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IntroductionUnderstanding violent criminality and its impact on health and eventually the risk of premature mortality is important for efficient future interventions. This study aimed to explore the effect violent criminality had on premature mortality (i.e., death before the age of 65) among individuals with substance use disorders (SUDs).MethodsThe cohort was created by identifying all Swedish patients diagnosed with SUD between the first of January 2013 and 31st of December 2014. The individuals were split into three age categories.ResultsThere were significant differences in standard mortality rates (SMR) in the cohort compared to the general Swedish population across the three age categories. We found differences between the SMRs for individuals convicted of violent and nonviolent crimes in the two younger age categories [age 15–29: violent crime (42.4) vs. non-violent crime (36.6), age 30–44: violent crime (28.0) vs. non-violent crime (23.0)]. A Cox regression analysis showed that each conviction of a violent crime increased the hazard ratio (HR) of premature mortality significantly [age 15–29; HR = 1.10 (95% CI: 1.04–1.17), age 30–44; HR =1.06 (95% CI: 1.03–1.09)]. After correcting for non-violent crimes, the increased risk only remained for the youngest group [HR = 1.06 (95% CI: 1.00–1.13)].DiscussionThis study suggests that criminal behavior constitutes a proxy for the risk behaviors that increase the risk of premature mortality among young individuals with SUD even after controlling for confounders. Longitudinal studies, examining time-dependent risks and protective influences, are needed to explain the different pathways and processes leading to the amplified premature mortality in the groups.

Background: Tinzaparin, a low-molecular weight heparin (LMWH), has shown anti-neoplastic properties in animal models and in in vitro studies of human cancer cell lines. The reduction of CA-125 levels during neoadjuvant chemotherapy (NACT) in patients with epithelial ovarian cancer (EOC) co-varies with the prognosis; the larger the decrease in CA-125, the better the prognosis. Purpose: This study aims to evaluate the potential anti-neoplastic effects of tinzaparin by investigating changes in serum CA-125 levels in advanced EOC patients who receive NACT. Material and methods: This is an open randomized multicenter pilot trial. Forty patients with EOC selected to receive NACT will be randomized 1:1 to receive daily addition of tinzaparin or no tinzaparin. The processing and treatment of the patients will otherwise follow the recommendations in the Swedish National Guidelines for Ovarian Cancer. Before every cycle of chemotherapy, preoperatively, and 3 weeks after the last cycle of chemotherapy, a panel of biomarkers, including CA-125, will be measured. Patients: Inclusion criteria are women aged 18 years or older, World Health Organization performance status 0–1, histologically confirmed high-grade serous, endometrioid or clear cell EOC, International Federation of Gynecology and Obstetrics (FIGO) stages III-IV. In addition, a CA-125 level of ≥ 250 kIE/L at diagnosis. Exclusion criteria are contraindications to LMWH, ongoing or recent treatment with unfractionated heparin, LMWH, warfarin or non-vitamin K antagonist oral anticoagulants. Interpretation: This study will make an important contribution to the knowledge of the anti-neoplastic effects of tinzaparin in EOC patients and may thus guide the planning of a future study on the impact of tinzaparin on survival in EOC.

Abstract Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated.

BackgroundTreatment seeking for gambling disorder is known to be low and there has been a lack of longitudinal research regarding treatment opportunities. The present study aimed to assess possible changes in treatment uptake after a formal introduction of gambling disorder in social services and health care legislations, by using register data, including patient characteristics with respect to socio-demographics and comorbidities.MethodsNationwide register data were collected for the years 2005–2019, describing diagnoses in specialized out-patient health care and in in-patient hospital care. Numbers and characteristics of patients with gambling disorder were followed longitudinally. Also, a new legislation for treatment by public institutions was introduced in 2018, and data were compared for the years before and after the shift in legislation, both nationally, for each of the three major urban regions, and for the rest of the country. Comparisons were made with respect to concurrent mental health comorbidities, age and gender.ResultsThe number of out-patient gambling disorder diagnoses increased over time, but without any significant step changes around the shift in legislation. Over time, patients were younger, became more likely to have gambling disorder as their primary diagnosis, and less likely to have mental health comorbidities, whereas gender distribution did not change. Among the smaller group of patients diagnosed in in-patient settings, mental health comorbidity increased over time. Despite gradual changes over time, no changes in demographics were seen around the actual shift in legislation, although the psychiatric comorbidity appeared to increase after this change.ConclusionAfter the introduction of gambling disorder in the responsibility of social services and health care settings in Sweden, the number of patients diagnosed with gambling disorder increased only modestly. Likely, further implementation of gambling disorder treatment is required in the health care services. Also, longer longitudinal studies are needed in order to understand to what extent patients not seeking health care treatment are received by municipal social services or remain outside the treatment system.

Abstract Background Periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome is an autoinflammatory disorder that primarily affects young children, and typically gives rise to fever episodes that recur monthly for several years. This study investigated the impact of PFAPA syndrome on the families of affected children, the health-related quality of life (HRQOL) of children with the syndrome, and how these factors were influenced by tonsillectomy. Methods This prospective cohort study included 24 children with typical PFAPA syndrome that were referred for tonsillectomy, of whom 20 underwent the procedure. The control group consisted of randomly selected children from the general population. Family impact and HRQOL were measured using the standardized, validated questionnaires Pediatric Quality of Life Inventory™ (PedsQL™) Family Impact Module (FIM) and PedsQL™ 4.0 Generic Core Scales (GCS). Parents to children with PFAPA completed the questionnaires before and 6 months after their child underwent tonsillectomy, and HRQOL was measured both between and during PFAPA episodes. The Wilcoxon signed-rank test was used to compare data before and after tonsillectomy in the patient group, while the Mann–Whitney test was used for comparison of the patient and control groups. Results Before tonsillectomy, children with PFAPA had significantly lower scores than the control group on the PedsQL™ FIM and the PedsQL™ 4.0 GCS during fever episodes. After tonsillectomy, all patients improved with diminished febrile episodes, which resulted in significantly higher scores regarding both family impact and HRQOL at the time of follow-up. HRQOL of in children with PFAPA improved after tonsillectomy even when compared to afebrile intervals before the procedure. The differences between PFAPA patients and controls were eliminated after tonsillectomy. Conclusion PFAPA syndrome has a profound negative impact on the families of affected children. Tonsillectomy that leads to cessation or reduction of fever episodes eases the impact of the disease on the family. HRQOL in children with PFAPA is low during febrile episodes and similar to healthy controls in between episodes. The improvement of HRQOL in patients with PFAPA after tonsillectomy compared to the afebrile intervals before tonsillectomy highlights that the constantly recurring fevers may affect the children’s well-being even between fever episodes.

