Necroptosis is a form of programmed cell death that depends on the activation of receptor interacting protein kinase-1 (RIPK1) and RIPK3 by receptors such as tumor necrosis factor (TNF) receptor-1. Structural studies indicate that activation of RIPK3 by RIPK1 involves the formation of oligomers via interactions of the RIP homotypic interaction motif (RHIM) domains shared by both proteins; however, the molecular mechanisms by which this occurs are not fully understood. To gain insight into this process, we constructed versions of RIPK3 that could be induced to dimerize or oligomerize in response to a synthetic drug. Using this system, we find that although the formation of RIPK3 dimers is itself insufficient to trigger cell death, this dimerization seeds a RHIM-dependent complex, the propagation and stability of which is controlled by caspase-8 and RIPK1. Consistent with this idea, we find that chemically enforced oligomerization of RIPK3 is sufficient to induce necroptosis, independent of the presence of the RHIM domain, TNF stimulation or RIPK1 activity. Further, although RIPK1 contributes to TNF-mediated RIPK3 activation, we find that RIPK1 intrinsically suppresses spontaneous RIPK3 activation in the cytosol by controlling RIPK3 oligomerization. Cells lacking RIPK1 undergo increased spontaneous RIPK3-dependent death on accumulation of the RIPK3 protein, while cells containing a chemically inhibited or catalytically inactive form of RIPK1 are protected from this form of death. Together, these data indicate that RIPK1 can activate RIPK3 in response to receptor signaling, but also acts as a negative regulator of spontaneous RIPK3 activation in the cytosol.
WONKA is a tool for the systematic analysis of an ensemble of protein–ligand structures. It makes the identification of conserved and unusual features within such an ensemble straightforward. WONKA uses an intuitive workflow to process structural co-ordinates. Ligand and protein features are summarised and then presented within an interactive web application. WONKA’s power in consolidating and summarising large amounts of data is described through the analysis of three bromodomain datasets. Furthermore, and in contrast to many current methods, WONKA relates analysis to individual ligands, from which we find unusual and erroneous binding modes. Finally the use of WONKA as an annotation tool to share observations about structures is demonstrated. WONKA is freely available to download and install locally or can be used online at http://wonka.sgc.ox.ac.uk.
Structure based drug design; Data driven drug design; Bromodomains
To examine the association between changes in procoagulants (fibrinogen factors VII and VIII and von Willebrand factor) and the risk of insulin resistance.
Using data from the Coronary Artery Risk Development in Young Adults study, we followed 2398 black and white adults without diabetes, aged 25–37 years at year 7, to year 20. Levels of fibrinogen factors VII and VIII and von Willebrand factor were divided in tertiles (low/middle/high) at years 7 and 20 and four groups reflecting changes were defined: ‘low’ (low at years 7 and 20), ‘stable’ (low/middle at years 7 and 20, but not both low at years 7 and 20), ‘high’ (high at year 7 and low/middle at year 20; or low/middle at year 7 and high at year 20) and ‘highest’ (high at years 7 and 20). Linear regression models were used to evaluate 13-year changes (year 20–year 7) in fibrinogen level and factors VII, VIII and von Willebrand change groups in relation to insulin resistance measures.
Homeostasis model assessment of insulin resistance (year 20) and changes in log homeostasis model assessment of insulin resistance (year 20–year 7) were significantly associated with the 13-year increase in fibrinogen (P < 0.001). Compared with participants in the low group, those in the high group had significantly higher levels of homeostasis model assessment of insulin resistance (year 20) and changes in homeostasis model assessment of insulin resistance (year 20–year 7) for fibrinogen factor VII and von Willebrand factor (P < 0.017). No significant associations were observed between fibrinogen VIII and insulin resistance measures.
An increase in fibrinogen level and persistently high levels of factor VII and von Willebrand factor are significantly associated with increased risk of insulin resistance. These findings provide new insight into the mechanisms to explain the heightened risk for thrombosis in adults with insulin resistance/diabetes.
Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.
The purpose of this study was to assess the prevalence of male infertility and treatment-related risk factors in childhood cancer survivors.
Within the Childhood Cancer Survivor Study, 1622 survivors and 274 siblings completed the Male Health Questionnaire. The analysis was restricted to survivors (938/1622; 57.8%) and siblings (174/274; 63.5%) who tried to become pregnant. Relative risks (RR) and 95% confidence intervals (CI) for the prevalence of self-reported infertility were calculated using generalized linear models for demographic variables and treatment-related factors to account for correlation among survivors and siblings of the same family. All statistical tests were two-sided.
