MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Precise mapping of hilar cholangiocarcinoma with a skip lesion by SpyGlass cholangioscopy: A case report.

BACKGROUND: Cholangiocarcinoma (CC) is a very aggressive cancer with a poor prognosis. As surgery is the only curative therapy, preoperative evaluation of the tumor extent is essential for surgical planning. Although high-quality image modalities such as computed tomography and magnetic resonance imaging have been used extensively in preoperative evaluation, the accuracy is low. To obtain precise localization of tumor spread arising from the hilar region preoperatively, the development of an acceptable imaging modality is still an unmet need. CASE SUMMARY: A 52-year-old female presented to our emergency department with jaundice, abdominal pain, and fever. Initially, she was treated for cholangitis. Endoscopic retrograde cholangiopancreatography with the cholangiogram showed long segment filling defect in the common hepatic duct with dilatation of bilateral intrahepatic ducts. Transpapillary biopsy was performed, and the pathology suggested intraductal papillary neoplasm with high-grade dysplasia. After treatment of cholangitis, contrasted-enhanced computed tomography revealed a hilar lesion with undetermined Bismuth-Corlette classification. SpyGlass cholangioscopy showed that the lesion involved the confluence of the common hepatic duct with one skip lesion in the posterior branch of the right intrahepatic duct, which was not detected by previous image modalities. The surgical plan was modified from extended left hepatectomy to extended right hepatectomy. The final diagnosis was hilar CC, pT2aN0M0. The patient has remained disease-free for more than 3 years. CONCLUSION: SpyGlass cholangioscopy may have a role in precision localization of hilar CC to provide surgeons with more information before the operation.

Rheumatic manifestations of hepatitis C virus infection are associated with autoantibodies but not viremia.

BACKGROUND: To investigate the associations between extrahepatic manifestations, autoantibodies, and viremia in patients with hepatitis C virus (HCV) infection. METHODS: This cross-sectional study recruited patients with HCV infection from the outpatient department of a tertiary medical center in Northern Taiwan between January 2017 and August 2019. Autoantibody profiles and the clinical parameters of HCV infection were evaluated using laboratory tests, and a questionnaire was used to record extrahepatic manifestations. HCV infection status, including inactive HCV infection, active hepatitis, and cirrhosis, was defined according to abdominal ultrasonography findings and alanine transaminase levels. RESULTS: A total of 77 patients with HCV were recruited, with 19.5% and 16.9% of patients, respectively, presenting with arthritis and dry eyes. Autoantibody screening revealed rheumatoid factor (RF), antinuclear antibody (ANA), anti-Ro antibody, and anti-La antibody positivity in 20.8%, 23.4%, 13.0%, and 2.6% of the patients, respectively. The presence of RF was associated with arthritis, whereas the presence of ANA was associated with dry eyes but not dry mouth. Active hepatitis and HCV-related cirrhosis were associated with viremia, but not autoantibody profiles. CONCLUSION: In this single-center study, the prevalence of extrahepatic manifestations and autoantibodies did not differ in patients stratified by the HCV infection status. Rheumatic manifestations were associated with the presence of autoantibodies but not with viremia.

The evolution and structure of snake venom phosphodiesterase (svPDE) highlight its importance in venom actions.

For decades, studies of snake venoms focused on the venom-ome-specific toxins (VSTs). VSTs are dominant soluble proteins believed to contribute to the main venomous effects and emerged into gene clusters for fast adaptation and diversification of snake venoms. However, the conserved minor venom components, such as snake venom phosphodiesterase (svPDE), remain largely unexplored. Here, we focus on svPDE by genomic and transcriptomic analysis across snake clades and demonstrate that soluble svPDE is co-opted from the ancestral membrane-attached ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) gene by replacing the original 5' exon with the exon encoding a signal peptide. Notably, the exons, promoters, and transcription/translation starts have been replaced multiple times during snake evolution, suggesting the evolutionary necessity of svPDE. The structural and biochemical analyses also show that svPDE shares the similar functions with ENPP family, suggesting its perturbation to the purinergic signaling and insulin transduction in venomous effects.

Correction: Multi-targeting of functional cysteines in multiple conserved SARS-CoV-2 domains by clinically safe Zn-ejectors.

[This corrects the article DOI: 10.1039/D0SC02646H.].

Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease.