We describe a female patient suffering from severe chronic non-bacterial osteomyelitis (CNO) with systemic inflammation and advanced malnutrition and complete deficiency of myeloperoxidase (MPO). CNO is a rare autoinflammatory bone disorder associated with dysregulation of the innate immune system. MPO deficiency is a genetic disorder with partial or complete absence of the phagocyte peroxidase MPO. MPO deficiency has no established clinical phenotype but reports indicate increased susceptibility to infection and chronic inflammation. The patient’s symptoms began at 10 years of age with pain in the thighs, systemic inflammation and malnutrition. She was diagnosed with CNO at 14 years of age. Treatment with nonsteroidal anti-inflammatory drugs, corticosteroids, bisphosphonates or IL1-receptor antagonists (anakinra) did not relieve the symptoms. However, the patient responded instantly and recovered from her clinical symptoms when treated with TNFα blockade (adalimumab). Three years after treatment initiation adalimumab was withdrawn, resulting in rapid symptom recurrence. When reintroducing adalimumab, the patient promptly responded and went into remission. In addition to clinical and laboratory profiles, neutrophil functions (reactive oxygen species, ROS; neutrophil extracellular traps, NETs; degranulation; apoptosis; elastase activity) were investigated both in a highly inflammatory state (without treatment) and in remission (on treatment). At diagnosis, neither IL1β, IL6, nor TNFα was significantly elevated in serum, but since TNFα blockade terminated the inflammatory symptoms, the disease was likely TNFα-driven. All neutrophil parameters were normal both during treatment and treatment withdrawal, except for MPO-dependent intracellular ROS- and NET formation. The role of total MPO deficiency for disease etiology and severity is discussed.

Epigenome‐wide gene–gene (G × G) interactions associated with non‐small‐cell lung cancer (NSCLC) survival may provide insights into molecular mechanisms and therapeutic targets. Hence, we proposed a three‐step analytic strategy to identify significant and robust G × G interactions that are relevant to NSCLC survival. In the first step, among 49 billion pairs of DNA methylation probes, we identified 175 775 G × G interactions with PBonferroni ≤ 0.05 in the discovery phase of epigenomic analysis; among them, 15 534 were confirmed with P ≤ 0.05 in the validation phase. In the second step, we further performed a functional validation for these G × G interactions at the gene expression level by way of a two‐phase (discovery and validation) transcriptomic analysis, and confirmed 25 significant G × G interactions enriched in the 6p21.33 and 6p22.1 regions. In the third step, we identified two G × G interactions using the trans‐omics analysis, which had significant (P ≤ 0.05) epigenetic cis‐regulation of transcription and robust G × G interactions at both the epigenetic and transcriptional levels. These interactions were cg14391855 × cg23937960 (βinteraction = 0.018, P = 1.87 × 10−12), which mapped to RELA × HLA‐G (βinteraction = 0.218, P = 8.82 × 10−11) and cg08872738 × cg27077312 (βinteraction = −0.010, P = 1.16 × 10−11), which mapped to TUBA1B × TOMM40 (βinteraction =−0.250, P = 3.83 × 10−10). A trans‐omics mediation analysis revealed that 20.3% of epigenetic effects on NSCLC survival were significantly (P = 0.034) mediated through transcriptional expression. These statistically significant trans‐omics G × G interactions can also discriminate patients with high risk of mortality. In summary, we identified two G × G interactions at both the epigenetic and transcriptional levels, and our findings may provide potential clues for precision treatment of NSCLC.

The interaction between DNA methylation of tripartite motif containing 27 (cg05293407TRIM27) and smoking has previously been identified to reveal histologically heterogeneous effects of TRIM27 DNA methylation on early‐stage non‐small‐cell lung cancer (NSCLC) survival. However, to understand the complex mechanisms underlying NSCLC progression, we searched three‐way interactions. A two‐phase study was adopted to identify three‐way interactions in the form of pack‐year of smoking (number of cigarettes smoked per day × number of years smoked) × cg05293407TRIM27 × epigenome‐wide DNA methylation CpG probe. Two CpG probes were identified with FDR‐q ≤ 0.05 in the discovery phase and P ≤ 0.05 in the validation phase: cg00060500KIAA0226 and cg17479956EXT2. Compared to a prediction model with only clinical information, the model added 42 significant three‐way interactions using a looser criterion (discovery: FDR‐q ≤ 0.10, validation: P ≤ 0.05) had substantially improved the area under the receiver operating characteristic curve (AUC) of the prognostic prediction model for both 3‐year and 5‐year survival. Our research identified the complex interaction effects among multiple environment and epigenetic factors, and provided therapeutic target for NSCLC patients.

Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.

Circulating antieosinophil antibodies (AEOSA) have been associated with various autoimmune conditions affecting the liver, kidneys, lungs, and joints but are not part of routine clinical diagnostics. While analyzing human sera for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence (IIF) on granulocytes, 0.8% of analyzed samples were found to be reactive with eosinophils. Our aim was to determine the diagnostic relevance and antigenic specificity of AEOSA. AEOSA were seen either in combination with an myeloperoxidase (MPO)-positive p-ANCA (44%; AEOSA+/ANCA+) or on their own (56%; AEOSA+/ANCA−). AEOSA/ANCA positivity was seen in patients with thyroid disease (44%) or vasculitis (31%), while AEOSA+/ANCA− pattern was more common in patients with autoimmune disorders of the gastrointestinal tract and/or liver. Eosinophil peroxidase (EPX) was the main target recognized in 66% of the AEOSA+ sera by enzyme-linked immunosorbent assay (ELISA). Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) were also identified as target antigens but less frequently and only in combination with EPX. In conclusion, we confirmed that EPX is a major target of AEOSA, illustrating the high antigenic potential of EPX. Our results also demonstrate the presence of concomitant AEOSA/ANCA positivity in a defined patient group. Further research should aim to elucidate the association of AEOSA with autoimmunity.

Abstract Background Slow degradation kinetics of long-chain fatty acids (LCFA) and their accumulation in anaerobic digesters disrupt methanogenic activity and biogas production at high loads of waste lipids. In this study, we evaluated the effect of effluent solids recirculation on microbial LCFA (oleate) degradation capacity in continuous stirred-tank sludge digesters, with the overall aim of providing operating conditions for efficient co-digestion of waste lipids. Furthermore, the impacts of LCFA feeding frequency and sulfide on process performance and microbial community dynamics were investigated, as parameters that were previously shown to be influential on LCFA conversion to biogas. Results Effluent solids recirculation to municipal sludge digesters enabled biogas production of up to 78% of the theoretical potential from 1.0 g oleate l−1 day−1. In digesters without effluent recirculation, comparable conversion efficiency could only be reached at oleate loading rates up to 0.5 g l−1 day−1. Pulse feeding of oleate (supplementation of 2.0 g oleate l−1 every second day instead of 1.0 g oleate l−1 every day) did not have a substantial impact on the degree of oleate conversion to biogas in the digesters that operated with effluent recirculation, while it marginally enhanced oleate conversion to biogas in the digesters without effluent recirculation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea revealed that pulse feeding resulted in prevalence of fatty acid-degrading Smithella when effluent recirculation was applied, whereas Candidatus Cloacimonas prevailed after pulse feeding of oleate in the digesters without effluent recirculation. Combined oleate pulse feeding and elevated sulfide level contributed to increased relative abundance of LCFA-degrading Syntrophomonas and enhanced conversion efficiency of oleate, but only in the digesters without effluent recirculation. Conclusions Effluent solids recirculation improves microbial LCFA degradation capacity, providing possibilities for co-digestion of larger amounts of waste lipids with municipal sludge.

