Combined Model of Quantitative Evaluation of Chest Computed Tomography and Laboratory Values for Assessing the Prognosis of Coronavirus Disease 2019.

PURPOSE: To assess the prognostic power of quantitative analysis of chest CT, laboratory values, and their combination in COVID-19 pneumonia. MATERIALS AND METHODS: Retrospective analysis of patients with PCR-confirmed COVID-19 pneumonia and chest CT performed between March 07 and November 13, 2020. Volume and percentage (PO) of lung opacifications and mean HU of the whole lung were quantified using prototype software. 13 laboratory values were collected. Negative outcome was defined as death, ICU admittance, mechanical ventilation, or extracorporeal membrane oxygenation. Positive outcome was defined as care in the regular ward or discharge. Logistic regression was performed to evaluate the prognostic value of CT parameters and laboratory values. Independent predictors were combined to establish a scoring system for prediction of prognosis. This score was validated on a separate validation cohort. RESULTS: 89 patients were included for model development between March 07 and April 27, 2020 (mean age: 60.3 years). 38 patients experienced a negative outcome. In univariate regression analysis, all quantitative CT parameters as well as C-reactive protein (CRP), relative lymphocyte count (RLC), troponin, and LDH were associated with a negative outcome. In a multivariate regression analysis, PO, CRP, and RLC were independent predictors of a negative outcome. Combination of these three values showed a strong predictive value with a C-index of 0.87. A scoring system was established which categorized patients into 4 groups with a risk of 7 %, 30 %, 67 %, or 100 % for a negative outcome. The validation cohort consisted of 28 patients between May 5 and November 13, 2020. A negative outcome occurred in 6 % of patients with a score of 0, 50 % with a score of 1, and 100 % with a score of 2 or 3. CONCLUSION: The combination of PO, CRP, and RLC showed a high predictive value for a negative outcome. A 4-point scoring system based on these findings allows easy risk stratification in the clinical routine and performed exceptionally in the validation cohort. KEY POINTS: · A high PO is associated with an unfavorable outcome in COVID-19. · PO, CRP, and RLC are independent predictors of an unfavorable outcome, and their combination has strong predictive power. · A 4-point scoring system based on these values allows quick risk stratification in a clinical setting. CITATION FORMAT: · Scharf G, Meiler S, Zeman F et al. Combined Model of Quantitative Evaluation of Chest Computed Tomography and Laboratory Values for Assessing the Prognosis of Coronavirus Disease 2019. Fortschr Röntgenstr 2022; 194: 737 - 746.

CT Features of COVID-19 Pneumonia Differ Depending on the Severity and Duration of Disease.

BACKGROUND: CT is important in the care of patients with COVID-19 pneumonia. However, CT morphology can change significantly over the course of the disease. To evaluate the CT morphology of RT-PCR-proven COVID-19 pneumonia in a German cohort with special emphasis on identification of potential differences of CT features depending on duration and severity of disease. METHOD: All patients with RT-PCR-proven COVID-19 pneumonia and chest CT performed between March 1 and April 15, 2020 were retrospectively identified. The CT scans were evaluated regarding the presence of different CT features (e. g. ground glass opacity, consolidation, crazy paving, vessel enlargement, shape, and margin of opacifications), distribution of lesions in the lung and extent of parenchymal involvement. For subgroup analyses the patients were divided according to the percentage of parenchymal opacification (0-33 %, 34-66 %, 67-100 %) and according to time interval between symptom onset and CT date (0-5 d, 6-10 d, 11-15 d, > 15 d). Differences in CT features and distribution between subgroups were tested using the Mantel-Haenszel Chi Squared for trend. RESULTS: The frequency of CT features (ground glass opacity, consolidation, crazy paving, bronchial dilatation, vessel enlargement, lymphadenopathy, pleural effusion) as well as pattern of parenchymal involvement differed significantly depending on the duration of disease and extent of parenchymal involvement. The early phase of disease was characterized by GGO and to a lesser extent consolidation. The opacifications tended to be round and to some extent with sharp margins and a geographic configuration. The vessels within/around the opacifications were frequently dilated. Later on, the frequency of consolidation and especially crazy paving increased, and the round/geographic shape faded. After day 15, bronchial dilatation occurred, and lymphadenopathy and pleural effusion were seen more frequently than before. CONCLUSION: The prevalence of CT features varied considerably during the course of disease and depending on the severity of parenchymal involvement. Radiologists should take into account the time interval between symptom onset and date of CT and the severity of disease when discussing the likelihood of COVID-19 pneumonia based on CT morphology. KEY POINTS: · The frequency of CT features and pattern of parenchymal involvement vary depending on the duration and extent of COVID-19 pneumonia.. · The early phase is characterized by GGO and consolidation which demonstrate a round shape and at least to some extent have sharp margins and a geographic configuration.. · The frequency of consolidation and especially crazy paving increases during the course of disease.. · Beyond day 15 after symptom onset, bronchial dilatation occurs.. · Radiologists should take into account the duration and severity of disease when considering COVID-19 pneumonia.. CITATION FORMAT: · Schaible J, Meiler S, Poschenrieder F et al. CT Features of COVID-19 Pneumonia Differ Depending on the Severity and Duration of Disease. Fortschr Röntgenstr 2021; 193: 672 - 682.

