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ObjectivePsychosocial interventions can reduce cancer-related fatigue effectively. However, it is still unclear if intervention effects differ across subgroups of patients. These meta-analyses aimed at evaluating moderator effects of (a) sociodemographic characteristics, (b) clinical characteristics, (c) baseline levels of fatigue and other symptoms, and (d) intervention-related characteristics on the effect of psychosocial interventions on cancer-related fatigue in patients with non-metastatic breast and prostate cancer.MethodsData were retrieved from the Predicting OptimaL cAncer RehabIlitation and Supportive care (POLARIS) consortium. Potential moderators were studied with meta-analyses of pooled individual patient data from 14 randomized controlled trials through linear mixed-effects models with interaction tests. The analyses were conducted separately in patients with breast (n = 1091) and prostate cancer (n = 1008).ResultsStatistically significant, small overall effects of psychosocial interventions on fatigue were found (breast cancer: β = -0.19 [95% confidence interval (95%CI) = -0.30; -0.08]; prostate cancer: β = -0.11 [95%CI = -0.21; -0.00]). In both patient groups, intervention effects did not differ significantly by sociodemographic or clinical characteristics, nor by baseline levels of fatigue or pain. For intervention-related moderators (only tested among women with breast cancer), statistically significant larger effects were found for cognitive behavioral therapy as intervention strategy (β = -0.27 [95%CI = -0.40; -0.15]), fatigue-specific interventions (β = -0.48 [95%CI = -0.79; -0.18]), and interventions that only targeted patients with clinically relevant fatigue (β = -0.85 [95%CI = -1.40; -0.30]).ConclusionsOur findings did not provide evidence that any selected demographic or clinical characteristic, or baseline levels of fatigue or pain, moderated effects of psychosocial interventions on fatigue. A specific focus on decreasing fatigue seems beneficial for patients with breast cancer with clinically relevant fatigue.

The literature on children’s active transportation has shown the influence of the built environment characteristics on walking and crashes. Various reviews have examined those two questions. One influence on walking is the perception of traffic safety. However, it is not clear how, or even if, the built environment affects such perceptions. This research aims to understand which traffic and built environment characteristics influence objective and subjective/perceived traffic safety for children based on the analysis of previous studies in the field. Two types of research were used: the first examines the association between traffic and built environment characteristics and child pedestrian and/or cyclist collisions/injuries; the second relates to the perception of safety by parents and children for active transportation and, where studied, its relationship with built environment characteristics. A systematic review was conducted using five electronic databases. The total number of articles retrieved was reduced to 38 following the eligibility criteria and quality assessment, where 25 articles relate to injuries among children and 13 articles pertain to perception of safety. The results showed that high traffic volume and high vehicle speed are the main reasons children and parents feel unsafe when children use active travel, which matches the main findings on objective safety. Few articles on perception of safety related to the objective built environment were found. However, consistent findings exist. The presence of sidewalk was related to the safety of children. The presence of a crossing guard was positively related to perceived safety but was associated with higher rates of injuries among children. Intersection density was related to unsafe perceptions but was not statistically associated with objective traffic safety. Additionally, population density was found to be positively related to injuries among children, but not to perception of safety. The results help policy strategy to enhance the safety of children when using active transport modes.Keywords: injury; perception of safety; children; active transportation; traffic; road design

BackgroundFood may trigger Attention-Deficit/Hyperactivity Disorder (ADHD) symptoms. Therefore, an elimination diet (ED) might be an effective treatment for children with ADHD. However, earlier studies were criticized for the nature of the control group, potential confounders explaining the observed effects, unsatisfactory blinding, potential risks of nutritional deficiencies and unknown long term and cost-effectiveness. To address these issues, this paper describes the rationale, study design and methods of an ongoing two arm randomized controlled trial (RCT) comparing the short (5 week) and long term (1 year) effects of an elimination diet and a healthy diet compared with care as usual (CAU) in children with ADHD.MethodsA total ofN = 162 children (5–12 years) with ADHD will be randomized to either an ED or a healthy diet. A comparator arm includingN = 60 children being solely treated with CAU (e.g. medication) is used to compare the effects found in both dietary groups. The two armed RCT is performed in two youth psychiatry centers in the Netherlands, with randomization within each participating center. The primary outcome measure is response to treatment defined as a ≥ 30% reduction on an ADHD DSM-5 rating scale (SWAN) and/or on an emotion dysregulation rating scale (SDQ: dysregulation profile). This is assessed after 5 weeks of dietary treatment, after which participants continue the diet or not. Secondary outcome measures include the Disruptive Behavior Diagnostic Observational Schedule (DB-DOS), parent and teacher ratings of comorbid symptoms, cognitive assessment (e.g. executive functions), school functioning, physical measurements (e.g. weight), motor activity, sleep pattern, food consumption, nutritional quality of the diet, adherence, parental wellbeing, use of health care resources and cost-effectiveness. Assessments take place at the start of the study (T0), after five weeks (T1), four months (T2), eight months (T3) and 12 months of treatment (T4). T0, T1 and T4 assessments take place at one of the psychiatric centers. T2 and T3 assessments consist of filling out online questionnaires by the parents only.DiscussionThis RCT will likely contribute significantly to clinical practice for ADHD by offering insight into the feasibility, nutritional quality, (cost-)effectiveness and long term effects of dietary treatments for ADHD.Trial registrationwww.trialregister.nl,NTR5434. Registered at October 11th, 2015.