Mohammad et al. show that subcutaneous injection of Staphylococcus aureus lipoproteins (Lpp) leads to the infiltration of neutrophils and monocytes/macrophages, inducing skin lesions in mice. They find that S. aureus Lpp promotes abscess formation, which protects bacteria from innate immune killing, suggesting an intriguing bacterial immune evasion mechanism.

Background and purpose: Few studies on magnetic resonance imaging (MRI) only head and neck radiation treatment planning exist, and none using a generally available software. The aim of this study was to evaluate the accuracy of absorbed dose for head and neck synthetic computed tomography data (sCT) generated by a commercial convolutional neural network-based algorithm. Materials and methods: For 44 head and neck cancer patients, sCT were generated and the geometry was validated against computed tomography data (CT). The clinical CT based treatment plan was transferred to the sCT and recalculated without re-optimization, and differences in relative absorbed dose were determined for dose-volume-histogram (DVH) parameters and the 3D volume. Results: For overall body, the results of the geometric validation were (Mean ± 1sd): Mean error −5 ± 10 HU, mean absolute error 67 ± 14 HU, Dice similarity coefficient 0.98 ± 0.05, and Hausdorff distance difference 4.2 ± 1.7 mm. Water equivalent depth difference for region Th1-C7, mid mandible and mid nose were −0.3 ± 3.4, 1.1 ± 2.0 and 0.7 ± 3.8 mm respectively. The maximum mean deviation in absorbed dose for all DVH parameters was 0.30% (0.12 Gy). The absorbed doses were considered equivalent (p-value

Tripartite motif containing 27 (TRIM27) is highly expressed in lung cancer, including non‐small‐cell lung cancer (NSCLC). Here, we profiled DNA methylation of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumours from 613 early‐stage NSCLC patients and evaluated associations between CpG methylation of TRIM27 and overall survival. Significant CpG probes were confirmed in 617 samples from The Cancer Genome Atlas. The methylation of the CpG probe cg05293407TRIM27 was significantly associated with overall survival in patients with LUSC (HR = 1.65, 95% CI: 1.30–2.09, P = 4.52 × 10−5), but not in patients with LUAD (HR = 1.08, 95% CI: 0.87–1.33, P = 0.493). As incidence of LUSC is associated with higher smoking intensity compared to LUAD, we investigated whether smoking intensity impacted on the prognostic effect of cg05293407TRIM27 methylation in NSCLC. LUSC patients had a higher average pack‐year of smoking (37.49LUAD vs 54.79LUSC, P = 1.03 × 10−19) and included a higher proportion of current smokers than LUAD patients (28.24%LUAD vs 34.09%LUSC, P = 0.037). cg05293407TRIM27 was significantly associated with overall survival only in NSCLC patients with medium–high pack‐year of smoking (HR = 1.58, 95% CI: 1.26–1.96, P = 5.25 × 10−5). We conclude that cg05293407TRIM27 methylation is a potential predictor of LUSC prognosis, and smoking intensity may impact on its prognostic value across the various types of NSCLC.

BRCA-deficient breast cancer is characterised by homologous recombination deficiency. Here, the authors show that hypermethylated BRCA1 phenotypically copies mutated BRCA1 in triple negative breast cancers.

Abstract Background Alzheimer’s disease (AD) is the most common form of age-related neurodegenerative diseases. Cerebral deposition of Aβ peptides, especially Aβ42, is considered the major neuropathological hallmark of AD and the putative cause of AD-related neurotoxicity. Aβ peptides are produced by sequential proteolytic processing of APP, with β-secretase (BACE) being the initiating enzyme. Therefore, BACE has been considered an attractive therapeutic target in AD research and several BACE inhibitors have been tested in clinical trials, but so far, all have had negative outcomes or even led to worsening of cognitive function. AD can be triggered by Aβ years before the first symptoms appear and one reason for the failures could be that the clinical trials were initiated too late in the disease process. Another possible explanation could be that BACE inhibition alters physiological APP processing in a manner that impairs synaptic function, causing cognitive deterioration. Methods The aim of this study was to investigate if partial BACE inhibition, mimicking the putative protective effect of the Icelandic mutation in the APP gene, could reduce Aβ generation without affecting synaptic transmission. To investigate this, we used an optical electrophysiology platform, in which effects of compounds on synaptic transmission in cultured neurons can be monitored. We employed this method on primary cortical rat neuronal cultures treated with three different BACE inhibitors (BACE inhibitor IV, LY2886721, and lanabecestat) and monitored Aβ secretion into the cell media. Results We found that all three BACE inhibitors tested decreased synaptic transmission at concentrations leading to significantly reduced Aβ secretion. However, low-dose BACE inhibition, resulting in less than a 50% decrease in Aβ secretion, did not affect synaptic transmission for any of the inhibitors tested. Conclusion Our results indicate that Aβ production can be reduced by up to 50%, a level of reduction of relevance to the protective effect of the Icelandic mutation, without causing synaptic dysfunction. We therefore suggest that future clinical trials aimed at prevention of Aβ build-up in the brain should aim for a moderate CNS exposure of BACE inhibitors to avoid side effects on synaptic function.

Thymidine kinase 2 (TK2) deficiency in humans leads to a myopathic form of mitochondrial DNA (mtDNA) deficiency. Here we present a skeletal and cardiac muscle specific TK2 knockout mouse (mTk2 KO). The mice showed dilated hearts and markedly reduced adipose tissue during week 12 to 16. A severe decrease of mtDNA was found only in skeletal muscle and heart tissue in mTk2 KO mice. Expression analysis of key metabolic genes of 16 weeks knockout mice showed significant changes of genes involved in lipid metabolism, with different patterns in heart and skeletal muscle. Our study further suggests that lipoprotein lipase (LPL) from liver supports the metabolism when heart and skeletal muscle were impaired due to mitochondrial dysfunction. The angiotensin-converting enzyme 2 (ACE2), which is involved in glucose homeostasis, was also affected by mtDNA deficiency in our study. Interestingly, both the gene and protein expression of ACE2 were increased in cardiac tissue of mTk2 KO mice. Since ACE2 is a receptor for the SARS-CoV-2 virus, its regulation in relation to mitochondrial function may have important clinical implications.