Sharp margin and geographic shape: systematic evaluation of two novel CT features in COVID-19 pneumonia.

OBJECTIVE: CT is important in the care of patients with COVID-19 pneumonia. However, specificity might be poor in the absence of a clinical and epidemiological context. The goal of this work was to systematically evaluate two novel CT features (sharp margin and geographic shape) of COVID-19 pneumonia. METHODS: All patients with reverse transcription polymerase chain reaction proven COVID-19 pneumonia and chest CT between March first and April 15, 2020 were retrospectively identified from two tertiary care hospitals in Germany. The CTs were evaluated regarding the presence of typical CT signs (e.g. ground glass opacitiy, consolidation, crazy paving). Moreover, the shape of the opacifications (round, geographic, curvilinear) and their margin (unsharp, sharp) was determined. RESULTS: The study population comprised 108 patients (64 male) with a mean age of 59.6 years. Ground glass opacities (96%) and consolidation (75%) were the most prevalent CT signs. Crazy paving was seen in 17%, bronchial dilatation in 21%, air bronchogram in 29%, vessel enlargement in 47%, cavitation in 0%, lymphadenopathy in 32%, pleural effusion in 16%. Round configuration of densities was present in 41% of CTs, geographic shape in 27% and curvilinear opacities in 44%. 79% of opacifications were at least partially sharply marginated. In almost all cases, the lung was affected bilaterally (94%). CONCLUSION: The CT pattern of COVID-19 pneumonia in a cohort from Germany was in accordance with prior studies. However, we identified two novel CT signs of COVID-19 pneumonia which have so far not been systematically evaluated. A sharp border and geographic shape of opacifications were frequently observed. ADVANCES IN KNOWLEDGE: The newly described CT features "sharp margin" and "geographic shape" of opacifications in patients with COVID-19 pneumonia might help to increase specificity of CT.

Computed tomography characterization and outcome evaluation of COVID-19 pneumonia complicated by venous thromboembolism.