The Lon AAA+ protease (LonA) is a ubiquitous ATP-dependent proteolytic machine, which selectively degrades damaged proteins or native proteins carrying exposed motifs (degrons). Here we characterize the structural basis for substrate recognition and discrimination by the N-terminal domain (NTD) of LonA. The results reveal that the six NTDs are attached to the hexameric LonA chamber by flexible linkers such that the formers tumble independently of the latter. Further spectral analyses show that the NTD selectively interacts with unfolded proteins, protein aggregates, and degron-tagged proteins by two hydrophobic patches of its N-lobe, but not intrinsically disordered substrate, α-casein. Moreover, the NTD selectively binds to protein substrates when they are thermally induced to adopt unfolded conformations. Collectively, our findings demonstrate that NTDs enable LonA to perform protein quality control to selectively degrade proteins in damaged states and suggest that substrate discrimination and selective degradation by LonA are mediated by multiple NTD interactions.

There are many different types of protein which each have different roles in biology. Most proteins are surrounded by water and are folded so that their water-attracting regions are on the outside and more fat-like regions, which repel water, are on the inside. When a protein becomes damaged or is assembled incorrectly, some of the fat-like regions end up on the outside of the protein and become exposed to water. This can prevent the protein from performing its role and harm the cell instead. LonA proteases are responsible for dismantling and recycling these harmful proteins, as well as proteins that have been labelled for destruction. They do this by unfolding the unwanted protein and transporting it into an enclosed chamber made of six LonA molecules. Once inside the chamber, the target protein is broken down into smaller fragments that can be used to build other structures. LonA proteases contain a region called the N-terminal domain, or NTD for short, which is thought to be responsible for identifying which proteins need degrading. Yet it remained unclear how the NTD recognizes and binds to these target proteins. To answer this question, Tzeng et al. studied the detailed structure of a LonA protease that had been purified from bacteria cells. This revealed that the NTD of LonA contains two water-repelling regions which bind to fat-like segments on the surface of proteins that have become unfolded or tagged for destruction. Further experiments showed that the NTD is bound to the main body of LonA via a 'flexible linker'. This led Tzeng et al. to propose that the NTD sways around loosely at the end of LonA searching for proteins with exposed water-repelling regions. Once an NTD identifies and attaches to a target, the NTDs of the other LonA molecules then bind to the protein and help insert it into the chamber. Proteases are a vital component of all biological systems. Controlling protein destruction and recycling is a key factor in how cells divide and respond to a changing environment. This study provides new insights into how LonA operates in bacteria, which may apply to proteases more widely. This contributes to our knowledge of fundamental biology and may also be relevant in a range of diseases where protein recycling is defective or inefficient.

Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B.

DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B-3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

Multi-targeting of functional cysteines in multiple conserved SARS-CoV-2 domains by clinically safe Zn-ejectors.

We present a near-term treatment strategy to tackle pandemic outbreaks of coronaviruses with no specific drugs/vaccines by combining evolutionary and physical principles to identify conserved viral domains containing druggable Zn-sites that can be targeted by clinically safe Zn-ejecting compounds. By applying this strategy to SARS-CoV-2 polyprotein-1ab, we predicted multiple labile Zn-sites in papain-like cysteine protease (PLpro), nsp10 transcription factor, and nsp13 helicase. These are attractive drug targets because they are highly conserved among coronaviruses and play vital structural/catalytic roles in viral proteins indispensable for virus replication. We show that five Zn-ejectors can release Zn2+ from PLpro and nsp10, and clinically-safe disulfiram and ebselen can not only covalently bind to the Zn-bound cysteines in both proteins, but also inhibit PLpro protease. We propose combining disulfiram/ebselen with broad-spectrum antivirals/drugs to target different conserved domains acting at various stages of the virus life cycle to synergistically inhibit SARS-CoV-2 replication and reduce the emergence of drug resistance.

Liver Transplantation After Downstagings of Ruptured Advanced Hepatocellular Carcinoma in Cirrhotic Liver: Is It Advisable? A Case Report.

Spontaneous rupture of hepatocellular carcinoma (HCC), defined as T4 in TNM stage by the American Joint Committee on Cancer (eighth edition), is a serious life-threatening complication. Effective treatment remains challenging because of a high 1-month mortality, a short median survival, and the potential of peritoneal metastasis. We reported on a case that received a living related donor liver transplantation (LDLT) after successful consecutive downstaging therapies. A 63-year-old man with alcohol-related liver cirrhosis and multiple HCC developed spontaneous rupture and hemoperitoneum. He received 3 sessions of transcatheter hepatic arterial chemoembolization and target therapy with sorafenib. Computed tomography scans and magnetic resonance imaging after 11 months of treatment showed that the patient's HCCs fulfilled the Milan criteria and the University of California San Francisco criteria prior to LDLT. The perioperative course was rather smooth. After discharge, interval follow-up computed tomography studies of chest and liver and a whole-body bone scan showed no tumor recurrence or metastasis up to 20 months post-operation. Successful downstaging therapies of ruptured HCC to fulfill Milan criteria to receive liver transplantation is advisable in highly selected patients.