Background: There is an increased risk of suicidality in gambling disorder (GD) and economic hardship is common in the population. Economic hardship itself is a risk factor for suicidality. This study aims to explore the risk of intentional self-harm in GD utilizing social welfare payment (SWP) as a proxy for economic hardship and exploring how economic hardship, gender, criminality, socioeconomic-, and psychiatric risk factors might contribute to intentional self-harm in GD.Methods: This is a nationwide register-based study of 848 individuals diagnosed with GD in the Swedish healthcare system during the years of 2011–2014 with an average follow up of 4.9 years. Pearson's Chi-square analyses were carried out for comparisons regarding psychiatric comorbidity and intentional self-harm with regards to gender and SWPs. Univariable and multivariable Cox regression were utilized to analyse risk factors for intentional self-harm.Results: A large part of the study population received SWPs (45.5% with an insignificant overrepresentation of women) and psychiatric disorders were more common in these individuals (p < 0.001). Conviction for crime in general (p < 0.001) as well as intentional self-harm (p = 0.025) were also more common amongst recipients of SWPs. Criminal conviction in general was abundant (26.5%). In the stepwise multivariable regression, substance-related diagnoses as well as anxiety, depressive, and personality disorders remained risk factors for intentional self-harm and no significant results were found with regards to gender, criminal history, or SWPs.Conclusions: Social welfare payment was common among GD patients and intentional self-harm was more common amongst recipients than GD patients as a whole. Social welfare payments were however not a significant risk factor for intentional self-harm. However, attention to suicidality and self-injurious behavior should be paid from social services controlling SWPs due to the large prevalence of intentional self-harm in this group. In accordance with previous studies, comorbid psychiatric disorders such as anxiety, depression, substance use, and personality disorders increased the risk of intentional self-harm.

Abstract Background Waste lipids are attractive substrates for co-digestion with primary and activated sewage sludge (PASS) to improve biogas production at wastewater treatment plants. However, slow conversion rates of long-chain fatty acids (LCFA), produced during anaerobic digestion (AD), limit the applicability of waste lipids as co-substrates for PASS. Previous observations indicate that the sulfide level in PASS digesters affects the capacity of microbial communities to convert LCFA to biogas. This study assessed the microbial community response to LCFA loads in relation to sulfide level during AD of PASS by investigating process performance and microbial community dynamics upon addition of oleate (C18:1) and stearate (C18:0) to PASS digesters at ambient and elevated sulfide levels. Results Conversion of LCFA to biogas was limited (30% of theoretical biogas potential) during continuous co-digestion with PASS, which resulted in further LCFA accumulation. However, the accumulated LCFA were converted to biogas (up to 66% of theoretical biogas potential) during subsequent batch-mode digestion, performed without additional substrate load. Elevated sulfide level stimulated oleate (but not stearate) conversion to acetate, but oleate and sulfide imposed a synergistic limiting effect on acetoclastic methanogenesis and biogas formation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea showed that differences in sulfide level and LCFA type resulted in microbial community alterations with distinctly different patterns. Taxonomic profiling of the sequencing data revealed that the phylum Cloacimonetes is likely a key group during LCFA degradation in PASS digesters, where different members take part in degradation of saturated and unsaturated LCFA; genus W5 (family Cloacimonadaceae) and family W27 (order Cloacimonadales), respectively. In addition, LCFA-degrading Syntrophomonas, which is commonly present in lipid-fed digesters, increased in relative abundance after addition of oleate at elevated sulfide level, but not without sulfide or after stearate addition. Stearate conversion to biogas was instead associated with increasing abundance of hydrogen-producing Smithella and hydrogenotrophic Methanobacterium. Conclusions Long-chain fatty acid chain saturation and sulfide level are selective drivers for establishment of LCFA-degrading microbial communities in municipal sludge digesters.

Background: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. Methods: We daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. Findings: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. Interpretation: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. Fund: This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Keywords: TK2, Deoxynucleoside therapy, mtDNA depletion, Encephalomyopathy, Thymidine, Deoxycytidine

Abstract We consider a hypothesis in which classical space-time emerges from information exchange (interactions) between quantum fluctuations in the gravity theory. In this picture, a line element would arise as a statistical average of how frequently particles interact, through an individual rate dt ∼ 1/f t and spatially interconnecting rates dl ∼ c/f . The question is if space-time can be modelled consistently in this way. The ansatz would be opposite to the standard treatment of space-time as insensitive to altered physics at event horizons (disrupted propagation of information) but by extension relate to the connection of space-time to entanglement (interactions) through the gauge/gravity duality. We make a first, rough analysis of the implications this type of quantization would have on the classical structure of flat space-time, and of what would be required of the interactions. Seeing no obvious reason for why the origin would be unrealistic, we comment on expected effects in the presence of curvature.

Smoking cessation prolongs survival and decreases mortality of patients with non‐small‐cell lung cancer (NSCLC). In addition, epigenetic alterations of some genes are associated with survival. However, potential interactions between smoking cessation and epigenetics have not been assessed. Here, we conducted an epigenome‐wide interaction analysis between DNA methylation and smoking cessation on NSCLC survival. We used a two‐stage study design to identify DNA methylation–smoking cessation interactions that affect overall survival for early‐stage NSCLC. The discovery phase contained NSCLC patients from Harvard, Spain, Norway, and Sweden. A histology‐stratified Cox proportional hazards model adjusted for age, sex, clinical stage, and study center was used to test DNA methylation–smoking cessation interaction terms. Interactions with false discovery rate‐q ≤ 0.05 were further confirmed in a validation phase using The Cancer Genome Atlas database. Histology‐specific interactions were identified by stratification analysis in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. We identified one CpG probe (cg02268510SIPA1L3) that significantly and exclusively modified the effect of smoking cessation on survival in LUAD patients [hazard ratio (HR)interaction = 1.12; 95% confidence interval (CI): 1.07–1.16; P = 4.30 × 10–7]. Further, the effect of smoking cessation on early‐stage LUAD survival varied across patients with different methylation levels of cg02268510SIPA1L3. Smoking cessation only benefited LUAD patients with low methylation (HR = 0.53; 95% CI: 0.34–0.82; P = 4.61 × 10–3) rather than medium or high methylation (HR = 1.21; 95% CI: 0.86–1.70; P = 0.266) of cg02268510SIPA1L3. Moreover, there was an antagonistic interaction between elevated methylation of cg02268510SIPA1L3 and smoking cessation (HRinteraction = 2.1835; 95% CI: 1.27–3.74; P = 4.46 × 10−3). In summary, smoking cessation benefited survival of LUAD patients with low methylation at cg02268510SIPA1L3. The results have implications for not only smoking cessation after diagnosis, but also possible methylation‐specific drug targeting.

