Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

Effects of Coronary Artery Revascularization with a Polymer-Free Biolimus A9-Coated BioFreedom Stent Versus Bypass Surgery before Noncardiac Surgery.

PURPOSE: The present study aimed to evaluate the efficacy and safety of polymer-free drug-coated BioFreedom stent implantation in comparison to coronary artery bypass graft (CABG) before major noncardiac surgery. MATERIALS AND METHODS: In a multicenter registry, 55 patients required revascularization before major noncardiac surgery that should not be delayed >6 months. Of them, 27 underwent BioFreedom stent implantation and 28 underwent CABG. Primary outcomes included rate of noncardiac surgery, time from revascularization to noncardiac surgery, and occurrence of composite outcomes (all-cause death, myocardial infarction, stent thrombosis, stroke, repeat revascularization, or major bleeding). RESULTS: The rate of major noncardiac surgery was significantly higher in the BioFreedom group (92.6%) than in the CABG group (64.3%; p=0.027). Time from revascularization to noncardiac surgery was significantly shorter in the BioFreedom group (38.0 days) than in the CABG group (73.0 days; p=0.042). During the hospitalization for revascularization period, the occurrence of primary outcomes did not differ between the groups. However, the BioFreedom group showed a shorter hospitalization period and lower total treatment cost than the CABG group. During the hospital stay for noncardiac surgery, the occurrence of composite outcome was not significantly different between groups (4% vs. 0%; p>0.999): stroke occurred in only 1 case, and there were no cases of death or stent thrombosis in the BioFreedom group. CONCLUSION: This study demonstrated that BioFreedom stenting as a revascularization strategy before major noncardiac surgery might be feasible and safe in selected patients with less severe coronary artery diseases.

Effective Killing of Cancer Cells Through ROS-Mediated Mechanisms by AMRI-59 Targeting Peroxiredoxin I.

AIMS: The intrinsic increase of reactive oxygen species (ROS) production in cancer cells after malignant transformation frequently induces redox adaptation, leading to enhanced antioxidant capacity. Peroxiredoxin I (PrxI), an enzyme responsible for eliminating hydrogen peroxide, has been found to be elevated in many types of cancer cells. Since overexpression of PrxI promoted cancer cells' survival and resistance to chemotherapy and radiotherapy, PrxI has been proposed as a therapeutic target for anticancer drugs. In this study, we aimed to investigate the anticancer efficacy of a small molecule inhibitor of PrxI. RESULTS: By a high-throughput screening approach, we identified AMRI-59 as a potent inhibitor of PrxI. AMRI-59 increased cellular ROS, leading to the activation of both mitochondria- and apoptosis signal-regulated kinase-1-mediated signaling pathways, resulting in apoptosis of A549 human lung adenocarcinoma. AMRI-59 caused no significant changes in ROS level, proliferation, and apoptosis of PrxI-knockdown A549 cells by RNA interference. PrxI overexpression or N-acetylcysteine pretreatment abrogated AMRI-59-induced cytotoxicity in A549 cells. AMRI-59 rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells. Moreover, significant antitumor activity of AMRI-59 was observed in mouse tumor xenograft model implanted with A549 cells with no apparent acute toxicity. INNOVATION: This study offers preclinical proof-of-concept for AMRI-59, a lead small molecule inhibitor of PrxI, as an anticancer agent. CONCLUSIONS: Our results highlight a promising strategy for cancer therapy that preferentially eradicates cancer cells by targeting the PrxI-mediated redox-dependent survival pathways.

Structure-Activity Relationships of Neplanocin A Analogues as S-Adenosylhomocysteine Hydrolase Inhibitors and Their Antiviral and Antitumor Activities.

On the basis of the potent inhibitory activity of neplanocin A (1) against S-adenosylhomocysteine (AdoHcy) hydrolase, we analyzed the comprehensive structure-activity relationships by modifying the adenine and carbasugar moiety of 1 to find the pharmacophore in the active site of the enzyme. The introduction of 7-deazaadenine instead of adenine eliminated the inhibitory activity against the AdoHcy hydrolase, while 3-deazaadenine maintained the inhibitory activity of the enzyme, indicating that N-7 is essential for its role as a hydrogen bonding acceptor. The substitution of hydrogen at the 6'-position with fluorine increased the inhibitory activity of the enzyme. The one-carbon homologation at the 5'-position generally decreased the inhibitory activity of the enzyme, indicating that steric repulsion exists. A molecular docking study also supported these experimental data. In this study, 6'-fluoroneplanocin A (2) was the most potent inhibitor of AdoHcy hydrolase (IC50 = 0.24 µM). It showed a potent anti-VSV activity (EC50 = 0.43 µM) and potent anticancer activity in all the human tumor cell lines tested.

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbß3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.

Reactive oxygen species play a critical role in collagen-induced platelet activation via SHP-2 oxidation.

