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Purpose We compared two imaging techniques to assess whether 3D VISTA imaging could replace 2D FSE in diagnosing PCL reconstruction complications. Materials and methods This retrospective study included 40 patients who underwent surgery of PCL reconstruction and follow-up knee MRI (3D VISTA and 2D FSE) for evaluation of PCL graft integrity. Each image was interpreted independently by two radiologists without knowledge of radiologic reports or clinical history. The diagnostic performance of the 2D FSE PCL view, 3D VISTA PCL view, orthogonal 2D FSE image, and combined sequences were evaluated in terms of sensitivity, specificity, and accuracy for diagnosing complications of PCL graft. The reference diagnoses were made arthroscopically or clinically. Results The sensitivities of the 3D VISTA PCL view were similar to those of the 2D PCL view. The sensitivities of the combination of the orthogonal view and the 3D VISTA PCL view were also similar to those of the combination of the orthogonal view and the 2D PCL view. The specificities and accuracies of each image exhibited similar results. There was no statistically significant difference in diagnostic performance between the 3D VISTA PCL view and the 2D PCL view (solitary or combined with the orthogonal view). Conclusions The diagnostic performance of the PCL views on 3D VISTA images is comparable to that of 2D FSE images in the diagnosis of PCL graft complications.

Summary Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021)., Graphical abstract, Highlights • An optimized protocol to measure the global translation rate in senescent cells • Use of L-AHA-based click chemistry enables a sensitive measurement of protein translation • Flow cytometry allows quantitative analysis of the global translation rate, Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances.

Summary Background CT radiation is arguably carcinogenic. Results from single-centre studies, mostly retrospective, have advocated lowering the CT radiation dose for the diagnosis of appendicitis. However, adoption of low-dose CT has been slow. We aimed to assess the effectiveness of low-dose CT compared with standard-dose CT in the diagnosis of appendicitis in adolescents and young adults. Methods We did this pragmatic, multicentre, randomi s ed controlled non-inferiority trial at 20 South Korean teaching hospitals with little experience with low-dose CT. Patients aged 15–44 years with suspected appendicitis were randomly assigned (1:1), via computer-generated random assignments (permuted block sizes of two, four, six, and eight) concealed in sequentially numbered envelopes, to receive low-dose CT (2 mSv) or standard-dose CT (≤8 mSv). Randomisation was stratified by site. Group allocation was concealed from patients, outcome assessors, and adverse event adjudicators; care providers, site pathologists, and data collectors were aware of allocation. The primary endpoint was the negative (unnecessary) appendectomy rate among all appendectomies, with a non-interiority margin of 4·5% for low-dose versus standard-dose CT. Primary analy s is was by modified intention to treat, which included all patients who received an appendectomy in the group to which they were assigned. This trial is registered with ClinicalTrials.gov, number NCT01925014. Findings Between Dec 4, 2013, and Aug 18, 2016, we assigned 1535 patients to the low-dose CT group and 1539 patients to the standard-dose CT group. 22 (3·9%) of 559 patients had a negative appendectomy in the low-dose group versus 16 (2·7%) of 601 patients in the standard-dose group (difference 1·3%, 95% CI −0·8 to 3·3; p=0·0022 for the non-inferiority test). We recorded 43 adverse events in 43 (2·8%) of 1535 patients in the low-dose group and 41 adverse events in 40 (2·6%) of 1539 patients in the standard-dose group. One life-threatening adverse event of anaphylaxis caused by an iodinated contrast material occurred in the low-dose group. Interpretation Radiation dose of appendiceal CT for adolescents and young adults can be reduced to 2 mSv without impairing clinical outcomes. In view of the vast number of appendiceal CT examinations done worldwide, use of low-dose CT could prevent a sizeable number of radiation-associated cancers in the future. Funding Korea Health Industry Development Institute, Seoul National University Bundang Hospital, Dasol Life Science, and Bracco Imaging Korea.