Hypoxia occurs frequently in human cancers and promotes stabilization and activation of hypoxia inducible factor (HIF). HIF-1α is specific for the hypoxia response, and its degradation mediated by three enzymes EGLN1, EGLN2 and EGLN3. Although EGLNs expression has been found to be related to prognosis of many cancers, few studies examined DNA methylation in EGLNs and its relationship to prognosis of early-stage non-small cell lung cancer (NSCLC). We analyzed EGLNs DNA methylation data from tumor tissue samples of 1,230 early-stage NSCLC patients, as well as gene expression data from The Cancer Genome Atlas. The sliding windows sequential forward feature selection method and weighted random forest were used to screen out the candidate CpG probes in lung adenocarcinomas (LUAD) and lung squamous cell carcinomas patients, respectively, in both discovery and validation phases. Then Cox regression was performed to evaluate the association between DNA methylation and overall survival. Among the 34 CpG probes in EGLNs, DNA methylation at cg25923056EGLN2 was identified to be significantly associated with LUAD survival (HR = 1.02, 95% CI: 1.01–1.03, P = 9.90 × 10–5), and correlated with EGLN2 expression (r = – 0.36, P = 1.52 × 10–11). Meanwhile, EGLN2 expression was negatively correlated with HIF1A expression in tumor tissues (r = – 0.30, P = 4.78 × 10–8) and significantly (P = 0.037) interacted with HIF1A expression on overall survival. Therefore, DNA methylation of EGLN2- HIF1A is a potential marker for LUAD prognosis and these genes are potential treatment targets for further development of HIF-1α inhibitors in lung cancer therapy.

Abstract Background Galectin-3 is a 32 kDa protein secreted by macrophages involved in processes such as cell activation, chemotaxis and phagocytosis. Galectin-3 has previously been shown to improve the ability of airway macrophages to ingest apoptotic cells (efferocytosis) in chronic obstructive pulmonary disease (COPD) and may be of interest in non-eosinophilic asthma (NEA) which is also characterised by impaired efferocytosis. It was hypothesised that the addition of exogenous galectin-3 to monocyte-derived macrophages (MDMs) derived from donors with NEA would enhance their ability to engulf apoptotic granulocytes. Methods Eligible non-smoking adults with asthma (n = 19), including 7 with NEA and healthy controls (n = 10) underwent a clinical assessment, venepuncture and sputum induction. MDMs were co-cultured with apoptotic granulocytes isolated from healthy donors with or without exogenous recombinant galectin-3 (50 μg/mL) and efferocytosis was assessed by flow cytometry. Galectin-3 expression and localisation in MDMs was visualised by immunofluorescence staining and fluorescence microscopy. Galectin-3, interleukin (IL)-6 and CXCL8 secretion were measured in cell culture supernatants by ELISA and cytometric bead array. Results Baseline efferocytosis (mean (±standard deviation)) was lower in participants with asthma (33.2 (±17.7)%) compared with healthy controls (45.3 (±15.9)%; p = 0.081). Efferocytosis did not differ between the participants with eosinophilic asthma (EA) (31.4 (±19.2)%) and NEA (28.7 (±21.5)%; p = 0.748). Addition of galectin-3 significantly improved efferocytosis in asthma, particularly in NEA (37.8 (±18.1)%) compared with baseline (30.4 (±19.7)%; p = 0.012). Efferocytosis was not associated with any of the clinical outcomes but was negatively correlated with sputum macrophage numbers (Spearman r = − 0.671; p = 0.017). Galectin-3 was diffusely distributed in most MDMs but formed punctate structures in 5% of MDMs. MDM galectin-3 secretion was lower in asthma (9.99 (2.67, 15.48) ng/mL) compared with the healthy controls (20.72 (11.28, 27.89) ng/mL; p = 0.044) while IL-6 and CXCL8 levels were similar. Conclusions Galectin-3 modulates macrophage function in asthma, indicating a potential role for galectin-3 to reverse impaired efferocytosis in NEA.

Thiamine (vitamin B1) is often deficient in oncopatients, particularly those undergoing chemotherapy. However, interaction between the thiamine deficiency and anticancer action of drugs has not been characterized. A major natural thiamine derivative, thiamine diphosphate (ThDP), is a coenzyme of central metabolism, also known to affect transcriptional activity of the master metabolic regulator and genome guardian p53. A direct transcriptional target of p53, p21, regulates cell cycle dynamics and DNA damage response. Our work focuses on dependence of the action of the DNA damaging anticancer drug cisplatin on metabolic regulation through p53/p21 axes and cellular thiamine status in human lung adenocarcinoma cells A549. These cells are used as a model of a hardly curable cancer, known to develop chemoresistance to platinum drugs, such as cisplatin. Compared to wild type (A549WT), a stable line with a 60% knockdown of p21 (A549p21–) is less sensitive to antiproliferative action of cisplatin. In contrast, in the thiamine-deficient medium, cisplatin impairs the viability of A549p21– cells more than that of A549WT cells. Analysis of the associated metabolic changes in the cells indicates that (i) p21 knockdown restricts the production of 2-oxoglutarate via glutamate oxidation, stimulating that within the tricarboxylic acid (TCA) cycle; (ii) cellular cisplatin sensitivity is associated with a 4-fold upregulation of glutamic-oxaloacetic transaminase (GOT2) by cisplatin; (iii) cellular cisplatin resistance is associated with a 2-fold upregulation of p53 by cisplatin. Correlation analysis of the p53 expression and enzymatic activities upon variations in cellular thiamine/ThDP levels indicates that p21 knockdown substitutes positive correlation of the p53 expression with the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) for that with the activity of glutamate dehydrogenase (GDH). The knockdown also changes correlations of the levels of OGDHC, GDH and GOT2 with those of the malate and isocitrate dehydrogenases. Thus, a p53/p21-dependent change in partitioning of the glutamate conversion to 2-oxoglutarate through GOT2 or GDH, linked to NAD(P)-dependent metabolism of 2-oxoglutarate in affiliated pathways, adapts A549 cells to thiamine deficiency or cisplatin treatment. Cellular thiamine deficiency may interfere with antiproliferative action of cisplatin due to their common modulation of the p53/p21-dependent metabolic switch between the glutamate oxidation and transamination.