Background The complexity of oesophageal surgery and the significant risk of morbidity necessitates that oesophagectomy is predominantly performed by a consultant surgeon, or a senior trainee under their supervision. The aim of this study was to determine the impact of trainee involvement in oesophagectomy on postoperative outcomes in an international multicentre setting. Methods Data from the multicentre Oesophago-Gastric Anastomosis Study Group (OGAA) cohort study were analysed, which comprised prospectively collected data from patients undergoing oesophagectomy for oesophageal cancer between April 2018 and December 2018. Procedures were grouped by the level of trainee involvement, and univariable and multivariable analyses were performed to compare patient outcomes across groups. Results Of 2232 oesophagectomies from 137 centres in 41 countries, trainees were involved in 29.1 per cent of them (n = 650), performing only the abdominal phase in 230, only the chest and/or neck phases in 130, and all phases in 315 procedures. For procedures with a chest anastomosis, those with trainee involvement had similar 90-day mortality, complication and reoperation rates to consultant-performed oesophagectomies (P = 0.451, P = 0.318, and P = 0.382, respectively), while anastomotic leak rates were significantly lower in the trainee groups (P = 0.030). Procedures with a neck anastomosis had equivalent complication, anastomotic leak, and reoperation rates (P = 0.150, P = 0.430, and P = 0.632, respectively) in trainee-involved versus consultant-performed oesophagectomies, with significantly lower 90-day mortality in the trainee groups (P = 0.005). Conclusion Trainee involvement was not found to be associated with significantly inferior postoperative outcomes for selected patients undergoing oesophagectomy. The results support continued supervised trainee involvement in oesophageal cancer surgery., The aim of this study was to determine the impact of trainee involvement in oesophagectomy on perioperative outcomes in the international multicentre Oesophago-Gastric Anastomosis Audit (OGAA). Analysis of 2232 oesophagectomies has shown that trainee involvement did not negatively impact perioperative outcomes.

Summary Human induced pluripotent stem cells (hiPSCs) are used to study organogenesis and model disease as well as being developed for regenerative medicine. Endothelial cells are among the many cell types differentiated from hiPSCs, but their maturation and stabilization fall short of that in adult endothelium. We examined whether shear stress alone or in combination with pericyte co-culture would induce flow alignment and maturation of hiPSC-derived endothelial cells (hiPSC-ECs) but found no effects comparable with those in primary microvascular ECs. In addition, hiPSC-ECs lacked a luminal glycocalyx, critical for vasculature homeostasis, shear stress sensing, and signaling. We noted, however, that hiPSC-ECs have dysfunctional mitochondrial permeability transition pores, resulting in reduced mitochondrial function and increased reactive oxygen species. Closure of these pores by cyclosporine A improved EC mitochondrial function but also restored the glycocalyx such that alignment to flow took place. These results indicated that mitochondrial maturation is required for proper hiPSC-EC functionality., Graphical Abstract, Highlights • hiPSC-ECs lack a functional glycocalyx and fail to align to flow • hiPSC-ECs have reduced mitochondrial function and increased leakage of ROS • Closing the mPTP with cyclosporine A induces mitochondrial maturation • Improved mitochondrial function restores the glycocalyx and alignment to flow, Closure of the mitochondrial permeability transition pore by cyclosporine A improved mitochondrial function and maturation of hiPSC-ECs but also restored the glycocalyx such that alignment to flow took place. This functional glycocalyx, necessary for growth factor signaling and anticoagulation, is a prerequisite for future hiPSC-EC applications in tissue engineering, organoid vascularization and therapeutic use of hiPSC-ECs.

Background The generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human kidney organoid protocols are available that generate organoids containing kidney structures. However, these kidney organoids are relatively small ranging up to 0.13 cm2 and therefore contain a small number of nephrons compared to an adult kidney, thus defying the exploration of future use for therapy. Method We have developed a scalable, easily accessible, and reproducible protocol to increase the size of the organoid up to a nephron sheet of 2.5 cm2 up to a maximum of 12.6 cm2 containing a magnitude of nephrons. Results Confocal microscopy showed that the subunits of the nephrons remain evenly distributed throughout the entire sheet and that these tissue sheets can attain ~ 30,000–40,000 glomerular structures. Upon transplantation in immunodeficient mice, such nephron sheets became vascularized and matured. They also show reuptake of injected low-molecular mass dextran molecules in the tubular structures, indicative of glomerular filtration. Furthermore, we developed a protocol for the cryopreservation of intermediate mesoderm cells during the differentiation and demonstrate that these cells can be successfully thawed and recovered to create such tissue sheets. Conclusion The scalability of the procedures, and the ability to cryopreserve the cells during differentiation are important steps forward in the translation of these differentiation protocols to future clinical applications such as transplantable auxiliary kidney tissue.

A common drawback of metabolic analyses of complex biological samples is the inability to consider cell-to-cell heterogeneity in the context of an organ or tissue. To overcome this limitation, we present an advanced high-spatial-resolution metabolomics approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) combined with isotope tracing. This method allows mapping of cell-type-specific dynamic changes in central carbon metabolism in the context of a complex heterogeneous tissue architecture, such as the kidney. Combined with multiplexed immunofluorescence staining, this method can detect metabolic changes and nutrient partitioning in targeted cell types, as demonstrated in a bilateral renal ischemia-reperfusion injury (bIRI) experimental model. Our approach enables us to identify region-specific metabolic perturbations associated with the lesion and throughout recovery, including unexpected metabolic anomalies in cells with an apparently normal phenotype in the recovery phase. These findings may be relevant to an understanding of the homeostatic capacity of the kidney microenvironment. In sum, this method allows us to achieve resolution at the single-cell level in situ and hence to interpret cell-type-specific metabolic dynamics in the context of structure and metabolism of neighboring cells.