Background: The prevalence of significant and advanced liver fibrosis in the general population has not been well characterized. Using magnetic resonance elastography (MRE), we investigated the prevalence of significant fibrosis in a general community-based cohort. Methods: The study analyzed 2,170 participants who underwent the MR chemical shift technique and MRE. We also conducted subgroup analyses for participants with non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, and diabetes mellitus (DM). Findings: The prevalence of F2 (≥3.0 kPa) and F3 fibrosis (≥3.6 kPa) in the general population was 5.1% and 1.3%, respectively. The prevalence of NAFLD (>5% hepatic fat fraction) was 20.8% in the average risk population (after excluding alcohol use and viral hepatitis), and the prevalence of significant and advanced fibrosis in NAFLD participants was 7.5% and 1.1%, respectively. The prevalence of DM was 7.7% in the general population, with significant fibrosis in 13.7% and advanced fibrosis in 4.8%. In participants with fatty liver (of any etiology) plus diabetes, 27.5% had ≥F2 and 7.2% ≥F3. Multivariate analyses indicated that older age, lower platelet count, higher AST/ALT ratio, diabetes, and fatty liver were independently associated with significant fibrosis. Interpretation: In the general population, the prevalence of significant and advanced fibrosis was 5.1% and 1.3%, respectively, and increased three- to four-fold in diabetes. Funding: None. Declaration of Interest: None declared. Ethical Approval: This study was approved by the Institutional Review Board of Kangbuk Samsung Hospital (IRB No. KBSMC 2018-06-008-001), and the protocol was registered at the Clinical Research Information Service (http://cris.nih.go.kr/) with registration number KCT0003782.

Phenol, a monosubstituted aromatic hydrocarbon with various commercial uses, is a major organic constituent in industrial wastewaters. The ecotoxic action of phenol for aquatic environment is well known. In this study, rapid phenol toxicity tests (1 h) were developed based on chlorophyll a (Chl a) fluorescence and the movement parameters of the freshwater flagellate, Euglena agilis Carter. Phenol significantly reduced the maximum quantum yield (Fv/Fm) of photosystem II (PS II) and the maximum photosynthetic electron transport rate (rETRmax) with median effective concentration (EC50) values of 8.94 and 4.67 mM, respectively. Phenol reduced the motility and triggered change in the swimming velocity of the test organism. Among the parameters tested, velocity was the most sensitive biomarker with an EC50 of 3.17 mM. The EC50 values for Fv/Fm, motility, and velocity appear to overlap the permitted levels of phenol. In conclusion, the photosynthesis and movement of E. agilis can be fast and sensitive risk assessment parameters for the evaluation of phenol toxicity in municipal and industrial effluents.

International audience; NLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases.

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.

To address how transforming growth factor (TGF)-beta and oncogenic H-ras signal transduction pathways interact with each other in the malignant progression of breast epithelial cells, we investigated the role of TGF-beta signaling pathway in invasive and migrative properties of H-ras-transformed MCF10A human breast epithelial cells in this study. Here we show that TGF-beta treatment significantly enhanced invasion and migration of H-ras MCF10A cells. H-ras-mediated activation of p38 MAPK and ERK-1/2 was stimulated by TGF-beta. TGF-beta increased expression of matrix metalloproteinase (MMP)-2 through transcriptional activation while TGF-beta-stimulated MMP-9 up-regulation did not occur at transcription level. Activation of p38 MAPK pathway was required for TGF-beta-induced cell migration, invasion and MMP-2/-9 up-regulation, indicating a critical role of p38 MAPK signaling in TGF-beta-promoted tumor progression of H-ras-activated cells. ERKs signaling was also crucial for TGF-beta-enhanced invasive and migrative phenotypes but the up-regulation of MMP-2/-9 was not dependent on ERKs activity. Taken together, we show that TGF-beta promotes H-ras-mediated cell migration and invasive phenotypes in which p38 MAPK and ERKs signaling pathways are involved. Our findings revealing how H-ras and TGF-beta signal pathways interact with each other in MCF10A human breast cells may provide an insight into molecular mechanisms for contribution of TGF-beta to a malignant progression of breast cancer in collaboration with activated H-ras.