Background: Gambling disorder is known to be associated with increased risk of suicidal behavior. However, relatively little is known about how the risk of suicide attempts in gambling disorder is influenced by comorbid alcohol or drug use disorders, as well as other psychiatric conditions.Methods: The present study is a nationwide, diagnostic register study assessing the risk of suicide attempts (including fatal ones) in gambling disorder in Sweden in 2005–2016.Results: In a total of 2,099 individuals (23 percent women) with gambling disorder, 417 individuals had a suicide attempt (including 10 fatal cases of suicide) during the study period. Suicidal behavior was more common in patients with substance use disorders at any time during the study period (50 percent if both alcohol and drug use disorders were present, and 10 percent if none of these were present). In logistic regression, suicidal behavior was significantly associated with female gender (OR 2.13 [1.63–2.78]), mood disorders (OR 2.65 [2.00–3.50]), anxiety disorders (OR 1.78 [1.34–2.35]), and with alcohol (OR 1.95 [1.51–2.51]) or drug use disorders (OR 3.60 [2.76–4.69]), respectively.Conclusions: Suicidal behavior in clinical gambling disorder patients is common, but markedly more common in the presence of substance use and other comorbid disorders.

Background:. Ulnar nerve entrapment at the elbow (UNE) is overrepresented in patients with diabetes, but the outcome of surgery is unknown. We aimed to evaluate patient-reported outcome in patients with and without diabetes, and to assess potential sex differences and compare surgical treatment methods. Methods:. Data on patients operated for UNE (2010–2016, n = 1354) from the Swedish National Registry for Hand Surgery were linked to the Swedish National Diabetes Register. Symptoms were assessed preoperatively (n = 389), and 3 (n = 283), and at 12 months postoperatively (n = 267) by QuickDASH and HQ-8 (specific hand surgery questionnaire—8 questions). Only simple decompressions were included when comparing groups. Results:. Men with diabetes reported higher postoperative QuickDASH scores than men without diabetes. Women scored their disability higher than men on all time-points in QuickDASH, but showed larger improvement between preoperative and 12 months postoperative values. Patients operated with transposition scored 10.8 points higher on QuickDASH than patients who had simple decompression at 12 months (95% confidence interval 1.98–19.6). Conclusions:. Women with diabetes benefit from simple decompression for UNE to the same extent as women without diabetes. Men with diabetes risk not to benefit from simple decompression as much as women do. Ulnar nerve transposition had a higher risk of residual symptoms compared to simple decompression.

Objectives: Mutation analysis by massive parallel sequencing (MPS) is routinely performed in the clinical management of lung cancer in Sweden. We describe the clinical and mutational profiles of lung cancer patients subjected to the first 1.5 years of treatment predictive MPS testing in an autonomous regional health care region. Methods: Tumors from all patients with lung cancer who had an MPS test from January 2015 to June 2016 in the Skåne health care region in Sweden (1.3 million citizens) were included. Six hundred eleven tumors from 599 patients were profiled using targeted sequencing with a 26-gene exon-focused panel. Data on disease patterns and characteristics of the patients subjected to testing were assembled, and correlations between mutational profiles and clinical features were analyzed. Results: MPS with the 26-gene panel revealed alterations in 92% of the 611 lung tumors, with the most frequent mutations detected in the nontargetable genes TP53 (62%) and KRAS (37%). Neither KRAS nor TP53 mutations were associated with disease pattern, chemotherapy response, progression-free survival, or overall survival in advanced-stage disease treated with platinum-based doublet chemotherapy as a first-line treatment. Among targetable genes, EGFR driver mutations were detected in 10% of the tumors, and BRAF p.V600 variants in 2.3%. For the 71 never smokers (12%), targetable alterations (EGFR mutations, BRAF p.V600, MET exon 14 skipping, or ALK/ROS1 rearrangement) were detected in 59% of the tumors. Conclusion: Although the increasing importance of MPS as a predictor of response to targeted therapies is indisputable, its role in prognostics or as a predictor of clinical course in nontargetable advanced stage lung cancer requires further investigation.

B‐cell translocation gene 2 (BTG2) is a tumour suppressor protein known to be downregulated in several types of cancer. In this study, we investigated a potential role for BTG2 in early‐stage non‐small cell lung cancer (NSCLC) survival. We analysed BTG2 methylation data from 1230 early‐stage NSCLC patients from five international cohorts, as well as gene expression data from 3038 lung cancer cases from multiple cohorts. Three CpG probes (cg01798157, cg06373167, cg23371584) that detected BTG2 hypermethylation in tumour tissues were associated with lower overall survival. The prognostic model based on methylation could distinguish patient survival in the four cohorts [hazard ratio (HR) range, 1.51–2.21] and the independent validation set (HR = 1.85). In the expression analysis, BTG2 expression was positively correlated with survival in each cohort (HR range, 0.28–0.68), which we confirmed with meta‐analysis (HR = 0.61, 95% CI 0.54–0.68). The three CpG probes were all negatively correlated with BTG2 expression. Importantly, an integrative model of BTG2 methylation, expression and clinical information showed better predictive ability in the training set and validation set. In conclusion, the methylation and integrated prognostic signatures based on BTG2 are stable and reliable biomarkers for early‐stage NSCLC. They may have new applications for appropriate clinical adjuvant trials and personalized treatments in the future.

Abstract Background Glutamate oxaloacetate transaminase 1 (GOT1) regulates cellular metabolism through coordinating the utilization of carbohydrates and amino acids to meet nutrient requirements. KRAS mutated cancer cells were recently shown to rely on GOT1 to support long-term cell proliferation. The aim of the present study was to address the role of GOT1 in the metabolic adaption of cancer cells. Methods GOT1-null and knockdown cell lines were established through CRISPR/Cas9 and shRNA techniques. The growth properties, colony formation ability, autophagy and selected gene expression profiles were analysed. Glucose deprivation decreased the viability of the GOT1-null cells and rescue experiments were conducted with selected intermediates. The redox NADH/NAD+ homeostasis as well as lactate secretion were determined. GOT1 expression levels and correlation with survival rates were analysed in selected tumor databases. Results Inhibition of GOT1 sensitized the cancer cells to glucose deprivation, which was partially counteracted by oxaloacetate and phosphoenol pyruvate, metabolic intermediates downstream of GOT1. Moreover, GOT1-null cells accumulated NADH and displayed a decreased ratio of NADH/NAD+ with nutrient depletion. The relevance of GOT1 as a potential target in cancer therapy was supported by a lung adenocarcinoma RNA-seq data set as well as the GEO:GSE database of metastatic melanoma where GOT1 expression was increased. High levels of GOT1 were further linked to poor survival as analysed by the GEPIA web tool, in thyroid and breast carcinoma and in lung adenocarcinoma. Conclusions Our study suggests an important role of GOT1 to coordinate the glycolytic and the oxidative phosphorylation pathways in KRAS mutated cancer cells. GOT1 is crucial to provide oxaloacetate at low glucose levels, likely to maintain the redox homeostasis. Our data suggest GOT1 as a possible target in cancer therapy.