BACKGROUND: COVID-19 is frequently complicated by venous thromboembolism (VTE). Computed tomography (CT) of the chest-primarily usually conducted as low-dose, non-contrast enhanced CT-plays an important role in the diagnosis and follow-up of COVID-19 pneumonia. Performed as contrast-enhanced CT pulmonary angiography, it can reliably detect or rule-out pulmonary embolism (PE). Several imaging characteristics of COVID-19 pneumonia have been described for chest CT, but no study evaluated CT findings in the context of VTE/PE. PURPOSE: In our retrospective study, we analyzed clinical, laboratory and CT imaging characteristics of 50 consecutive patients with RT-PCR proven COVID-19 pneumonia who underwent contrast-enhanced chest CT at two tertiary care medical centers. MATERIAL AND METHODS: All patients with RT-PCR proven COVID-19 pneumonia and contrast-enhanced chest CT performed at two tertiary care hospitals between March 1st and April 20th 2020 were retrospectively identified. Patient characteristics (age, gender, comorbidities), symptoms, date of symptom onset, RT-PCR results, imaging results of CT and leg ultrasound, laboratory findings (C-reactive protein, differential blood count, troponine, N-terminal pro-B-type natriuretic peptide (NT-proBNP), fibrinogen, interleukin-6, D-dimer, lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase muscle-brain (CKmb) and lactate,) and patient outcome (positive: discharge or treatment on normal ward; negative: treatment on intensive care unit (ICU), need for mechanical ventilation, extracorporeal membrane oxygenation (ECMO), or death) were analyzed. Follow-up was performed until May 10th. Patients were assigned to two groups according to two endpoints: venous thromboembolism (VTE) or no VTE. For statistical analysis, univariate logistic regression models were calculated. RESULTS: This study includes 50 patients. In 14 out of 50 patients (28%), pulmonary embolism was detected at contrast-enhanced chest CT. The majority of PE was detected on CTs performed on day 11-20 after symptom onset. Two patients (14%) with PE simultaneously had evidence of deep vein thrombosis. 15 patients (30%) had a negative outcome (need for intensive care, mechanical ventilation, extracorporeal membrane oxygenation, or death), and 35 patients (70%) had a positive outcome (transfer to regular ward, or discharge). Patients suffering VTE had a statistically significant higher risk of an unfavorable outcome (p = 0.028). In univariate analysis, two imaging characteristics on chest CT were associated with VTE: crazy paving pattern (p = 0.024) and air bronchogram (n = 0.021). Also, elevated levels of NT-pro BNP (p = 0.043), CK (p = 0.023) and D-dimers (p = 0.035) were significantly correlated with VTE. CONCLUSION: COVID-19 pneumonia is frequently complicated by pulmonary embolism (incidence of 28% in our cohort), remarkably with lacking evidence of deep vein thrombosis in nearly all thus affected patients of our cohort. As patients suffering VTE had an adverse outcome, we call for a high level of alertness for PE and advocate a lower threshold for contrast-enhanced CT in COVID-19 pneumonia. According to our observations, this might be particularly justified in the second week of disease and if a crazy paving pattern and / or air bronchogram is present on previous non-enhanced CT.

Can CT performed in the early disease phase predict outcome of patients with COVID 19 pneumonia? Analysis of a cohort of 64 patients from Germany.

PURPOSE: The aim of this study was to investigate if CT performed in the early disease phase can predict the course of COVID-19 pneumonia in a German cohort. METHOD: All patients with RT-PCR proven COVID-19 pneumonia and chest CT performed within 10 days of symptom onset between March 1st and April 15th 2020 were retrospectively identified from two tertiary care hospitals. 12 CT features, their distribution in the lung and the global extent of opacifications were evaluated. For analysis of prognosis two compound outcomes were defined: positive outcome was defined as either discharge or regular ward care; negative outcome was defined as need for mechanical ventilation, treatment on intensive care unit, extracorporeal membrane oxygenation or death. Follow-up was performed until June 19th. For statistical analysis uni- und multivariable logistic regression models were calculated. RESULTS: 64 patients were included in the study. By univariable analysis the following parameters predicted a negative outcome: consolidation (p = 0.034), crazy paving (p = 0.004), geographic shape of opacification (p = 0.022), dilatation of bronchi (p = 0.002), air bronchogram (p = 0.013), vessel enlargement (p = 0.014), pleural effusion (p = 0.05), bilateral disease (p = 0.004), involvement of the upper lobes (p = 0.004, p = 0.015) or the right middle lobe (p < 0.001) and severe extent of opacifications (p = 0.002). Multivariable analysis revealed crazy paving and severe extent of parenchymal involvement to be independently predictive for a poor outcome. CONCLUSIONS: Easy to assess CT features in the early phase of disease independently predicted an adverse outcome of patients with COVID-19 pneumonia.

Adherence to pan-genotypic glecaprevir/pibrentasvir and efficacy in HCV-infected patients: A pooled analysis of clinical trials.