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular, extracellular compositions and developmental plateau needs to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on a transwell filter. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.HIGHLIGHTSProlonging kidney organoid culture results in a developmental plateau instead of improved in vitro maturation.Proteomic analyses point to an increased expression of specific collagen subtypes associated with renal fibrosis.Encapsulating kidney organoids using a soft thiol-ene cross-linked alginate hydrogel reduces collagen type 1a1 and αSMA deposition.

Background and Objectives:Early identification of child mental health problems (MHPs) is important to provide adequate, timely treatment. Dutch preventive youth healthcare monitors all aspects of a child's healthy development. We explored the usefulness of their electronic health records (EHRs) in scientific research and aimed to develop prediction models for child MHPs.Methods:Population-based cohort study with anonymously extracted electronic healthcare data from preventive youth healthcare centers in the Leiden area, the Netherlands, from the period 2005–2015. Data was analyzed with respect to its continuity, percentage of cases and completeness. Logistic regression analyses were conducted to develop prediction models for the risk of a first recorded concern for MHPs in the next scheduled visit at age 3/4, 5/6, 10/11, and 13/14 years.Results:We included 26,492 children. The continuity of the data was low and the number of concerns for MHPs varied greatly. A large number of determinants had missing data for over 80% of the children. The discriminatory performance of the prediction models were poor.Conclusions:This is the first study exploring the usefulness of EHRs from Dutch preventive youth healthcare in research, especially in predicting child MHPs. We found the usefulness of the data to be limited and the performance of the developed prediction models was poor. When data quality can be improved, e.g., by facilitating accurate recording, or by data enrichment from other available sources, the analysis of EHRs might be helpful for better identification of child MHPs.

Obesity adversely affects health and is associated with subclinical systemic inflammation and features of accelerated aging, including the T-cell immune system. The presence of metabolic syndrome (MetS) may accelerate, while bariatric surgery might reverse these phenomena. To examine the effects of MetS and bariatric surgery on T-cell aging, we measured relative telomere length (RTL) and T-cell differentiation status in obese patients before and after bariatric surgery. WHO II/III classified obese patients scheduled for bariatric surgery were included: 41 without MetS and 67 with MetS. RTL and T-cell differentiation status were measured in circulating CD4+ and CD8+ T cells via flow cytometry. T-cell characteristics were compared between patients with and without MetS prior to and at 3, 6, and 12 months after surgery considering effects of age, cytomegalovirus-serostatus, and weight loss. Thymic output, represented by numbers of CD31-expressing naive T cells, showed an age-related decline in patients with MetS. MetS significantly enhanced CD8+ T-cell differentiation. Patients with MetS had significant lower CD4+ RTL than patients without MetS. Within the first 6 months after bariatric surgery, RTL increased in CD4+ T cells after which it decreased at month 12. A decline in both thymic output and more differentiated T cells was seen following bariatric surgery, more pronounced in the MetS group and showing an association with percentage of body weight loss. In obese patients, MetS results in attrition of RTL and accelerated T-cell differentiation. Bariatric surgery temporarily reverses these effects. These data suggest that MetS is a risk factor for accelerated aging of T cells and that MetS should be a more prominent factor in the decision making for eligibility for bariatric surgery.

Generational differences in health-related factors will affect forecasts of future disease patterns and health care needs. We studied whether participation in sports activity and being physically active are different between 10-year generations over a part of their life course. We used three different datasets on Dutch adults: a cohort study running for 20 years, the Doetinchem Cohort Study (DCS), multiple yearly databases (2001-2015) from the Netherlands health interview study (HIS), and a retrospective cohort study on life time sports careers, the Sports Participation Monitor (SPM). Based on a different questionnaire in each study, frequencies of weekly sport participation and being physically active according to recommended levels were determined by generation and sex. All data sets showed that self-reported sport participation has been increasing with every 10-year generation already for many decades. Especially for those generations born in the 1930s up to the 1960s, sport participation is higher compared to their predecessors. For instance at age 50, 43% of those born in the 1940s engaged in sports activities compared to 55% of those born in the 1950s (DCS data). Physical activity according to recommended levels showed no systematic differences by generation. In conclusion, favourable generation-specific trends in participation in sports activities are found: recent generations are doing better than the older generations. It is unclear whether this also reflects higher levels of physical activity or lower levels of inactivity. Future research may reveal whether these developments also hold for other countries, and whether these developments continue for the younger generations.