Among those who provided self-report data, the prevalence of infertility was 46.0% in survivors versus 17.5% in siblings (RR=2.64, 95% CI 1.88-3.70, p < 0.001). Of survivors who met the definition for infertility, 37% had reported at least one pregnancy with a female partner that resulted in a live birth. In a multivariable analysis, risk factors for infertility included an alkylating agent dose score (AAD) ≥ 3 (RR= 2.13, 95% CI 1.69-2.68 for AAD ≥ 3 versus AAD<3), surgical excision of any organ of the genital tract (RR=1.63, 95% CI 1.20-2.21), testicular radiation ≥ 4Gy (RR=1.99, 95% CI 1.52-2.61), and exposure to bleomycin (RR=1.55, 95% CI 1.20-2.01).
Many survivors who experience infertility father their own children suggesting episodes of both fertility and infertility. This and the novel association of infertility with bleomycin warrant further investigation.
Implications for Cancer Survivors
Though infertility is common, male survivors reporting infertility often father their own children. Bleomycin may pose some fertility risk.
infertility; cancer; male; long-term survivors; pediatrics
Despite being the most evolutionarily conserved of the mammalian caspases, little is understood about the cellular function of caspase-2 in normal tissues or what role caspase-2 may have in the progression of human disease. It has been reported that deletion of the caspase-2 gene (Casp2), accelerates Eμ-myc lymphomagenesis in mice, and thus caspase-2 may act as a tumor suppressor in hematological malignancies. Here, we sought to extend these findings to epithelial cancers by examining the potential role of caspase-2 as a tumor suppressor in the mouse mammary carcinogenesis model; MMTV/c-neu. The rate of tumor acquisition was significantly higher in multiparous Casp2−/−/MMTV mice compared with Casp2+/+/MMTV and Casp2+/−/MMTV mice. Cells from Casp2−/−/MMTV tumors were often multinucleated and displayed bizarre mitoses and karyomegaly, while cells from Casp2+/+/MMTV and Casp2+/−/MMTV tumors never displayed this phenotype. Tumors from Casp2−/−/MMTV animals had a significantly higher mitotic index than tumors from Casp2+/+/MMTV and Casp2+/−/MMTV animals. Cell cycle analysis of Casp2−/− E1A/Ras-transformed mouse embryonic fibroblasts (MEF) also indicated a higher proliferative rate in the absence of caspase-2. In vitro assays further illustrated that MEF had increased genomic instability in the absence of caspase-2. This appears to be due to disruption of the p53 pathway because we observed a concomitant decrease in the induction of the p53 target genes, Pidd, p21 and Mdm2. Thus caspase-2 may function as a tumor suppressor, in part, through regulation of cell division and genomic stability.
caspase; apoptosis; oncogene
Symptomatic osteoarthritis poses a major challenge to primary health care but no studies have related accessing primary care (‘detection’), receiving recommended treatments (‘treatment’), and achieving adequate control (‘control’).
To provide estimates of detection, treatment, and control within a single population adapting the approach used to determine a Rule of Halves for other long-term conditions.
400 adults aged 50+ years with prevalent symptomatic knee osteoarthritis.
Prospective cohort with baseline questionnaire, clinical assessment, and plain radiographs, and questionnaire follow-up at 18 and 36 months and linkage to primary care medical records.
‘Detection’ was defined as at least one musculoskeletal knee-related GP consultation between baseline and 36 months. ‘Treatment’ was self-reported use of at least one recommended treatment or physiotherapy/hospital specialist referral for their knee problem at all three measurement points. Pain was ‘controlled’ if characteristic pain intensity <5 out of 10 on at least two occasions.
In 221 cases (55.3%; 95%CI: 50.4, 60.1) there was evidence that the current problem had been detected in general practice. Of those detected, 164 (74.2% (68.4, 80.0)) were receiving one or more of the recommended treatments at all three measurement points. Of those detected and treated, 45 (27.4% (20.5, 34.3)) had symptoms under control on at least two occasions. Using narrower definitions resulted in substantially lower estimates.
Osteoarthritis care does not conform to a Rule of Halves. Symptom control is low among those accessing health care and receiving treatment.
Osteoarthritis; Rule of Halves; Primary care
In pT1-T3N0 urothelial carcinoma of the bladder (UCB) patients, multi-modal therapy is inconsistently recommended. The aim of the study was to develop a prognostic tool to help decision-making regarding adjuvant therapy.
We included 2145 patients with pT1-3N0 UCB after radical cystectomy (RC), naive of neoadjuvant or adjuvant therapy. The cohort was randomly split into development cohort based on the US patients (n=1067) and validation cohort based on the Europe patients (n=1078). Predictive accuracy was quantified using the concordance index.
With a median follow-up of 45 months, 5-year recurrence-free and cancer-specific survival estimates were 68% and 73%, respectively. pT-stage, ge, lymphovascular invasion, and positive margin were significantly associated with both disease recurrence and cancer-specific mortality (P-values⩽0.005). The accuracies of the multivariable models at 2, 5, and 7 years for predicting disease recurrence were 67.4%, 65%, and 64.4%, respectively. Accuracies at 2, 5, and 7 years for predicting cancer-specific mortality were 69.3%, 66.4%, and 65.5%, respectively. We developed competing-risk, conditional probability nomograms. External validation revealed minor overestimation.