Abstract Background KDM lysine demethylase family members are related to lung cancer clinical outcomes and are potential biomarkers for chemotherapeutics. However, little is known about epigenetic alterations in KDM genes and their roles in lung cancer survival. Methods Tumor tissue samples of 1230 early-stage non-small cell lung cancer (NSCLC) patients were collected from the five independent cohorts. The 393 methylation sites in KDM genes were extracted from epigenome-wide datasets and analyzed by weighted random forest (Ranger) in discovery phase and validation dataset, respectively. The variable importance scores (VIS) for the sites in top 5% of both discovery and validation sets were carried forward for Cox regression to further evaluate the association with patient’s overall survival. TCGA transcriptomic data were used to evaluate the correlation with the corresponding DNA methylation. Results DNA methylation at sites cg11637544 in KDM2A and cg26662347 in KDM1A were in the top 5% of VIS in both discovery phase and validation for squamous cell carcinomas (SCC), which were also significantly associated with SCC survival (HR cg11637544 = 1.32, 95%CI, 1.16–1.50, P = 1.1 × 10−4; HR cg26662347 = 1.88, 95%CI, 1.37–2.60, P = 3.7 × 10−3), and correlated with corresponding gene expression (cg11637544 for KDM2A, P = 1.3 × 10−10; cg26662347 for KDM1A P = 1.5 × 10−5). In addition, by using flexible criteria for Ranger analysis followed by survival classification tree analysis, we identified four clusters for adenocarcinomas and five clusters for squamous cell carcinomas which showed a considerable difference of clinical outcomes with statistical significance. Conclusions These findings highlight the association between somatic DNA methylation in KDM genes and early-stage NSCLC patient survival, which may reveal potential epigenetic therapeutic targets.

Rapid bone destruction often leads to permanent joint dysfunction in patients with septic arthritis, which is mainly caused by Staphylococcus aureus (S. aureus). Staphylococcal cell wall components are known to induce joint inflammation and bone destruction. Here, we show that a single intra-articular injection of S. aureus lipoproteins (Lpps) into mouse knee joints induced chronic destructive macroscopic arthritis through TLR2. Arthritis was characterized by rapid infiltration of neutrophils and monocytes. The arthritogenic effect was mediated mainly by macrophages/monocytes and partially via TNF-α but not by neutrophils. Surprisingly, a S. aureus mutant lacking Lpp diacylglyceryl transferase (lgt) caused more severe joint inflammation, which coincided with higher bacterial loads of the lgt mutant in local joints than those of its parental strain. Coinjection of pathogenic S. aureus LS-1 with staphylococcal Lpps into mouse knee joints caused improved bacterial elimination and diminished bone erosion. The protective effect of the Lpps was mediated by their lipid moiety and was fully dependent on TLR2 and neutrophils. The blocking of CXCR2 on neutrophils resulted in total abrogation of the protective effect of the Lpps. Our data demonstrate that S. aureus Lpps elicit innate immune responses, resulting in a double-edged effect. On the one hand, staphylococcal Lpps boost septic arthritis. On the other hand, Lpps act as adjuvants and activate innate immunity, which could be useful for combating infections with multiple drug-resistant strains.

Human neutrophils are filled with intracellular storage organelles, called granules and secretory vesicles, which differ in their content of soluble matrix proteins and membrane-bound molecules. To date, at least four distinct granule/vesicle subsets have been identified. These organelles may secrete their content extracellularly following mobilization to and fusion with the plasma membrane, but some of them may also fuse with internal membrane-enclosed organelles, typically a plasma membrane-derived phagosome. There are also instances where different granules appear to fuse with one another, a process that would enable mixing of their matrix and membrane components. Such granule fusion enables e.g., myeloperoxidase-processing of intragranular oxygen radicals, a key event in the formation of neutrophil extracellular traps (Björnsdottir et al., 2015) [1]. Described herein are data that show the quantification of such heterotypic granule–granule fusion by the use of imaging flow cytometry, a technique that combines flow cytometry with microscopy. The analysis described is based on immunofluorescent staining of established granule markers (lactoferrin and/or NGAL for one granule subset; the specific granules, and CD63 for another granule subset, the azurophil granules) and calculation of a colocalization score for resting and PMA-stimulated neutrophils.

Background: Psychiatric comorbidity is common in gambling disorder, a condition with low rates of treatment seeking. There is a paucity of documented nationwide data on gambling disorder and its co-occurring psychiatric comorbidities in the health care system.Methods: This is a nationwide register-based study of all patients aged above 18 years who were diagnosed with gambling disorder (corresponding to pathological gambling, code F63.0, in the ICD-10) in Swedish specialized out-patient health care or in-patient care, from 2005 through 2016. All psychiatric disorders co-occurring with the diagnoses were recorded, along with age, gender and the type of medical specialty.Results: A total of 2,099 patients were included (1,784 in out-patient care and 629 patients in in-patient care), among whom 77 percent were men. Treatment uptake during the study period increased significantly in out-patient care, with an increasing uptake of younger individuals, whereas in-patient treatment uptake remained stable. A co-occurring psychiatric diagnosis was registered in 73 percent of patients, more commonly in females (77 vs. 71 percent, p < 0.01). Several diagnostic subgroups were more common in women, with anxiety and affective disorders being the most common subgroups. Prevalence of substance use disorders did not differ with respect to gender.Conclusions: Despite a large gap between probable population prevalence of gambling disorder and the number of treated patients, the number of patients treated in out-patient health care with a gambling disorder diagnosis increased over time, with an increasing treatment uptake in younger individuals. Psychiatric comorbidity is common in gambling disorder patients in the health care system, with a higher prevalence in women.

Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD.

Autoinflammatory disease was introduced as a concept in 1999, demarcating an entirely new group of diseases in clinical, immunological, and conceptual terms. During recent years, the preconditions for the definition of autoinflammatory conditions have changed. This includes the recent discovery of a number of monogenic autoinflammatory conditions with complex phenotypes that combine autoinflammation with defects of the adaptive and/or innate immune system, resulting in the occurrence of infection, autoimmunity, and/or uncontrolled hyperinflammation in addition to autoinflammation. Further, there are strong indications that classical IL-1-driven autoinflammatory diseases are associated with activation of adaptive immunity. As suggested by this development, we are of the opinion that an all-encompassing definition of autoinflammatory diseases should regard autoinflammatory conditions and innate dysregulation as inseparable and integral parts of the immune system as a whole. Hence, in this article, we try to advance the conceptual understanding of autoinflammatory disease by, proposing a modification of the definition by Daniel Kastner et al., which allows for a congruent and precise description of conditions that expand the immunological spectrum of autoinflammatory disease.

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.

