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Necroptotic signaling converges in the assembly of a cytosolic signaling platform, the necrosome, with the activation of its downstream effector, MLKL. RIPK1 and RIPK3, key components of the necrosome, act as signaling intermediates for the activation of MLKL. We report that RIPK3 and MLKL continuously shuttle between the nucleus and the cytoplasm, whereas RIPK1 is constitutively present in both compartments. During TNF-induced necroptosis, nuclear RIPK1 becomes ubiquitinated, after which nuclear MLKL becomes phosphorylated and oligomerized. Pharmacological inhibition of the nuclear export machinery leads to retention of RIPK3 and MLKL in the nucleus, prevents the nucleation of cytosolic RIPK3/MLKL oligomerization, and reduces cell death. Our results suggest that passage of necroptotic signaling components through the nucleus is a mechanism for regulating cytosolic necrosome formation and consequently necroptotic cell death. Kathrin Weber et al . report that the necrosome components RIPK3 and MLKL constitutively shuttle between the nucleus and cytoplasm. They find that increasing ratios of nuclear:cytosolic RIPK3 and MLKL prevents necrotic cell death, suggesting a mechanism by which the cell regulates necrosome formation and death.

TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders can, in certain conditions, be attributed to RIPK1 kinase-dependent cell death. Survival, however, is the default response of most cells to TNF stimulation, indicating that cell demise is normally actively repressed and that specific checkpoints must be turned off for cell death to proceed. We identified RIPK1 as a direct substrate of MK2 in the TNFR1 signalling pathway. Phosphorylation of RIPK1 by MK2 limits cytosolic activation of RIPK1 and the subsequent assembly of the death complex that drives RIPK1 kinase-dependent apoptosis and necroptosis. In line with these in vitro findings, MK2 inactivation greatly sensitizes mice to the cytotoxic effects of TNF in an acute model of sterile shock caused by RIPK1-dependent cell death. In conclusion, we identified MK2-mediated RIPK1 phosphorylation as an important molecular mechanism limiting the sensitivity of the cells to the cytotoxic effects of TNF. Dondelinger et al. and Menon et al. show that MAPKAP kinase-2 (MK2) phosphorylates RIPK1 to regulate TNF-mediated cell death as well as RIPK1 signalling in inflammation and bacterial infection.

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13 , the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis . These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals. Hair is the main skin appendage of mammals. Here, the authors show that claws of clawed frogs and hair contain homologous keratins and depend on the same transcription factor, Hoxc13, suggesting a common evolutionary origin of these skin appendages.

A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.

The sensitivity of cells to death receptor-induced apoptosis is commonly controlled by multiple checkpoints in order to limit induction of excessive or unnecessary death. Although cytotoxic in various cancer cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) does not trigger apoptosis in most non-transformed cells. The molecular nature of the checkpoints that normally protect the cells from TRAIL-induced death are not fully understood. Endoplasmic reticulum (ER) stress has been reported to switch the sensitivity of human cells to the cytotoxic effect of TRAIL, suggesting that this cellular state perturbs some of these protective mechanisms. We found that tunicamycin (TU), but no other ER stress inducers, sensitized mouse fibroblasts and hippocampal neuronal cells to TRAIL-induced apoptosis. Importantly, the sensitization was specific to TRAIL and not caused by differences in ER stress induction. Instead, it relied on the inhibition of N -glycosylation of the mouse TRAIL receptor (mTRAIL-R). Inhibition of N -glycosylation did not alter cell surface expression of mTRAIL-R but enhanced its ability to bind TRAIL, and facilitated mTRAIL-R oligomerization, which resulted in enhanced death-inducing signaling complex (DISC) formation and caspase-8 activation. Remarkably, reconstitution of mTRAIL-R-deficient cells with a version of mTRAIL-R mutated for the three N -glycosylation sites identified in its ectodomain confirmed higher sensitivity to TRAIL-induced apoptosis. Together, our results demonstrate that inhibition of N -glycosylation of mTRAIL-R, and not ER stress induction, sensitizes mouse cells to TRAIL-induced apoptosis. We therefore reveal a new mechanism restraining TRAIL cytotoxicity in mouse cells.

Background. Acyclovir resistance of varicella zoster virus (VZV) may arise in stem cell transplant (SCT) recipients with VZV disease and is usually a result of mutations in VZV thymidine kinase (TK), which is the target protein of acyclovir. Early detection of such mutations is necessary to enable timely therapy adaptation, for example, to foscarnet. We aimed to investigate whether TK mutations arise over time, and what sample types might be the most useful for this method. Methods. Spatially and temporally distinct samples from 3 SCT recipients with VZV disease unresponsive to acyclovir treatment were retrospectively investigated for the presence of TK mutations by polymerase chain reaction and sequence analysis. Results. In all 3 patients, a mutation in the VZV TK coding region was found resulting in an amino acid substitution. TK mutations were not only temporally but also spatially compartmentalized. In particular, plasma samples frequently showed wild-type TK sequences, whereas cerebrospinal fluid or skin vesicle fluid acquired on the same day contained mutant sequences. Conclusions. This study shows the importance of careful sampling for molecular diagnostics of acyclovir resistance in VZV disease. All affected body sites should be sampled and plasma samples may not be representative for the viral mutation status.