ObjectivesTo investigate associations of job demands and resources with patient-related burnout among physicians.DesignMulticentre observational study.SettingFifty medical departments at 14 (academic and non-academic) hospitals in the Netherlands.ParticipantsFour hundred sixty-five physicians (71.6% response rate), comprising 385 (82.8%) medical specialists and 80 (17.2%) residents.Main outcome measuresJob demands (workload and bureaucratic demands), job resources (participation in decision making, development opportunities, leader’s inspiration, relationships with colleagues and patients)—measured with the validated Questionnaire of Experience and Evaluation of Work and Physician Worklife Survey—and patient-related burnout, measured using the validated Copenhagen Burnout Inventory.ResultsPatient-related burnout was positively associated with workload (b=0.36; 95% CI, 0.25 to 0.48; pConclusionsPhysicians with high workloads and few development opportunities reported higher levels of patient-related burnout. Those with positive patient relationships were less likely to experience patient-related burnout, even in the presence of excessive bureaucracy. Therefore, positive physician–patient relationships may be supported to reduce the likelihood of physicians’ patient-related burnout. However, the specific support needed to effectively reduce patient-related burnout may vary per healthcare context and thus requires intensified research across healthcare systems and settings.

Centromeres are chromatin domains maintained by a self‐templating feedback loop based on nucleosomes bearing the histone H3 variant CENP‐A. The underlying centromeric DNA sequence is largely dispensable, yet paradoxically, it has highly conserved features. Hoffmann et al (2020) now uncover that when the epigenetic chromatin cycle falters, a genetically hardwired mechanism offers robustness to a dynamic epigenetic feedback loop ensuring long‐term centromere inheritance.

Differentiation of human‐induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial‐like cells (hiPSC‐derived endothelial cells [hiPSC‐ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC‐ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel‐Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC‐ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC‐ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC‐ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC‐ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC‐EC. Mechanistically, pro‐VWF dimers require the lower pH environment of the trans‐Golgi network for maturation and tubulation. These data show that while hiPSC‐ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function., Formation of mature Weibel‐Palade bodies in human‐induced pluripotent stem cell‐derived endothelial cells is limited by the increased intracellular pH (pHi) around the Golgi apparatus and trans‐Golgi network. This increase in pHi is caused by reduced intracellular lactate accompanied by reduced H+, caused by reduced glycolysis, and reduced the uptake of lactate and H+ via monocarboxylate transporter member 1.

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling., CENP-A is a stable centromere mark, although active transcription poses a potential threat for retaining CENP-A through chromatin remodeling and nucleosome eviction. Here, the authors show that maintenance of the centromeric mark is preserved by Spt6, which recycles CENP-A nucleosomes.

Contains fulltext : 225951.pdf (Publisher’s version ) (Closed access) PURPOSE: This study evaluated the effects of coping skills training (CST) on symptoms of depression and anxiety in cancer patients, and investigated moderators of the effects. METHODS: Overall effects and intervention-related moderators were studied in meta-analyses of pooled aggregate data from 38 randomized controlled trials (RCTs). Patient-related moderators were examined using linear mixed-effect models with interaction tests on pooled individual patient data (n = 1953) from 15 of the RCTs. RESULTS: CST had a statistically significant but small effect on depression (g = -0.31,95% confidence interval (CI) = -0.40;-0.22) and anxiety (g = -0.32,95%CI = -0.41;-0.24) symptoms. Effects on depression symptoms were significantly larger for interventions delivered face-to-face (p = .003), led by a psychologist (p = .02) and targeted to patients with psychological distress (p = .002). Significantly larger reductions in anxiety symptoms were found in younger patients (p(interaction)

Recently, new physical activity (PA) guidelines were adopted in the Netherlands consisting of two components: (1) addressing duration of moderate and vigorous PA, (2) bone and muscle strengthening activities. The aim of this study is to retrospectively assess the long-term trend in fulfilling the criteria of the new PA guidelines and to gain insight into which activities contribute to changes over time. Data were available for 2001&ndash, 2018 of a nationally representative sample of approximately 7000 Dutch citizens aged 12 years and over using the Short Questionnaire to Assess Health-enhancing physical activity (SQUASH). Multiple logistic regression analysis was performed by age, sex, and level of education. Overall, a positive trend was found from 39.9% adherence in 2001 to 46.0% in 2018. Adherence levels among adolescents decreased and increased among adults and seniors. Intermediate and higher educated groups showed positive trends over time whereas a stable trend was observed among lower educated. Activities contributing most to changes over time were sports, leisure time walking, and strenuous occupational activities. In the period 2001&ndash, 2018, though an increasing trend was found, less than half of the population was sufficiently active. Special effort is necessary to reach adolescents, seniors, and lower educated groups in PA promotion programs.

Kidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great potential for disease modeling, drug screening, and possibly future therapeutic application. At the same time, use of these PSC-derived organoids is hampered by lack of maturation and off-target differentiation. Here, we review the main protocols for the generation of kidney organoids from human-induced PSCs, discussing their advantages and limitations. In particular, we will focus on the vascularization of the kidney organoids, which appears to be one of the critical factors to achieve maturation and functionality of the organoids.

Bothrops lanceolatussnake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability ofB. lanceolatusvenom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show thatB. lanceolatusvenom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations thatB. lanceolatusvenom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, furtherin vivostudies may support the idea that therapeutic management of systemicB. lanceolatusenvenomation could include the use of Complement inhibitors as adjunct therapy.