BACKGROUND & AIMS: Adequate adherence to hepatitis C virus (HCV) treatment is believed to be a key component of treatment success because non-adherence can potentially result in treatment failure and the emergence of resistant viral variants. This analysis assessed factors associated with non-adherence to glecaprevir/pibrentasvir (G/P) therapy and the impact of non-adherence on sustained virological response at post-treatment week 12 (SVR12) rates in HCV genotype (GT) 1-6-infected patients. METHODS: Adherence was calculated by pill counts at study visits during treatment, and defined as having a lowest treatment adherence of ≥80% and ≤120% at each study visit. Exploratory logistic regression modelling assessed predictors of non-adherence to G/P therapy. SVR12 rates by treatment adherence were assessed in the intent-to-treat (ITT) population and modified ITT (mITT) population, which excludes non-virological failures. RESULTS: Overall, 97% (2024/2091) of patients were adherent to G/P therapy at all consecutive study visits. Alcohol use was the only baseline characteristic independently associated with non-adherence to G/P therapy (OR: 2.38; 95% CI: 1.13-5.01; P = .022). In the mITT population, overall SVR12 rates were high both in patients who were adherent to G/P therapy and those who were not (99% [1983/2008] and 95% [58/61] respectively; P = .047). Corresponding SVR12 rates in the ITT population were 98% (1983/2024) and 87% (58/67) respectively. CONCLUSIONS: Most patients adhered to G/P therapy. SVR12 rates were high both in patients who were adherent to G/P treatment and those who were not. Patient education on treatment adherence should remain an important part of HCV treatment. CLINICAL TRIALS REGISTRATION: NCT02604017, NCT02640482, NCT02640157, NCT02636595, NCT02642432, NCT02651194, NCT02243293, NCT02446717.

Real-world safety and effectiveness of ombitasvir/paritaprevir/ritonavir ± dasabuvir ± ribavirin in hepatitis C virus genotype 1- and 4-infected patients with diverse comorbidities and comedications: A pooled analysis of post-marketing observational studies from 13 countries.

Ombitasvir/paritaprevir/ritonavir ± dasabuvir ± ribavirin (OBV/PTV/r ± DSV ± RBV) regimens show high efficacy and good tolerability in clinical trials for chronic hepatitis C virus (HCV) genotypes (GT) 1 or 4. To evaluate whether these results translate to clinical practice, data were pooled from observational studies across 13 countries. Treatment-naïve or -experienced patients, with or without cirrhosis, received OBV/PTV/r ± DSV ± RBV according to approved local labels and clinical practice. Sustained virologic response at post-treatment Week 12 (SVR12), adverse events (AEs) and comedication management were assessed for patients initiating treatment before 1 June 2017. The safety population included 3850 patients who received ≥1 dose of study drug. The core population (N = 3808) further excluded patients with unknown GT or cirrhosis status, or who received off-label treatment. Patients had HCV GT1a (n = 732; 19%), GT1b (n = 2619; 69%) or GT4 (n = 457; 12%). In 3546 patients with sufficient follow-up data at post-treatment Week 12, the SVR12 rate was 96% (n/N = 3401/3546 [95% CI 95.2-96.5]). In patients with or without cirrhosis, SVR12 was comparable (96%). In patients with HCV GT1a, GT1b or GT4, SVR12 rates were 93%, 97% and 94%. In GT1b-infected patients with planned treatment for 8 weeks, SVR12 was 96%. In patients with ≥1 comorbidity (67%), SVR12 was 95%. 58% of patients received ≥1 comedication, and there was minimal impact on SVR12 rates using comedications for peptic ulcers and gastro-esophageal reflux disease, statins, antipsychotics or antiepileptics. Most comedications were maintained during treatment although 58% of patients changed their statin medication. AEs and serious AEs occurred in 26% and 3% of patients. Post-baseline Grade 3-4 laboratory abnormalities were rare (<3%), and discontinuation rates were low (<4%). Real-world evidence confirms the effectiveness of OBV/PTV/r ± DSV ± RBV in patients with HCV GT1 or GT4, regardless of common comorbidities or comedications, and is consistent with clinical trial results. Adverse safety outcomes may be limited by underreporting in the real-world setting.

Improvement of Hepatic and Extrahepatic Complications from Chronic Hepatitis C After Antiviral Treatment: A Retrospective Analysis of German Sickness Fund Data.

INTRODUCTION: German data regarding the economic burden of chronic hepatitis C (CHC) and potential benefits of CHC treatment are limited. To address this issue, we evaluated the role of treatment in mitigating the economic burden of hepatic and extrahepatic complications (EHCs) from CHC virus infection in Germany. METHODS: This retrospective, cross-sectional study used claims data from the Betriebskrankenkasse German sickness fund (2007-2014) to assess the medical costs of hepatic complications and EHCs, including conditions that are prevalent and behavioral factors associated with CHC. All-cause costs, medical costs related to hepatic and EHCs, and CHC-related and non-CHC-related pharmacy costs (adjusted to the 2016 euro rate) were calculated and compared between CHC patients' treated (n = 1714) and untreated time (n = 7124) and CHC patients that initiated treatment early (i.e., without cirrhosis; n = 1552) vs. late (i.e., with cirrhosis; n = 162). RESULTS: CHC treatment was associated with an average adjusted savings of €1885 in annual all-cause medical costs per patient, with a significant proportion attributed to EHC-related cost savings (adjusted difference, €1363; P < 0.01). Although initiating CHC treatment early was economically beneficial compared with initiating treatment late, the total cost savings were not significantly different (annual average adjusted difference, €3831; P = 0.27). However, nearly 60% of these savings were EHC related (adjusted difference, €2255; P < 0.01). CONCLUSION: CHC is associated with a significant economic burden in Germany, largely due to EHCs. Antiviral treatment may reduce the burden of CHC and result in significant cost savings, even when initiated at earlier stages of liver disease. FUNDING: AbbVie Inc.