Reappraisal of Failures in Downstaging Treatment of Hepatocellular Carcinoma Prior to Liver Transplant-Preliminary Report on the Impact of Underestimations of Tumor Numbers and Tumor Sizes as Measured From Imaging Before Transplant.

Downstaging treatment of hepatocellular carcinoma (HCC) prior to liver transplant (LT) is an accepted strategy to meet the Milan criteria. However, after transplant surgery, a reality is noted that the number or/and the size of some HCCs measured from the liver explants is different from that measured from the pre-LT imaging. If tumor number or tumor size measured from the liver explants was beyond that measured from pre-LT imaging, we define it as "failed downstaging." Among 27 patients who received downstaging therapies, there are 11 "number reduction failures" and 6 "size reduction failures." We attribute the discrepancy to 2 possible reasons; one is that the pre-LT imaging after downstaging could not completely detect all the HCC; the other is that the time interval between the downstaging and LT is long enough to develop new HCCs. After follow-up, 6 patients developed HCC recurrence. The significant factors affecting recurrence include tumor size from postdownstaging imaging (P = .048), tumor number ≥ 2 (P = .007), multiple sessions of downstaging (P = .03), ratio of neutrophil to lymphocyte (P = .047), and tumor number from liver explant (borderline P = .05). Tumor recurrence after LT is significantly higher in those with "size reduction failure" (P = .048). The interval between LT and tumor recurrence is significantly shorter in those with "size reduction failure" (P = .04). To decrease underestimations of HCC, combining various imaging studies including the computed tomographic scan, magnetic resonance imaging, and contrast ultrasonography is needed to increase the accuracy before LT. Repeated imaging studies at short intervals of no more than 3 months are necessary during a long wait. How to minimize the underestimations of HCC to determine the appropriate candidacy for LT is an important goal for transplantation surgeons.

Graft calcification caused by a torsion of the hepatic vein after a living-related donor liver transplantation.

 The torsion of vessels after liver transplantation rarely occurs. Likewise, calcification of a liver graft has seldom been reported. This report details a case which had torsion of the left hepatic vein on the seventh day after living-related donor liver transplantation. The torsion was reduced soon after re-exploration; however, congestion with partial necrosis of the graft occurred. On the follow-up imaging studies, some resolution of necrosis and graft regeneration were found, yet geographic calcification of the liver graft appeared.The patient died of pneumonia after 13 weeks, post-operation. The avoidance such torsion of vessels is necessary and important.

Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease.

The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85 Å crystal structure of Mg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA.

Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine.

Distal M domain of cobra ADAM-like metalloproteinase mediates the binding of positively charged cysteine-rich domain to αvß3 integrin in the suppression of cell migration.

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvß3 integrin in the surface-immobilized form. Atragin binds to αvß3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvß3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in a αvß3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvß3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

Similar rebleeding rate in 3-day and 7-day intravenous ceftriaxone prophylaxis for patients with acute variceal bleeding.

BACKGROUND/PURPOSE: Although prophylactic antibiotics have been recommended for cirrhotic patients with upper gastrointestinal bleeding, the duration of its use remains an inconclusive issue. We designed this study to investigate the duration of antibiotic prophylaxis for cirrhotic patients with acute esophageal variceal bleeding. METHODS: We enrolled those patients suffering from acute esophageal variceal bleeding and receiving band ligation. They were randomly allocated to two groups to receive prophylactic antibiotics; Group I: receiving intravenous ceftriaxone 500 mg every 12 hours for 3 days, and Group II: same regimen for 7 days. We used rebleeding rate within 14 days as the primary end point and also evaluated the survival rate within 28 days and the amount of transfusion during admission. RESULTS: There were 38 patients in Group I and 33 patients in Group II that completed the study course for analysis. Overall, there was no significant difference in the baseline characteristics between these two groups. There were three patients both in Group I and Group II who developed rebleeding within 14 days (8% vs. 9%, p > 0.99). There was also no difference between Group I and Group II in transfusion amount (2.71 ± 2.84 units vs. 3.18 ± 4.07, p = 0.839) and survival rate in 28 days (100 vs. 97%, p = 0.465). CONCLUSION: Our small scale study demonstrated that there was no difference in the rebleeding rate between 3-day and 7-day ceftriaxone prophylaxis for cirrhotic patients with acute esophageal variceal bleeding. There was also no difference in 28 day survival rate between these two groups.