Higher-educated people often have healthier diets, but it is unclear whether specific dietary patterns exist within educational groups. We therefore aimed to derive dietary patterns in the total population and by educational level and to investigate whether these patterns differed in their composition and associations with the incidence of fatal and non-fatal CHD and stroke. Patterns were derived using principal components analysis in 36 418 participants of the European Prospective Investigation into Cancer and Nutrition-Netherlands cohort. Self-reported educational level was used to create three educational groups. Dietary intake was estimated using a validated semi-quantitative FFQ. Hazard ratios were estimated using Cox Proportional Hazard analysis after a mean follow-up of 16 years. In the three educational groups, similar ‘Western’, ‘prudent’ and ‘traditional’ patterns were derived as in the total population. However, with higher educational level a lower population-derived score for the ‘Western’ and ‘traditional’ patterns and a higher score on the ‘prudent’ pattern were observed. These differences in distribution of the factor scores illustrate the association between education and food consumption. After adjustments, no differences in associations between population-derived dietary patterns and the incidence of CHD or stroke were found between the educational groups (Pinteraction between 0·21 and 0·98). In conclusion, although in general population and educational groups-derived dietary patterns did not differ, small differences between educational groups existed in the consumption of food groups in participants considered adherent to the population-derived patterns (Q4). This did not result in different associations with incident CHD or stroke between educational groups.

Summary Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids., Graphical Abstract, Highlights • PSC-derived kidney organoids remain disorganized and immature upon prolonged culture • Renal subcapsular transplantation in mice induces vascularization of kidney organoids • Intravital imaging shows perfusion of glomeruli and pre-existing vascular networks • Subcapsular transplantation results in progressive maturation of nephron structures, In this article, Van den Berg and colleagues show that PSC-derived kidney organoids contain nephron structures but remain disorganized and immature after prolonged culture. Upon transplantation, the organoids develop host-derived vascularization, functional glomerular perfusion, and connection to pre-existing vascular networks. The authors conclude that patent vasculature is required for ongoing morphogenesis and maturation of these kidney organoids.

Failed laparoscopic adjustable gastric banding (LAGB) can be converted to laparoscopic Roux-and-Y gastric bypass (LRYGB), which is currently the gold standard for bariatric surgery. Revisional LRYGB (rLRYGB) is associated with inferior results compared with primary LRYGB (pLRYGB), but the exact influence of the initial response to LAGB is unclear.To compare follow-up outcomes after pLRYGB with rLRYGB in nonresponders of LAGB and rLRYGB in responders of LAGB.General-community teaching hospital, Rotterdam, the Netherlands.All patients who underwent pLRYGB and rLRYGB after LAGB were reviewed in an observational study. Postoperative outcomes, excess weight loss, total weight loss, and success and failure rate were compared in patients after pLRYGB and rLRYGB (both responders and nonresponders of LAGB) at 12, 24, and 36 months.A total of 1285 primary patients, 96 nonresponders, and 120 responders were included. The median follow-up was 33.9±18.0 months. After 36 months, the mean percentage excess weight loss was significantly lower in the nonresponding group compared with the responding and primary groups (48.1% versus 58.2% versus 72.8%, P.001); the total weight loss showed the same trend. The success rate was 38.2% versus 61.0% versus 81.6% respectively, P.001. The failure rate was significantly higher after rLRYGB compared with pLRYGB (10.9% nonresponders, 8.5% responders, and 2.5% primary, P = .001).Nonresponders of LAGB show inferior weight loss results after rLRYGB compared with responders of LAGB and pLRYGB at all moments of follow-up.

CONTEXT Social support helps prevent burnout and promotes its positive opposite, work engagement. With higher work engagement performance increases. The context-specific aspects of social support for medical educators, in their educator role, are unknown. To help facilitate social support our study describes the essential elements of social support and their meaning for medical educators.METHODS We held interviews with medical educators purposefully sampled for diverse backgrounds and working circumstances and who spent a considerable amount of time on education. Both clinicians and basic scientists participated. The Pictor technique guided the interviews. Participants were invited to talk about the breadth of social support and elaborate on meaningful experiences. Template analysis was used for a descriptive phenomenological approach.RESULTS Thirteen medical educators were interviewed. We identified four themes: (i) sources of support and their intent (e.g. a superior with the intent to stimulate personal growth); (ii) the materialisation of support (e.g. sought or offered); (iii) its manifestation (e.g. the act of providing protected time); and (iv) the overarching effect of social support, both in terms of practical effects and the meaning of support. We identified three sorts of meanings of social support for educators. Receiving support could lead to (i) feeling reassured and confident; (ii) feeling encouraged and determined and (iii) a sense of relatedness and acknowledgement of the educator role.CONCLUSION Support for education comes from a wide range of sources because it is both sought and offered beyond the boundaries of the educational role. The resulting differences in support provided necessitate that educational leaders and policymakers consider the sources available to each educator, connecting educators where necessary. When facilitating or designing social support it is important that the need to feel reassured, encouraged or related is met.

We present two patients who were treated for an intentional overdose of sodium nitrite. When ingested sodium nitrite leads to severe methaemoglobinaemia, resulting in severe hypoxia (as methaemoglobin does not transport oxygen), vasodilation and hypotension. Symptoms include cyanosis, headache, nausea, convulsions, coma and death. When measured by pulse oximetry, patients with a sodium nitrite intoxication and severe methaemoglobinaemia generally have an oxygen saturation of around 85%. This value is unreliable as the oxygen content of the blood is often extremely low - this can be confirmed by arterial blood gas analysis. Treatment of sodium nitrite intoxication consists of intravenous administration of methylthioninium chloride 1-2 mg/kg. Methylthioninium chloride converts the methaemoglobin back to haemoglobin. Due to the pharmacokinetics of methylthioninium chloride and sodium nitrite, a rebound effect is not to be expected. The only contra-indication for methylthioninium chloride is glucose-6-phosphate dehydrogenase deficiency, which is extremely rare in the Netherlands.