Clinical and Economic Burden of Hepatic and Extrahepatic Complications from Chronic Hepatitis C: A Retrospective Analysis of German Sickness Fund Data.

INTRODUCTION: German data regarding the burden of complications from chronic hepatitis C (CHC) virus infection are limited. To address this issue, this study evaluates the clinical and economic burden of hepatic and extrahepatic complications (EHCs) associated with CHC in Germany. METHODS: This retrospective, cross-sectional study used claims data from the Betriebskrankenkasse German sickness fund (2007-2014) to assess the risks and medical costs of hepatic complications and EHCs, including conditions that are prevalent and behavioral factors associated with CHC. Prevalence, incidence, and risks were calculated for 1:1 matched patients with and without CHC (n = 3994). All-cause cost, medical costs related to hepatic and EHCs, as well as CHC-related and non-CHC-related pharmacy costs (adjusted to the 2016 Euro rate), were calculated and compared between 1:5 matched patients with (n = 8425) and without CHC (n = 42,125). RESULTS: Patients with CHC had a 3-fold higher risk for any EHC (OR = 3.0; P < 0.05) and higher EHC-related medical costs (adjusted difference, €1606; P < 0.01) compared with patients without CHC. Total costs (€10,108 vs. €5430), hepatic complication-related medical costs (€1425 vs. €556), EHC-related costs (€3547 vs. €1921), CHC-related pharmacy costs (€577 vs. €116), and non-CHC-related pharmacy costs (€3719 vs. €1479) were all significantly greater for patients with CHC compared with patients without CHC. EHC-related medical costs were a major contributor to the higher all-cause medical (84.4%) and total (44.3%) cost differences between patients with CHC and the matched sample of patients without CHC. CONCLUSION: CHC is associated with substantial clinical and economic burden in Germany, largely due to hepatic complications and EHCs. FUNDING: Abbvie Inc.

GAR22ß regulates cell migration, sperm motility, and axoneme structure.

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22ß), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22ß(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22ß(-/-) cells showed a more prominent focal adhesion turnover. GAR22ß overexpression or its reexpression in GAR22ß(-/-) cells reduced cell motility and focal adhesion turnover. GAR22ß-actin interaction was stronger than GAR22ß-microtubule interaction, resulting in GAR22ß localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22ß interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22ß-EB1 interaction was required for the ability of GAR22ß to modulate cell motility. We found that GAR22ß is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22ß as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes.

Use of clinical scores to guide therapeutic decisions in patients with rheumatoid arthritis in daily care.

OBJECTIVES: This study focuses on the application and impact of different clinical scores for treatment changes in daily practice in patients with rheumatoid arthritis (RA), as achieving remission is a feasible aim due to considerable improvements in therapeutic options. METHODS: In this prospective study, 1467 RA patients aged 15 to 88 years (72.5% female, 27.5% male) who had undergone treatment change or were treated with a disease-modifying antirheumatic drug (DMARD) for the first time were analysed. At three consecutive visits (T-1, T0, T1), scores were used to assess disease activity, loss of function, quality of life and imaging. In addition, the impact of the scores on treatment change was addressed (numerical rating scale, 1-10). RESULTS: The most commonly used scores were the DAS28 (65% of all visits), the Hanover functional ability questionnaire (FFbH, 36%) and the HAQ (11%). Other scores for evaluating RA are of little relevance in daily practice. No scores were calculated in only 10% of visits. Among the commonly used scores, the DAS28 had the highest influence on therapy decisions, followed by HAQ and FFbH (mean weight 6.62, 4.99 and 4.41, respectively). CONCLUSIONS: In daily practice, rheumatologists very often take scores for disease activity (especially DAS28) and loss of physical function into consideration when deciding on treatment for patients with RA. However, scores for measuring structural changes or quality of life, are not yet very well established with German rheumatologists.