BACKGROUND:The entry of HIV into its host cell is an interesting target for chemotherapeutic intervention in the life-cycle of the virus. During entry, reduction of disulfide bridges in the viral envelope glycoprotein gp120 by cellular oxidoreductases is crucial. The cellular thioredoxin reductase-1 plays an important role in this oxidoreduction process by recycling electrons to thioredoxin-1. Therefore, thioredoxin reductase-1 inhibitors may inhibit gp120 reduction during HIV-1 entry. In this present study, tellurium-based thioredoxin reductase-1 inhibitors were investigated as potential inhibitors of HIV entry. RESULTS:The organotellurium compounds inhibited HIV-1 and HIV-2 replication in cell culture at low micromolar concentrations by targeting an early event in the viral infection cycle. Time-of-drug-addition studies pointed to virus entry as the drug target, more specifically: the organotellurium compound TE-2 showed a profile similar or close to that of the fusion inhibitor enfuvirtide (T-20). Surface plasmon resonance-based interaction studies revealed that the compounds do not directly interact with the HIV envelope glycoproteins gp120 and gp41, nor with soluble CD4, but instead, dose-dependently bind to thioredoxin reductase-1. By inhibiting the thioredoxin-1/thioredoxin reductase-1-directed oxidoreduction of gp120, the organotellurium compounds prevent conformational changes in the viral glycoprotein which are necessary during viral entry. CONCLUSION:Our findings revealed that thioredoxin-1/thioredoxin reductase-1 acts as a cellular target for the inhibition of HIV entry.

Inflammation induced by hypoxia–ischemia (HI) contributes to the development of injury in the newborn brain. In this study, we investigated the role of galectin-3, a novel inflammatory mediator, in the inflammatory response and development of brain injury in a mouse model for neonatal HI. Galectin-3 gene and protein expression was increased after injury and galectin-3 was located in activated microglia/macrophages. Galectin-3-deficient mice (gal3−/−) were protected from injury particularly in hippocampus and striatum. Microglia accumulation was increased in the gal3−/− mice but accompanied by decreased levels of total matrix metalloproteinase (MMP)-9 and nitrotyrosine. The protection and increase in microglial infiltration was more pronounced in male gal3−/− mice. Trophic factors and apoptotic markers did not significantly differ between groups. In conclusion, galectin-3 contributes to neonatal HI injury particularly in male mice. Our results indicate that galectin-3 exerts its effect by modulating the inflammatory response.

Background: Inflammation and reactive oxygen species (ROS) are important in the development of perinatal brain injury. The ROS-generating enzyme NADPH oxidase (Nox2) is present in inflammatory cells and contributes to brain injury in adult animal models. Hypothesis: NADPH oxidase contributes to ROS formation and development of injury in the immature brain and inhibition of NADPH oxidase attenuates perinatal brain injury. Methods: We used animal models of term hypoxia–ischemia (HI) (P9 mice) as well as ibotenate-induced excitotoxic injury (P5 mice) mimicking features of periventricular leukomalacia in preterm infants. In vitro microglia cell cultures were used to investigate NADPH oxidase-dependent ROS formation. In vivo we determined the impact 1) of HI on NADPH oxidase gene expression 2) of genetic (gp91-phox/Nox2 knock-out) and 3) of pharmacological NADPH oxidase inhibition on HI-induced injury and NMDA receptor-mediated excitotoxic injury, respectively. Endpoints were ROS formation, oxidative stress, apoptosis, inflammation and extent of injury. Results: Hypoxia–ischemia increased NADPH oxidase subunits mRNA expression in total brain tissue in vivo. In vitro ibotenate increased NADPH oxidase-dependent formation of reactive oxygen species in microglia. In vivo the inhibition of NADPH oxidase did not reduce the extent of brain injury in any of the animal models. In contrast, the injury was increased by inhibition of NADPH oxidase and genetic inhibition was associated with an increased level of galectin-3 and IL-1β. Conclusion: NADPH oxidase is upregulated after hypoxia–ischemia and activated microglia cells are a possible source of Nox2-derived ROS. In contrast to findings in adult brain, NADPH oxidase does not significantly contribute to the pathogenesis of perinatal brain injury. Results obtained in adult animals cannot be transferred to newborns and inhibition of NADPH oxidase should not be used in attempts to attenuate injury.

Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPRdes) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPRdes back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPRdes as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPRdes also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPRdes initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.

Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. In vitro, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites in vivo, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon in vivo transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue in vivo. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

BACKGROUND: CXC chemokines are induced by inflammatory stimuli in epithelial cells and some, like MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, are antibacterial for Streptococcus pyogenes. METHODOLOGY/PRINCIPAL FINDINGS: SpeB from S. pyogenes degrades a wide range of chemokines (i.e. IP10/CXCL10, I-TAC/CXCL11, PF4/CXCL4, GROalpha/CXCL1, GRObeta/CXCL2, GROgamma/CXCL3, ENA78/CXCL5, GCP-2/CXCL6, NAP-2/CXCL7, SDF-1/CXCL12, BCA-1/CXCL13, BRAK/CXCL14, SRPSOX/CXCL16, MIP-3alpha/CCL20, Lymphotactin/XCL1, and Fractalkine/CX3CL1), has no activity on IL-8/CXCL8 and RANTES/CCL5, partly degrades SRPSOX/CXCL16 and MIP-3alpha/CCL20, and releases a 6 kDa CXCL9 fragment. CXCL10 and CXCL11 loose receptor activating and antibacterial activities, while the CXCL9 fragment does not activate the receptor CXCR3 but retains its antibacterial activity. CONCLUSIONS/SIGNIFICANCE: SpeB destroys most of the signaling and antibacterial properties of chemokines expressed by an inflamed epithelium. The exception is CXCL9 that preserves its antibacterial activity after hydrolysis, emphasizing its role as a major antimicrobial on inflamed epithelium.

Purpose. Patients with soft tissue sarcomas (STS) are at increased risk of second primary malignancies, including a second STS, but distinction between metastases and a second primary STS is difficult. Patients and Methods. Array-based comparative genomic hybridization (aCGH) was applied to 30 multiple STS of the extremities and the trunk wall from 13 patients. Different histotypes were present with malignant fibrous histiocytomas/undifferentiated pleomorphic sarcomas being the predominant subtype. Results. aCGH profiling revealed genetic complexity with multiple gains and losses in all tumors. In an unsupervised hierarchical cluster analysis, similar genomic profiles and close clustering between the first and subsequent STS were identified in 5 cases, suggesting metastatic disease, whereas the tumors from the remaining 8 patients did not cluster and showed only weak pairwise correlation, suggesting development of second primary STS. Discussion. The similarities and dissimilarities identified in the first and second STS suggest that genetic profiles can be used to distinguish soft tissue metastases from second primary STS. The demonstration of genetically different soft tissue sarcomas in the same patient suggests independent tumor origin and serves as a reminder to consider development of second primary STS, which has prognostic and therapeutic implications.