Loxosceles spiders’ venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 µM and 0.59 µM, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 µM. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re‐endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re‐endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell –derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re‐endothelialized scaffold could, in contrast to the non‐re‐endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney., Human decellularized kidney scaffolds can be functionally re‐endothelialized using expanded human pluripotent stem cell–derived endothelial cells.

A 49-year-old man came to the emergency room with abdominal pain, vomiting, diarrhoea and rectal bleeding. Both colonoscopy and CT of the abdomen showed cecocolic intussusception caused by an appendiceal mucocele. An ileocecal resection was performed via a laparoscopic approach and microscopy of the tissue showed a low-grade mucinous neoplasm.

Background High levels of work engagement protect against burnout. This can be supported through the work environment and by faculty themselves when they try to improve their work environment. As a result, they can become more engaged and better performers. We studied the relationship between adaptations by physicians to improve their teaching work environment, known as job crafting, and their energy levels, or work engagement, in their work as care provider and teacher. Job crafting encompasses seeking social (i) and structural (ii) resources and challenges (iii) and avoiding hindrances (iv). Methods We established a cross-sectional questionnaire survey in a cohort of physicians participating in classroom and clinical teaching. Job crafting and work engagement were measured separately for physicians’ clinical and teaching activities. We analyzed our data using structural equation modelling controlling for age, gender, perceived levels of autonomy and participation in decision making. Results 383 physicians were included. Physicians’ work engagement for patient care was negatively associated with two job crafting behaviors in the teaching roles: seeking structural resources (classroom teaching: ß = − 0.220 [95% CI: -0.319 to − 0.129]; clinical teaching: ß = − 0.148 [95% CI: -0.255 to − 0.042]); seeking challenges (classroom teaching: ß = − 0.215 [95% CI: -0.317 to − 0.113]; clinical teaching:, ß = − 0.190 [95% CI: -0.319 to − 0.061]). Seeking social resources and avoiding hindrances were unaffected by physicians’ work engagement for patient care. Conclusions High engagement for teaching leads to job crafting in teaching. High engagement for patient care does not lead to job crafting in teaching. Electronic supplementary material The online version of this article (10.1186/s12909-018-1411-z) contains supplementary material, which is available to authorized users.

Work engagement deserves more attention in health professions education because of its positive relations with personal well-being and performance at work. For health professions education, these outcomes have been studied on various levels. Consider engaged clinical teachers, who are seen as better clinical teachers; consider engaged residents, who report committing fewer medical errors than less engaged peers. Many topics in health professions education can benefit from explicitly including work engagement as an intended outcome such as faculty development programs, feedback provision and teacher recognition. In addition, interventions aimed at strengthening resources could provide teachers with a solid foundation for well-being and performance in all their work roles. Work engagement is conceptually linked to burnout. An important model that underlies both burnout and work engagement literature is the job demands-resources (JD-R) model. This model can be used to describe relationships between work characteristics, personal characteristics and well-being and performance at work. We explain how using this model helps identifying aspects of teaching that foster well-being and how it paves the way for interventions which aim to increase teacher's well-being and performance.

Objectives The importance of self-regulated learning (SRL) has been broadly recognised by medical education institutions and regulatory bodies. Supporting the development of SRL skills has proven difficult because self-regulation is a complex interactive process and we know relatively little about the factors influencing this process in real practice settings. The aim of our study was therefore to identify factors that support or hamper medical students' SRL in a clinical context. MethodsWe conducted a constructivist grounded theory study using semi-structured interviews with 17 medical students from two universities enrolled in clerkships. Participants were purposively sampled to ensure variety in age, gender, experience and current clerkship. The Day Reconstruction Method was used to help participants remember their activities of the previous day. The interviews were transcribed verbatim and analysed iteratively using constant comparison and open, axial and interpretive coding. ResultsSelf-regulated learning by students in the clinical environment was influenced by the specific goals perceived by students, the autonomy they experienced, the learning opportunities they were given or created themselves, and the anticipated outcomes of an activity. All of these factors were affected by personal, contextual and social attributes. ConclusionsSelf-regulated learning of medical students in the clinical environment is different for every individual. The factors influencing this process are affected by personal, social and contextual attributes. Some of these are similar to those known from previous research in classroom settings, but others are unique to the clinical environment and include the facilities available, the role of patients, and social relationships pertaining to peers and other hospital staff. To better support students' SRL, we believe it is important to increase students' metacognitive awareness and to offer students more tailored learning opportunities. Discuss ideas arising from the article at discuss'.

Envenomation by Loxosceles spiders can result in severe systemic and local reactions, which are mainly triggered by Sphingomyelinase D (SMase D), a toxic component of Loxosceles venom. SMase D induces a systemic inflammatory condition similar to the reaction observed during an endotoxic shock. Considering the potent pro-inflammatory potential of Loxosceles venom and the SMase D, in this study we have used the whole human blood model to study the endotoxic-like shock triggered by SMase D. Recombinant purified SMase D from L. intermedia venom, similarly to LPS, induced activation of blood leukocytes, as observed by the increase in the expression of CD11b and TLR4, production of reactive oxygen and nitrogen species (superoxide anion and peroxynitrite) and release of TNF-α. Complement consumption in the plasma was also detected, and complement inhibition by compstatin decreased the SMase D and LPS-induced leukocyte activation, as demonstrated by a reduction in the expression of CD11b and TLR4 and superoxide anion production. Similar results were found for the L. intermedia venom, except for the production of TNF-α. These findings indicate that SMase D present in Loxosceles venom is able to activate leukocytes in a partially complement-dependent manner, which can contribute to the systemic inflammation that follows envenomation by this spider. Thus, future therapeutic management of systemic Loxosceles envenomation could include the use of complement inhibitors as adjunct therapy.