The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification.

The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L-KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin.

Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10.

Adenosine diphosphate-ribosylation is a post-translational modification mediated by intracellular and membrane-associated extracellular enzymes and many bacterial toxins. The intracellular enzymes modify their substrates either by poly-ADP-ribosylation, exemplified by ARTD1/PARP1, or by mono-ADP-ribosylation. The latter has been discovered only recently, and little is known about its physiological relevance. The founding member of mono-ADP-ribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitin-interaction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-κB signalling. We therefore studied the role of ARTD10 in this pathway. ARTD10 inhibits the activation of NF-κB and downstream target genes in response to interleukin-1ß and tumour necrosis factor-α, dependent on catalytic activity and poly-ubiquitin binding of ARTD10. Mechanistically ARTD10 interferes with poly-ubiquitination of NEMO, which interacts with and is a substrate of ARTD10. Our findings identify a novel regulator of NF-κB signalling and provide evidence for cross-talk between K63-linked poly-ubiquitination and mono-ADP-ribosylation.

Recognition of mono-ADP-ribosylated ARTD10 substrates by ARTD8 macrodomains.

ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribose from NAD(+) onto substrates. Some ARTs generate in an iterative process ADP-ribose polymers that serve as adaptors for distinct protein domains. Other ARTs, exemplified by ARTD10, function as mono-ADP-ribosyltransferases, but it has been unclear whether this modification occurs in cells and how it is read. We observed that ARTD10 colocalized with ARTD8 and defined its macrodomains 2 and 3 as readers of mono-ADP-ribosylation both in vitro and in cells. The crystal structures of these two ARTD8 macrodomains and isothermal titration calorimetry confirmed their interaction with ADP-ribose. These macrodomains recognized mono-ADP-ribosylated ARTD10, but not poly-ADP-ribosylated ARTD1. This distinguished them from the macrodomain of macroH2A1.1, which interacted with poly- but not mono-ADP-ribosylated substrates. Moreover, Ran, an ARTD10 substrate, was also read by ARTD8 macrodomains. This identifies readers of mono-ADP-ribosylated proteins, defines their structures, and demonstrates the presence of this modification in cells.

Caspase-dependent cleavage of the mono-ADP-ribosyltransferase ARTD10 interferes with its pro-apoptotic function.

ADP-ribosylation is a post-translational modification that regulates various physiological processes, including DNA damage repair, gene transcription and signal transduction. Intracellular ADP-ribosyltransferases (ARTDs or PARPs) modify their substrates either by poly- or mono-ADP-ribosylation. Previously we identified ARTD10 (formerly PARP10) as a mono-ADP-ribosyltransferase, and observed that exogenous ARTD10 but not ARTD10-G888W, a catalytically inactive mutant, interferes with cell proliferation. To expand on this observation, we established cell lines with inducible ARTD10 or ARTD10-G888W. Consistent with our previous findings, induction of the wild-type protein but not the mutant inhibited cell proliferation, primarily by inducing apoptosis. During apoptosis, ARTD10 itself was targeted by caspases. We mapped the major cleavage site at EIAMD406↓S, a sequence that was preferentially recognized by caspase-6. Caspase-dependent cleavage inhibited the pro-apoptotic activity of ARTD10, as ARTD10(1-406) and ARTD10(407-1025), either alone or together, were unable to induce apoptosis, despite catalytic activity of the latter. Deletion of the N-terminal RNA recognition motif in ARTD10(257-1025) also resulted in loss of pro-apoptotic activity. Thus our findings indicate that the RNA recognition motif contributes to the pro-apoptotic effect, together with the catalytic domain. We suggest that these two domains must be physically linked to stimulate apoptosis, possibly targeting ARTD10 through the RNA recognition motif to specific substrates that control cell death. Moreover, we established that knockdown of ARTD10 reduced apoptosis in response to DNA-damaging agents. Together, these findings indicate that ARTD10 is involved in the regulation of apoptosis, and that, once apoptosis is activated, ARTD10 is cleaved as part of negative feedback regulation.

ARTD10 substrate identification on protein microarrays: regulation of GSK3ß by mono-ADP-ribosylation.

