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.

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.].

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.

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.

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.

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.

Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

Is less than 5 mm as the narrowest surgical margin width in central resections of hepatocellular carcinoma justified?

BACKGROUND: The aim of this study was to investigate whether <5 mm as the narrowest margin width may negatively affect a patient's outcome. METHODS: A prospective cohort study was designed. From January 1994 to July 2010, 196 patients with hepatocellular carcinoma undergoing central hepatectomy were divided into group A (n = 172; narrowest margin, ≥5 to <10 mm) and group B (n = 24; narrowest margin, <5 mm), and outcomes were compared. RESULTS: Significant differences between groups A and B included tumor size (P = .057), infiltrative border (P = .021), satellite lesions (P = .021), and major perivascular abutment (P = .028). Marginal recurrence occurred in 50% of the patients in group B but none of those in group A (P < .001). There were no significant differences between the groups regarding recurrence, recurrence-related death, disease-free survival, and speed of recurrence, but a borderline significant difference was found regarding the cumulative probability of overall survival. After excluding early recurrence (within 1 year), group B had significantly lower cumulative probabilities of disease-free survival (P = .020) and overall survival (P < .001). CONCLUSIONS: In central resections, narrowest margin width of <5 mm does not negatively affect recurrence and overall survival. However, it increases perimargin recurrence and inversely affects late outcomes.

High expression of patched homolog-1 messenger RNA and glioma-associated oncogene-1 messenger RNA of sonic hedgehog signaling pathway indicates a risk of postresection recurrence of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has a high recurrence rate after resection. Abnormal activation of the sonic hedgehog (SHH) signaling pathway contributing to the carcinogenesis of some organs had been reported. We hypothesize that activation of SHH pathway contributes to the recurrence of HCC after surgical resection. METHODS: In a prospective study, from January 2006 to June 2010, a total of 50 consecutive patients with HCC after curative resection were enrolled. The ratio of the expression of messenger RNA (mRNA) of SHH, patched homolog-1 (PTCH-1) and glioma-associated oncogene-1 (GLI-1) between HCC tissues and the paired noncancerous liver tissues were measured. Both the clinicopathologic characteristics and these ratios were compared between those with recurrence and those without recurrence. They were also compared between those with and without survival. RESULTS: There was a statistically significant correlation among the ratios of PTCH-1 mRNA and serum AFP level (p = 0.045), tumor size (p = 0.001), vascular permeation (p = 0.043) and tumor, node, metastasis staging system (TNM) stage (p = 0.003). A borderline significant correlation was found among ratios of GLI-1 mRNA and tumor size (p = 0.062) and TNM stage (p = 0.051). There was no such significant correlation between SHH mRNA and any parameter. Both the ratios of PTCH-1 mRNA and GLI-1 mRNA significantly adversely affected recurrence (p = 0.003 and 0.005, respectively) and survival (p < 0.001 and <0.001, respectively). CONCLUSIONS: Expression of PTCH-1 mRNA and GLI-1 mRNA in HCC tissues is a potential biomarker to predict postresection disease recurrence.

Clinical manifestations and survival of hepatocellular carcinoma patients with peritoneal metastasis.

BACKGROUND AND AIM: Peritoneal metastasis is an uncommon manifestation of hepatocellular carcinoma (HCC). The aim of the present paper was to investigate the characteristics and survival of HCC patients with peritoneal metastases. METHODS: From January 1985 to December 2004, we retrospectively reviewed the records of 53 Taiwanese HCC patients with peritoneal metastases. RESULTS: Peritoneal metastases were detected at the time of HCC diagnosis (synchronously) in 10 patients and after the initial therapy for the primary tumors (metachronously) in 43 patients. The mean time for development of the metachronous peritoneal metastases was similar whether the primary cancer was treated with surgery (24 months) or transarterial chemoembolization (22.2 months). The single patient whose primary cancer was treated with supportive care alone developed peritoneal metastasis only 7.5 months after detection of the primary cancer. Surgical resection of the peritoneal metastases was possible in two-thirds of the 43 metachronous patients. The median survival for those who received surgery for these metastases was 12.5 months vs. 2.1 months for those without surgery (P = 0.0013). However, there was no difference in survival if patients were stratified to Child-Pugh grade. CONCLUSIONS: Peritoneal metastases of HCC are rare and can occur synchronously or metachronously. Though increased long-term survival was found in patients who had surgical removal of peritoneal metastases, the main determinant of better survival is Child-Pugh grade.

Diffuse intraperitoneal metastasis after spontaneous rupture of hepatocellular carcinoma.

Rupture of hepatocellular carcinoma (HCC) is a fatal complication. Intraperitoneal metastasis after rupture of HCC is rare. We report a case of diffuse intraperitoneal metastases after rupture of HCC. A previously asymptomatic 32-year-old man was admitted because of massive ascites due to ruptured HCC. Poor liver reserve limited the therapeutic options. Transarterial chemoembolization was performed to stop tumor bleeding. Abdominal computed tomography demonstrated multiple large peritoneal metastases 3 months after the rupture episode. Echo-guided fine needle aspiration from the suprapubic area was performed. Cytology was positive for HCC. It is rare for HCC to develop intraperitoneal metastases in as short as 3 months.