© 2018 The Authors. Psycho-Oncology Published by John Wiley & Sons Ltd. Objective: This individual patient data (IPD) meta-analysis aimed to evaluate the effects of psychosocial interventions (PSI) on quality of life (QoL), emotional function (EF), and social function (SF) in patients with cancer, and to study moderator effects of demographic, clinical, personal, and intervention-related characteristics. Methods: Relevant studies were identified via literature searches in 4 databases. We pooled IPD from 22 (n = 4217) of 61 eligible randomized controlled trials. Linear mixed-effect model analyses were used to study intervention effects on the post-intervention values of QoL, EF, and SF (z-scores), adjusting for baseline values, age, and cancer type. We studied moderator effects by testing interactions with the intervention for demographic, clinical, personal, and intervention-related characteristics, and conducted subsequent stratified analyses for significant moderator variables.Results: PSI significantly improved QoL (β = 0.14,95%CI = 0.06;0.21), EF (β = 0.13,95%CI = 0.05;0.20), and SF (β = 0.10,95%CI = 0.03;0.18). Significant differences in effects of different types of PSI were found, with largest effects of psychotherapy. The effects of coping skills training were moderated by age, treatment type, and targeted interventions. Effects of psychotherapy on EF may be moderated by cancer type, but these analyses were based on 2 randomized controlled trials with small sample sizes of some cancer types. Conclusions: PSI significantly improved QoL, EF, and SF, with small overall effects. However, the effects differed by several demographic, clinical, personal, and intervention-related characteristics. Our study highlights the beneficial effects of coping skills training in patients treated with chemotherapy, the importance of targeted interventions, and the need of developing interventions tailored to the specific needs of elderly patients.

Background Patients with lymphoma are at risk of experiencing adverse physical and psychosocial problems from their cancer and its treatment. Regular screening of these symptoms by the use of patient-reported outcomes (PROs) could increase timely recognition and adequate symptom management. Moreover, self-management interventions intend to enhance knowledge and skills and empower patients to better manage their disease and related problems. The objective of the Lymphoma InterVEntion (LIVE) trial is to examine whether feedback to patients on their PROs and access to a web-based, self-management intervention named Living with lymphoma will increase self-management skills and satisfaction with information, and reduce psychological distress. Methods/design The LIVE randomised controlled trial consists of three arms: (1) standard care, (2) PRO feedback, and (3) PRO feedback and the Living with lymphoma intervention. Patients who have been diagnosed with Hodgkin lymphoma, non-Hodgkin lymphoma, including chronic lymphocytic leukaemia, as registered in the Netherlands Cancer Registry in various hospitals will be selected for participation. Patients are invited via their haemato-oncologist 6 to 15 months after diagnosis. The PRO feedback includes a graphical overview of patients’ own symptom and functioning scores and an option to compare their scores with those of other patients with lymphoma and a normative population of the same age and sex. The Living with lymphoma intervention is based on cognitive behavioural therapy components and includes information, assignments, assessments, and videos. Changes in outcomes from baseline to 16 weeks, 12, and 24 months post intervention will be measured. Primary outcomes are self-management skills, satisfaction with information, and psychological distress. Secondary outcomes are health-related quality of life, illness perceptions, fatigue, and health care use. Discussion/design The results of the LIVE trial will provide novel insights into whether access to PRO feedback and the Living with lymphoma intervention will be effective in increasing self-management skills and satisfaction with information, and reducing distress. The LIVE trial is embedded in a population-based registry, which provides a unique setting to ascertain information on response, uptake, and characteristics of patients with lymphoma in web-based intervention(s). When effective, PRO feedback and Living with lymphoma could serve as easily and widely accessible interventions for coping with lymphoma. Trial registration Netherlands Trial Register, identifier NTR5953. Registered on 14 July 2016. Electronic supplementary material The online version of this article (doi:10.1186/s13063-017-1943-2) contains supplementary material, which is available to authorized users.

Item does not contain fulltext BACKGROUND: In 9% to 34% of cancer patients, the fear of cancer recurrence becomes so overwhelming that it affects quality of life. Clinicians need a brief questionnaire with a cutoff point that is able to differentiate between high- and low-fearful survivors. OBJECTIVE: This study investigated if the Cancer Worry Scale (CWS) could serve as an instrument to detect high levels of fear of recurrence in female breast cancer survivors. METHODS: One hundred ninety-four female breast cancer patients were assessed up to 11 years after their primary treatment for cancer. The women returned the questionnaires including the 8-item CWS, 2 items of the Cancer Acceptance Scale, the Checklist Individual Strength-Fatigue subscale, and the Cancer Empowerment Questionnaire. RESULTS: A cutoff score of 13 versus 14 (low: /=14) on the CWS was optimal for detecting severe levels of fear of recurrence. A cutoff score of 11 versus 12 (low: /=12) was optimal for screening. The Cronbach alpha coefficient of the CWS was .87; evidence to support the convergent and divergent validity of the CWS was also obtained. The CWS is able to detect high levels of fear of recurrence. CONCLUSION: The CWS is a reliable and valid questionnaire to assess fear of recurrence in breast cancer survivors. IMPLICATIONS FOR PRACTICE: With the CWS, it is possible for nurses to screen breast cancer survivors for severe levels of fear of cancer recurrence. Thereby, nurses can screen and assist survivors in accessing appropriate and available support.