BACKGROUND: Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification. RESULTS: We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3ß revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3ß activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling. CONCLUSIONS: Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3ß, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.

Dynamic subcellular localization of the mono-ADP-ribosyltransferase ARTD10 and interaction with the ubiquitin receptor p62.

BACKGROUND: ADP-ribosylation is a posttranslational modification catalyzed in cells by ADP-ribosyltransferases (ARTD or PARP enzymes). The ARTD family consists of 17 members. Some ARTDs modify their substrates by adding ADP-ribose in an iterative process, thereby synthesizing ADP-ribose polymers, the best-studied example being ARTD1/PARP1. Other ARTDs appear to mono-ADP-ribosylate their substrates and are unable to form polymers. The founding member of this latter subclass is ARTD10/PARP10, which we identified as an interaction partner of the nuclear oncoprotein MYC. Biochemically ARTD10 uses substrate-assisted catalysis to modify its substrates. Our previous studies indicated that ARTD10 may shuttle between the nuclear and cytoplasmic compartments. We have now addressed this in more detail. RESULTS: We have characterized the subcellular localization of ARTD10 using live-cell imaging techniques. ARTD10 shuttles between the cytoplasmic and nuclear compartments. When nuclear, ARTD10 can interact with MYC as measured by bimolecular fluorescence complementation. The shuttling is controlled by a Crm1-dependent nuclear export sequence and a central ARTD10 region that promotes nuclear localization. The latter lacks a classical nuclear localization sequence and does not promote full nuclear localization. Rather this non-conventional nuclear localization sequence results in an equal distribution of ARTD10 between the cytoplasmic and the nuclear compartments. ARTD10 forms discrete and dynamic bodies primarily in the cytoplasm but also in the nucleus. These contain poly-ubiquitin and co-localize in part with structures containing the poly-ubiquitin receptor p62/SQSTM1. The co-localization depends on the ubiquitin-associated domain of p62, which mediates interaction with poly-ubiquitin. CONCLUSIONS: Our findings demonstrate that ARTD10 is a highly dynamic protein. It shuttles between the nuclear and cytosolic compartments dependent on a classical nuclear export sequence and a domain that mediates nuclear uptake. Moreover ARTD10 forms discrete bodies that exchange subunits rapidly. These bodies associate at least in part with the poly-ubiquitin receptor p62. Because this protein is involved in the uptake of cargo into autophagosomes, our results suggest a link between the formation of ARTD10 bodies and autophagy. LAY Post-translational modifications refer to changes in the chemical appearance of proteins and occur, as the name implies, after proteins have been synthesized. These modifications frequently affect the behavior of proteins, including alterations in their activity or their subcellular localization. One of these modifications is the addition of ADP-ribose to a substrate from the cofactor NAD+. The enzymes responsible for this reaction are ADP-ribosyltransferases (ARTDs or previously named PARPs). Presently we know very little about the role of mono-ADP-ribosylation of proteins that occurs in cells. We identified ARTD10, a mono-ADP-ribosyltransferase, as an interaction partner of the oncoprotein MYC. In this study we have analyzed how ARTD10 moves within a cell. By using different live-cell imaging technologies that allow us to follow the position of ARTD10 molecules over time, we found that ARTD10 shuttles constantly in and out of the nucleus. In the cytosol ARTD10 forms distinct structures or bodies that themselves are moving within the cell and that exchange ARTD10 subunits rapidly. We have identified the regions within ARTD10 that are required for these movements. Moreover we defined these bodies as structures that interact with p62. This protein is known to play a role in bringing other proteins to a structure referred to as the autophagosome, which is involved in eliminating debris in cells. Thus our work suggests that ARTD10 might be involved in and is regulated by ADP-riboslyation autophagosomal processes.

SH3TC2, a protein mutant in Charcot-Marie-Tooth neuropathy, links peripheral nerve myelination to endosomal recycling.

Patients with Charcot-Marie-Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.

Learning how to read ADP-ribosylation.

ADP-ribosylation is a posttranslational modification that is emerging as a broadly used mechanism to regulate the functions of proteins and their interactions. Recent findings by three groups (Ahel et al., 2009; Gottschalk et al., 2009; Timinszky et al., 2009) establish that proteins with macrodomains bind poly-ADP-ribose to mediate the cellular response to DNA damage.