Despite RC, a significant number of patients with pT1-3N0 UCB experience disease recurrence and ultimately die of UCB. We developed and externally validated competing-risk, conditional probability post-RC nomograms for prediction of disease recurrence and cancer-specific mortality.
bladder cancer; radical cystectomy; T1-3 N0; prediction; nomogram; chemotherapy
Using genetically modified mouse models, we report here that p53 upregulated modulator of apoptosis (Puma) and Bcl-2 interacting mediator of cell death (Bim), two pro-apoptotic members of the B-cell lymphoma protein-2 (Bcl-2) family of proteins, cooperate in causing bone marrow and gastrointestinal tract toxicity in response to chemo and radiation therapy. Deletion of both Puma and Bim provides long-term survival without evidence of increased tumor susceptibility following a lethal challenge of carboplatin and ionizing radiation. Consistent with these in vivo findings, studies of primary mast cells demonstrated that the loss of Puma and Bim confers complete protection from cytokine starvation and DNA damage, similar to that observed for Bax/Bak double knockout cells. Biochemical analyses demonstrated an essential role for either Puma or Bim to activate Bax, thereby leading to mitochondrial outer membrane permeability, cytochrome c release and apoptosis. Treatment of cytokine-deprived cells with ABT-737, a BH3 mimetic, demonstrated that Puma is sufficient to activate Bax even in the absence of all other known direct activators, including Bim, Bid and p53. Collectively, our results identify Puma and Bim as key mediators of DNA damage-induced bone marrow failure and provide mechanistic insight into how BH3-only proteins trigger cell death.
Puma; Bim; apoptosis; myelosuppression; ABT-737
In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis', ‘necrosis' and ‘mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.
autophagy; mitochondrial membrane permeabilization; necroptosis; parthanatos; TNFR1; TP53
Despite an abundance of literature on the role of caspase-2 in apoptosis, there exists much controversy about this protease making it difficult to place caspase-2 correctly in the apoptotic cascade, and hence its role in apoptosis remains unclear. The identification of the PIDDosome as a signaling platform for caspase-2 activation prompted intense investigation into the true role of this orphan caspase. What has emerged is the idea that caspase-2 may not be mandatory for apoptosis and that activation of this caspase in response to some forms of stress has other effects on the cell such as regulation of cell cycle progression. This idea is particularly relevent to the discovery that caspase-2 may act as a tumor suppressor. Here, we discuss the proposed mechanisms through which caspase-2 signals, in particular those involving PIDD, and their impact on cellular fate.
caspase-2; apoptosis; PIDD; PIDDosome; cell cycle
The diagnosis of intraoperative anaphylaxis is important but can be difficult as the symptoms can be varying and dependent on patient factors.
PRESENTATION OF CASE
We describe an acute, unexpected and life threatening cardiovascular (CV) collapse, presumed to be due to an acute anaphylactic reaction secondary to gelatin administration, following induction of general anaesthesia (GA), in an ASA 3 patient scheduled for axillo-bifemoral bypass.
The management of the profound cardiovascular (CV) collapse was greatly assisted by sophisticated haemodynamic, depth of anaesthesia and cerebral oximetry monitoring.1 As far as we are aware this is the first such case where the full haemodynamic, depth of anaesthesia and cerebral oxygenation changes during CV collapse, presumed due to an acute anaphylactic reaction under GA have been fully documented.
The use of advanced monitoring intraoperatively proved extremely useful in guiding the resuscitation of a life threatening allergic reaction under general anaesthesia.
General anaesthesia; Analphylaxis; Haemodynamic monitoring; Cerebral oximetry; Depth of anaesthesia monitoring
► We dissociate structural correlates for two different non-native language skills. ► Number of languages spoken was associated with the posterior supramarginal gyrus. ► The efficiency of word use was associated with the left pars opercularis.
We used structural magnetic resonance imaging (MRI) and voxel based morphometry (VBM) to investigate whether the efficiency of word processing in the non-native language (lexical efficiency) and the number of non-native languages spoken (2+ versus 1) were related to local differences in the brain structure of bilingual and multilingual speakers. We dissociate two different correlates for non-native language processing. Firstly, multilinguals who spoke 2 or more non-native languages had higher grey matter density in the right posterior supramarginal gyrus compared to bilinguals who only spoke one non-native language. This is interpreted in relation to previous studies that have shown that grey matter density in this region is related to the number of words learnt in bilinguals relative to monolinguals and in monolingual adolescents with high versus low vocabulary. Our second result was that, in bilinguals, grey matter density in the left pars opercularis was positively related to lexical efficiency in second language use, as measured by the speed and accuracy of lexical decisions and the number of words produced in a timed verbal fluency task. Grey matter in the same region was also negatively related to the age at which the second language was acquired. This is interpreted in terms of previous findings that associated the left pars opercularis with phonetic expertise in the native language.