Nitric oxide is an endogenous thiol-reactive molecule that modulates the functions of many regulatory proteins by a thiol-redox mechanism. NO has now been shown to inhibit the activation of apoptosis signal-regulating kinase 1 (ASK1) in murine fibrosarcoma L929 cells through such a mechanism. Exposure of L929 cells to interferon-gamma resulted in the endogenous production of NO and in inhibition of the activation of ASK1 by hydrogen peroxide. The interferon-gamma-induced inhibition of ASK1 activity was blocked by N(G)-nitro-l-arginine, an inhibitor of NO synthase. Furthermore, the NO donor S-nitro-N-acetyl-dl-penicillamine (SNAP) inhibited ASK1 activity in vitro, and this inhibition was reversed by thiol-reducing agents such as dithiothreitol and beta-mercaptoethanol. SNAP did not inhibit the kinase activities of MKK3, MKK6, or p38 in vitro. The inhibition of ASK1 by interferon-gamma was not changed by 1H- (1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one, an inhibitor of guanylyl cyclase nor was it mimicked by 8-bromo-cyclic GMP. Site-directed mutagenesis revealed that replacement of cysteine 869 of ASK1 by serine rendered this protein resistant to the inhibitory effects both of interferon-gamma in intact cells and of SNAP in vitro. Co-immunoprecipitation data showed that NO production inhibited a binding of ASK1, but not ASK1(C869S), to MKK3 or MKK6. Moreover, interferon-gamma induced the S-nitrosylation of endogenous ASK1 in L929 cells. Together, these results suggest that NO mediates the interferon-gamma-induced inhibition of ASK1 in L929 cells through a thiolredox mechanism.

Cancer metastasis represents the most important cause of cancer death and agents that may inhibit tumor cell invasion have been extensively pursued. In the present study, we have examined the anti-invasive effect of apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl -L-2-amin o-8-oxodecanoyl)], a fungal metabolite that was identified as an antiprotozoal agent known to inhibit parasite histone deacetylase (HDAC). We show that apicidin significantly inhibits H-ras-induced invasive phenotype of MCF10A human breast epithelial cells in parallel with a specific downregulation of matrix metalloproteinase (MMP)-2, but not MMP-9. We also show that apicidin induces a morphological reversal and growth inhibition of H-ras MCF10A cells similar to that induced by other HDAC inhibitors. Taken in conjunction with the fact that uncontrolled ras activation is probably the most common genetic defect in human cancer cells, our data showing the anti-invasive and detransforming activities of apicidin in H-ras-transformed MCF10A cells may suggest a potential use of HDAC inhibitors for treatment of cancer.

Selenium, an essential biological trace element, has been shown to modulate functions of many regulatory proteins involved in signal transduction and to affect a variety of cellular activities including cell growth, survival, and death. The molecular mechanism by which selenium exerts its action on the cellular events, however, remains unclear. In our present study, we observed that selenite suppresses both the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase pathway in 293T cells. In contrast, selenite had little effect on the extracellular signal-regulated kinase pathway. Furthermore, selenite directly inhibited JNK/SAPK activity in vitro but not the p38 activity. The in vitro inhibition of JNK/SAPK by selenite was reversed by the addition of reducing agents such as dithiothreitol and beta-mercaptoethanol. Replacement of cysteine 116 in JNK1 by serine abolished the inhibitory effect of selenite on JNK1 activity both in vitro and in vivo. Selenite also suppressed a c-Jun-dependent luciferase reporter activity stimulated through the JNK signaling pathway. Taken together, our findings strongly suggest that selenite differentially modulates the mammalian mitogen-activated protein kinase pathways and that it can repress the JNK/SAPK signaling pathway by inhibiting JNK/SAPK through a thiol redox mechanism.