Stress ulcer prophylaxis in patients being weaned from the ventilator in a respiratory care center: A randomized control trial.

BACKGROUND/PURPOSE: No data has been available on prophylaxis for stress ulcer development during the process of weaning patients off mechanical ventilators. We conducted a randomized study to evaluate the efficacy of stress ulcer prophylaxis with lansoprazole OD in patients being weaned from mechanical ventilators. METHODS: A total of 120 patients were randomly allocated into two groups using blocked randomization, with 60 patients in each group. Group A was the treatment group, receiving lansoprazole OD 30 mg from a nasogastric tube for 14 days, while Group B, the control group, received no proton pump inhibitors or other medications for treating peptic ulcers. The primary end point of our study was apparent upper gastrointestinal bleeding within 2 weeks of enrollment. RESULTS: Apparent upper gastrointestinal bleeding occurred in zero patients and five patients in Groups A and B, respectively (Group A: 0% vs. Group B: 8.3%, p = 0.057). There was no significant difference between the two groups in ventilator-associated pneumonia (Group A: 6.7% vs. Group B: 10.0%, p = 0.509) and 30-day survival rates (Group A: 96.7% vs. Group B: 100%, p = 0.496). CONCLUSION: Stress ulcer prophylaxis with lansoprazole in patients being weaned from mechanical ventilators led to a lower but not statistically significant incidence of apparent upper gastrointestinal bleeding. There was no significant increase of incidence of ventilator-associated pneumonia in the prophylaxis group. Further larger scale studies are needed to clarify the benefit of stress ulcer prophylaxis in such patients.

Increasing biopsy number and sampling from gastric body improve the sensitivity of rapid urease test in patients with peptic ulcer bleeding.

BACKGROUND: Previous studies demonstrated that the sensitivity of rapid urease test (RUT) for diagnosis of Helicobacter pylori infection decreased during peptic ulcer bleeding. AIM: We designed this study and tried to find a better method to improve the detection rate of H. pylori infection at the same session of endoscopic diagnosis of peptic ulcer bleeding. METHODS: We prospectively enrolled 116 patients with peptic ulcer bleeding. These patients received intravenous proton pump inhibitor and then received upper gastrointestinal endoscopy within 24 h after arrival. We took one piece of biopsy from gastric antrum (Group 1), four pieces from gastric antrum (Group 2), and one piece from the gastric body (Group 3) for three separate RUTs, respectively. (13)C-urease breath test was used as gold standard for diagnosis of H. pylori infection. RESULTS: There were 74 patients (64 %) with positive (13)C-urease breath test. Among these 74 patients, 45 patients had positive RUT (sensitivity: 61 %) in Group 1; 55 patients had positive RUT (sensitivity: 74 %) in Group 2; 54 patients had positive RUT (sensitivity: 73 %) in Group 3. There were significant differences between Group 1 and Group 2 (p = 0.02) and between Group 1 and Group 3 (p = 0.022). CONCLUSIONS: The sensitivity of RUT was 61 % during peptic ulcer bleeding. The sensitivity of RUT can be increased significantly by increased biopsy number from gastric antrum or biopsy from gastric body.

Liver fat contents, abdominal adiposity and insulin resistance in non-diabetic prevalent hemodialysis patients.

BACKGROUND: The liver fat contents and abdominal adiposity correlate well with insulin resistance (IR) in the general population. However, the relationship between liver fat content, abdominal adiposity and IR in non-diabetic hemodialysis (HD) patients remains unclear. This study aimed to clarify the associations among these factors. METHODS: This is a cross-sectional, observational study. All patients received abdominal ultrasound for liver fat content. Abdominal adiposity was quantified with the conicity index (Ci) and waist circumference (WC). We checked the homeostasis model assessment for insulin resistance index (HOMA-IR) for IR. RESULTS: A total of 112 patients (60 women) were analyzed. Subjects with higher liver fat contents and WC had higher IR indices. But Ci did not correlate with IR indices. In both the multi-variable linear regression model and the logistic regression model, only higher liver fat content predicted a severe IR status. CONCLUSIONS: Liver fat contents have a remarkable correlation with IR; however, abdominal adiposity, measured either by Ci or WC, dose not independently correlate with IR in non-diabetic prevalent HD patients.