Language; Efficiency; Lexical; MRI; Bilingual
Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1−/− hosts repopulated with Bim−/− conventional CD4+ T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1−/− hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.
iTreg; apoptosis; Bim; Bcl-2
Although mechanisms of modern military wounding may be distinct from those of ancient conflicts, the infectious sequelae of ballistic trauma and the evolving microbial flora of war wounds remain a considerable burden on both the injured combatant and their deployed medical systems. Battlefield surgeons of ancient times favoured suppuration in war wounding and as such Galenic encouragement of pus formation would hinder progress in wound care for centuries. Napoleonic surgeons eventually abandoned this mantra, embracing radical surgical intervention, primarily by amputation, to prevent infection. Later, microscopy enabled identification of microorganisms and characterization of wound flora. Concurrent advances in sanitation and evacuation enabled improved outcomes and establishment of modern military medical systems. Advances in medical doctrine and technology afford those injured in current conflicts with increasing survivability through rapid evacuation, sophisticated resuscitation and timely surgical intervention. Infectious complications in those that do survive, however, are a major concern. Addressing antibiotic use, nosocomial transmission and infectious sequelae are a current clinical management and research priority and will remain so in an era characterized by a massive burden of combat extremity injury. This paper provides a review of infection in combat wounding from a historical setting through to the modern evidence base.
war; wound; infection; trauma; antibiotic; nosocomial
Bats are natural reservoirs for the majority of lyssaviruses globally, and are unique among mammals in having exceptional sociality and longevity. Given these facets, and the recognized status of bats as reservoirs for rabies viruses (RABVs) in the Americas, individual bats may experience repeated exposure to RABV during their lifetime. Nevertheless, little information exists with regard to within-host infection dynamics and the role of immunological memory that may result from abortive RABV infection in bats. In this study, a cohort of big brown bats (Eptesicus fuscus) was infected intramuscularly in the left and right masseter muscles with varying doses [10−0.1–104.9 median mouse intracerebral lethal doses (MICLD50)] of an E. fuscus RABV variant isolated from a naturally infected big brown bat. Surviving bats were infected a second time at 175 days post-(primary) infection with a dose (103.9–104.9 MICLD50) of the same RABV variant. Surviving bats were infected a third time at either 175 or 305 days post-(secondary) infection with a dose (104.9 MICLD50) of the same RABV variant. When correcting for dose, similar mortality was observed following primary and secondary infection, but reduced mortality was observed following the third and last RABV challenge, despite infection with a high viral dose. Inducible RABV-neutralizing antibody titres post-infection were ephemeral among infected individuals, and dropped below levels of detection in several bats between subsequent infections. These results suggest that long-term repeated infection of bats may confer significant immunological memory and reduced susceptibility to RABV infection.
To determine the injury patterns, complications, and mortality after alcohol consumption in trauma patients.
The Trauma Registry at an American College of Surgeons (ACS) level I center was queried for all patients with a toxicology screen admitted between 1st January 2002 and 31st December 2005. Alcohol-positive (AP) patients were matched to control patients who had a completely negative screen (AN) using age, gender, mechanism, Injury Severity Score (ISS), head Abbreviated Injury Scale (AIS), chest AIS, abdominal AIS, and extremity AIS. Injuries and outcomes were compared between the groups.
As many as 5,317 patients had toxicology data, of which 471 (8.9%) had a positive alcohol screen (AP). A total of 386 AP patients were then matched to 386 control (AN) patients. The AP group had a significantly higher mortality than the AN group overall (23 vs. 13%; p < 0.001), and by ISS stratification: ISS < 16 (6 vs. 0.4%; p < 0.001), ISS 16–25 (53 vs. 28%; p = 0.01), and ISS > 25 (90 vs. 67%; p = 0.01). AP patients had a higher incidence of admission systolic blood pressure < 90 (18 vs. 10%; p < 0.001) and Glasgow Coma Scale (GCS) score ≤ 8 (25 vs. 17%; p = 0.002). AN patients had a significantly higher incidence of hemopneumothorax (11 vs. 7%; p = 0.03), while AP patients had a higher incidence of cardiac arrest (8 vs. 3%; p = 0.004). There was no difference in intensive care unit (ICU) and hospital length of stay.
In a mixed population of trauma patients, an AP screen is associated with an increased incidence of admission hypotension and depressed GCS score. In this case-matched study, alcohol exposure appeared to increase mortality after injury.
Alcohol; Injury; Trauma; Complications; Mortality