Objective Most primary care clinical guidelines recommend restrictive antibiotic use for childhood infections. We investigated antibiotic prescription rates over time for oral and topical antibiotics for children (≤12 years) in the period 2000–2010. Design, setting and patients Longitudinal observational study among children (≤12 years) in a large Dutch general practice database in the period 2000–2010. Main outcome measures Oral and topical antibiotic prescribing rates per year and independent factors influencing antibiotic prescriptions. Results We analysed 108 555 patient-years during 2000–2010. At least one chronic disease was recorded in 15.8% of patient-years, with asthma most commonly registered. In 14.8% of the patient-years at least one antibiotic was prescribed, while 26.3% of these received two or more prescriptions. Young age and chronic disease had a significant effect on antibiotic prescriptions. Prescriptions for oral and topical antibiotics increased 4.9% and 1.8%, respectively, during 2000–2005 (p<0.001). Prescription rates for oral antibiotics decreased 3.3% during 2006–2010 (p<0.001), while topical prescribing rates remained stable. Conclusions One in six children received at least one oral antibiotic prescription per year during 2000–2010. While topical prescription rates steadily increased during 2005–2010 and remained stable during 2006–2010, prescription rates for oral antibiotics increased significantly during the period 2000–2005 and then significantly decreased during the period 2006–2010. As clinical guidelines remained the same over this period, the effects could be contributed to the initiation of the Dutch nationwide pneumococcal vaccination campaign in 2006.

BACKGROUNDThe optimal algorithm for serological syphilis screening is still a matter of debate. We have previously evaluated the performance of the Bioelisa Syphilis 3.0, using a selection of archived sera, and in this study compare these results with the Bioelisa results after clinical implementation. METHODSAll Bioelisa Syphilis 3.0 results obtained since clinical implementation were analyzed. Bioelisa-positive or borderline samples were retested using Treponema pallidum particle agglutination, rapid plasma reagin test, fluorescent treponemal antibody-absorption test, and/or immunoblot. On sera sent in together with cerebrospinal fluid, occasionally both the T. pallidum particle agglutination and Bioelisa were performed. RESULTSThe Bioelisa was performed on 14,622 sera. Bioelisa-positive samples, which were not retested by the previously described assays, were withdrawn from the database (n = 36). In 1.3% of the samples (187/14,586), the Bioelisa was positive or borderline and, ultimately, 115 sera were considered true positive (prevalence 0.8%). The specificity of the Bioelisa was 99.5%. CONCLUSIONSBased on the results of all performed diagnostic assays, the specificity of the Bioelisa of 99.5% is very consistent with that found in the initial study (100%; 95% confidence interval was 98.0%–100%). Interpreting (positive) test results is difficult in the absence of a gold standard, especially when the disease prevalence is low. Results should be viewed in the light of the patients’ characteristics.

IntroductionPatients with a first venous thromboembolism (VTE) are at risk of recurrence. Recurrent VTE (rVTE) can be prevented by extended anticoagulant therapy, but this comes at the cost of an increased risk of bleeding. It is still uncertain whether patients with an intermediate recurrence risk or with a high recurrence and high bleeding risk will benefit from extended anticoagulant treatment, and whether a strategy where anticoagulant duration is tailored on the predicted risks of rVTE and bleeding can improve outcomes. The aim of the Leiden Thrombosis Recurrence Risk Prevention (L-TRRiP) study is to evaluate the outcomes of tailored duration of long-term anticoagulant treatment based on individualised assessment of rVTE and major bleeding risks.Methods and analysisThe L-TRRiP study is a multicentre, open-label, cohort-based, randomised controlled trial, including patients with a first VTE. We classify the risk of rVTE and major bleeding using the L-TRRiP and VTE-BLEED scores, respectively. After 3 months of anticoagulant therapy, patients with a low rVTE risk will discontinue anticoagulant treatment, patients with a high rVTE and low bleeding risk will continue anticoagulant treatment, whereas all other patients will be randomised to continue or discontinue anticoagulant treatment. All patients will be followed up for at least 2 years. Inclusion will continue until the randomised group consists of 608 patients; we estimate to include 1600 patients in total. The primary outcome is the combined incidence of rVTE and major bleeding in the randomised group after 2 years of follow-up. Secondary outcomes include the incidence of rVTE and major bleeding, functional outcomes, quality of life and cost-effectiveness in all patients.Ethics and disseminationThe protocol was approved by the Medical Research Ethics Committee Leiden-Den Haag-Delft. Results are expected in 2028 and will be disseminated through peer-reviewed journals and during (inter)national conferences.Trial registration numberNCT06087952.

ObjectivesDespite the many proven advantages of a physically active lifestyle in patient populations, prescription of exercise is currently not widely implemented in routine clinical practice. The aims of this study were twofold: (1) to assess perceptions of clinicians on the current practice of exercise is medicine (E=M) prescription in two Dutch university medical centres and (2) to determine their perceived barriers and facilitators for the implementation of E=M in routine clinical care in Dutch university medical centres.DesignA mixed methodologies study, using both online questionnaires and semi-structured interviews.SettingDutch university medical centres.ParticipantsClinicians working within the departments of medical oncology, orthopaedics and rehabilitation medicine of two university medical centres.ResultsForty-five clinicians (response rate of 51%) completed the questionnaire, and 19 clinicians were interviewed. The results showed that even though clinicians had a positive attitude towards prescribing E=M, only a few reported to regularly prescribe E=M to their patients. The 52 identified facilitators and barriers for implementation of E=M were categorised into four main themes: (1) beliefs toward the implementation of E=M (eg, clinicians knowledge and skills, and social support), (2) factors related to the patient perspective (eg, patient priorities or motivation), (3) factors related to the referral options (eg, knowledge of and trust in local referral options) and (4) practical considerations when implementing E=M (eg, time constraints).ConclusionsOur study showed that even though many clinicians have a positive attitude toward an active lifestyle, many are not prescribing E=M on a regular basis. In order for clinicians to effectively implement E=M, strategies should focus on increasing clinicians E=M referral skills, improving clinicians knowledge of E=M referral options and develop a support system to ensure that E=M is high on the priority list of clinicians.