean 387 vs. 442 min), shorter hospital stay (mean 17 vs. 24 days), and less blood loss (mean 219 vs. 334 ml) than those in the LPD group. No statistical difference was observed between the two groups in terms of complication rate, mortality rate, R0 resection rate, and number of harvested lymph node. CONCLUSIONS: RPD is more efficient and secure process than LPD among properly selected patients. RPD is therefore a feasible alternative to the laparoscopic procedure. Further studies are needed to evaluate the cost effectiveness of the robotic approach for PD.

Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins.

Biochemical studies of cellular processes involving polyubiquitin have gained increasing attention. More tools are needed to identify ubiquitin (Ub)-binding proteins. We report diazirine-based photoaffinity probes that can capture Ub-binding proteins in cell lysates, and show that diazirines are preferable to aryl azides as the photo-crosslinking group, since they decrease non-selective capture. Photoaffinity probes containing at least two Ub units were required to effectively capture Ub-binding proteins. Different capture selectivity was observed for probes containing diubiquitin moieties with different types of linkages, thus indicating the potential to develop linkage-dependent probes for selectively profiling Ub-binding proteins under various cellular conditions.

Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis.

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.

Monomer/Oligomer Quasi-Racemic Protein Crystallography.

Racemic or quasi-racemic crystallography recently emerges as a useful technology for solution of the crystal structures of biomacromolecules. It remains unclear to what extent the biomacromolecules of opposite handedness can differ from each other in racemic or quasi-racemic crystallography. Here we report a finding that monomeric d-ubiquitin (Ub) has propensity to cocrystallize with different dimers, trimers, and even a tetramer of l-Ub. In these cocrystals the unconnected monomeric d-Ubs can self-assemble to form pseudomirror images of different oligomers of l-Ub. This monomer/oligomer cocrystallization phenomenon expands the concept of racemic crystallography. Using the monomer/oligomer cocrystallization technology we obtained, for the first time the X-ray structures of linear M1-linked tri- and tetra-Ubs and a K11/K63-branched tri-Ub.

The ubiquitin ligase RNF43 downregulation increases membrane expression of frizzled receptor in pancreatic ductal adenocarcinoma.

RNF43 is a novel tumor suppressor protein and known to be expressed in a multitude of tissue and dysregulated in cancers of these organs including ovarian and colorectal tissues. RNF43 expression has been shown to be expressed in mutated forms in several pancreatic cell lines. RNF43, by virtue of being an ubiquitin ligase, has the potential to ubiquitinylate membrane receptors like frizzled that subserves sensing Wnt soluble signals at the cell membrane. Thus, normally, RNF43 downregulates Wnt signaling by removing frizzled receptor from the membrane. In the present study, the expression of the tumor suppressor RNF43 was examined in human patient samples of pancreatic ductal adenocarcinoma (PDAC). Reduced levels of expression of RNF43 in PDAC were demonstrated by Western blotting. We incorporated membrane biotinylation assay to examine the expression of frizzled6 receptor in the membrane and demonstrated that it is significantly increased in PDAC tissues. This may be responsible for enhanced Wnt/beta-catenin signaling and provides the first level of evidence of a possible role of this well-known pathway in pancreatic exocrine carcinogenesis. We have utilized appropriate controls to ensure the true positivity of the findings of the present study. The contribution of Wnt/beta-catenin/RNF43 pathway in pancreatic carcinogenesis may provide for utilization of pharmacologic resources for precision-based approaches to treat pancreatic ductal adenocarcinoma.