AIMS: The collagen-stimulated generation of reactive oxygen species (ROS) regulates signal transduction in platelets, although the mechanism is unclear. The major targets of ROS include protein tyrosine phosphatases (PTPs). ROS-mediated oxidation of the active cysteine site in PTPs abrogates the PTP catalytic activity. The aim of this study was to elucidate whether collagen-induced ROS generation leads to PTP oxidation, which promotes platelet stimulation. RESULTS: SH2 domain-containing PTP-2 (SHP-2) is oxidized in platelets by ROS produced upon collagen stimulation. The oxidative inactivation of SHP-2 leads to the enhanced tyrosine phosphorylation of spleen tyrosine kinase (Syk), Vav1, and Bruton's tyrosine kinase (Btk) in the linker for the activation of T cells signaling complex, which promotes the tyrosine phosphorylation-mediated activation of phospholipase Cγ2 (PLCγ2). Moreover, we found that, relative to wild-type platelets, platelets derived from glutathione peroxidase 1 (GPx1)/catalase double-deficient mice showed enhanced cellular ROS levels, oxidative inactivation of SHP-2, and tyrosine phosphorylation of Syk, Vav1, Btk, and PLCγ2 in response to collagen, which subsequently led to increased intracellular calcium levels, degranulation, and integrin αIIbß3 activation. Consistent with these findings, GPx1/catalase double-deficiency accelerated the thrombotic response in FeCl3-injured carotid arteries. INNOVATION: The present study is the first to demonstrate that SHP-2 is targeted by ROS produced in collagen-stimulated platelets and suggests that a novel mechanism for the regulation of platelet activation by ROS is due to oxidative inactivation of SHP-2. CONCLUSION: We conclude that collagen-induced ROS production leads to SHP-2 oxidation, which promotes platelet activation by upregulating tyrosine phosphorylation-based signal transduction.

Percutaneous cardiopulmonary support-supported percutaneous coronary intervention: a single center experience.

BACKGROUND AND OBJECTIVES: Percutaneous cardiopulmonary support (PCPS) has proven to be a valuable technique in high-risk coronary patients undergoing percutaneous coronary intervention (PCI). However, there have been few studies on PCI associated with PCPS in Korea. We summarized our experience with PCPS-supported PCI. SUBJECTS AND METHODS: We retrospectively reviewed 19 patients with PCPS-supported PCI between August 2005 and June 2009. PCPS was used as an elective procedure for 10 patients with at least two of the following conditions: left-ventricular ejection fraction <35%, target vessel(s) supplying more than 50% of the viable myocardium, high risk surgical patients, and patients who refused coronary bypass surgery. In the remaining 9 patients PCPS was used as an emergency procedure, to stabilize and even resuscitate patients with acute myocardial infarction and cardiogenic shock, in order to attempt urgent PCI. RESULTS: Among the 19 patients who were treated with PCPS-supported PCI, 11 (57.9%) survived and 8 (42.1%) patients did not. ST elevation myocardial infarction with cardiogenic shock was more prevalent in the non-survivors than in the survivors (75% vs. 27.3%, p=0.04). The elective PCPS-supported PCI was practiced more frequently in the survivors than in the non-survivors (72.7% vs. 25%, p=0.04). In the analysis of the event-free survival curve between elective and emergency procedures, there was a significant difference in the survival rate (p=0.025). Among the survivors there were more patients with multi-vessel disease, but a lower Thrombolysis in Myocardial Infarction grade in the culprit lesions was detected in the non-survivors, before PCI. Although we studied high-risk patients, there was no procedure-related mortality. CONCLUSION: Our experience suggests that PCPS may be helpful in high risk patients treated with PCI, especially in elective cases. More aggressive and larger scale studies of PCPS should follow.

Gender-based differences in the management and prognosis of acute coronary syndrome in Korea.

PURPOSE: Gender-based differences exist in the characteristics, management, and prognosis of acute coronary syndrome (ACS). However, their impact on prognosis remains unclear. We aimed to identify factors causing these differences in Koreans. MATERIALS AND METHODS: We examined 6,636 ACS patients (66.2% males) visiting 72 Korean hospitals between April-2007 and December-2008. Gender-based differences in clinical demographics, therapy, and outcomes were analyzed over 6 months. RESULTS: Women were older than men [mean (standard deviation, SD) age, 67.6 (9.8) vs. 60.6 (11.2) years; p<0.001]; had higher rates of hypertension, diabetes mellitus, and lack of exercise (p<0.001 for all); and lower rates of obesity, familial history of cardiovascular disease (CVD), and smoking (p<0.05 for all). Atypical symptoms were more common in women (20.5% vs. 15.1% in men, p<0.001), whereas myocardial infarction with ST-segment elevation was less common (17.1% vs. 27.8%, p<0.001). Mean (SD) time lapse from symptom onset to arrival at hospital was longer in women [11.44 (18.19) vs. 8.26 (14.89) hours in men, p<0.001], as was the duration of hospitalization [7.58 (7.61) vs. 7.04 (7.72) days, p=0.007]. Fewer women underwent revascularization procedures, including thrombolytic therapy, balloon angioplasty, stent implantation, and coronary artery bypass grafting (79.4% vs. 83.3% men, p<0.001). No significant differences were observed in CVD-related death, recurrent ACS, stroke, refractory angina, or rehospitalization for angina. CONCLUSION: Female ACS patients were older than male subjects and had more atypical presentation. They arrived at the hospital later than men and had longer hospital stays, but less often required revascularization therapy. However, no gender-based differences were noted in ACS-related mortality and morbidity.