BACKGROUND The difficulties adolescents and young adults (AYAs) encounter during a cancer experience may result in a reduction in or absence of empowerment. The aims of the current study were to assess levels of empowerment and associated (demographic, clinical, or psychological) factors and examine the association between empowerment and health‐related quality of life (HRQOL) among AYA patients with cancer. METHODS Patients aged 18 to 35 years at time of cancer diagnosis and who were seen by 1 of the members of the specialized multidisciplinary AYA team of the Radboud University Medical Center were invited to complete questionnaires regarding empowerment; HRQOL; and sociodemographic, clinical, and psychological characteristics (autonomy, coping, unmet social support needs, and psychological distress). RESULTS A total of 83 AYA patients completed the questionnaires. The mean age of the participants at the time of diagnosis was 27.5 years. The vast majority had been treated with chemotherapy (86%), had a more advanced stage of disease, and had completed treatment at the time of participation (74%). The mean empowerment level was 154.1 (standard deviation, 17.8) with a range of 114 to 200. Multivariate analysis demonstrated that the autonomy subscales of self‐awareness (β = .35), capacity for managing new situations (β = .19), and social support (β = .35) were found to be positively associated with empowerment. Coping difficulties (β = ‐.19) were found to be negatively associated with empowerment. Empowerment was independently associated with physical (β = .31), psychological (β = .50), social (β = .39), religious (β = .33), and total HRQOL (β = .52; all P, Empowerment is positively associated with health‐related quality of life in adolescent and young adult patients with cancer. Interventions aiming at empowering these patients can improve their health‐related quality of life.

Background Dilation is the standard of care for recurrent benign esophageal strictures (BES). Biodegradable stents may prolong the effect of dilation and reduce recurrences. Efficacy and safety of dilation and biodegradable stent placement early in the treatment algorithm of recurrent BES were compared. Methods This multicenter, randomized study enrolled patients with BES treated with previous dilations to ≥ 16 mm. The primary end point was number of repeat endoscopic dilations for recurrent stricture within 3 and 6 months. Secondary outcomes through 12 months included safety, time to first dilation for recurrent stricture, dysphagia, and level of activity. Results At 3 months, the biodegradable stent group (n = 32) underwent significantly fewer endoscopic dilations for recurrent stricture compared with the dilation group (n = 34; P Conclusion Biodegradable stent placement is associated with temporary reduction in number of repeat dilations and prolonged time to recurrent dysphagia compared with dilation. Additional studies are needed to better define the exact role of biodegradable stent placement to treat recurrent BES.

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

BACKGROUND AND STUDY AIMS Enteral stent placement has emerged as a safe and effective palliative treatment option for patients with malignant gastric outlet obstruction (GOO). In an attempt to further optimize this treatment new enteral stents have been designed. This study is the first to describe the results regarding technical success, clinical success, complication rate, and stent dysfunction of the Evolution duodenal stent (Cook Medical, Limerick, Ireland). PATIENTS AND METHODS A total of 46 patients with symptomatic malignant GOO were included in this prospective multicenter cohort study. All patients who successfully received an Evolution duodenal stent were followed until death. RESULTS The technical and clinical success rates were 89 % (95 % confidence interval [CI] 77 % - 95 %) and 72 % (95 %CI 58 % - 83 %), respectively. The GOO Scoring System score, the Global Health Score, and the EuroQol visual analog scale improved significantly (GOOSS and Global Health Score P < 0.0001; EuroQol P = 0.005) when scores before stenting were compared with scores after stent placement. Median survival was 87 days, and stent patency was observed in 67 % for up to 395 days, accounting for death unrelated to the stent as a competing risk. Stent dysfunction occurred in 14 patients (30 %) (stent ingrowth n = 9; stent migration n = 2; extrinsic compression on the stent n = 2; food impaction n = 1). CONCLUSION These first data on the new Evolution duodenal stent show that it is safe and effective for the palliative treatment of symptomatic malignant GOO.

Human erythrocyte CD59 contains N- and O-glycans and a glycosylphosphatidylinositol (GPI) anchor, all of which have been analyzed in this study. The anchor consists principally of the minimum core glycan sequence Manalpha1-2Manalpha1-6Manalpha1-4GlcN-linked to a phosphatidylinositol moiety with the structure sn-1-O-alkyl(C18:0 and C18:1)-2-O-acyl(C20:4)glycerol-3-phospho-1-(2-O-palmitoyl(C16:0))myo- inositol. This structure is essentially identical to that of human erythrocyte cholinesterase (Deeg, M. A., Humphrey, D. R., Yang, S. H. , Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573-18580). This first comparison of GPI anchors from different proteins expressed in the same tissue suggests that human reticulocytes produce only one type of anchor structure. The N- and O-glycans were sequenced using a novel approach involving digestion of the total glycan pool with multiple enzyme arrays. The N-glycan pool contained families of bi-antennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. The predominant O-glycans were NeuNAcalpha2-3Galbeta1-3GalNAc and Galbeta1-3[NeuNAcalpha2-3]GalNAc. Inspection of a molecular model of CD59, based on the NMR solution structure of the extracellular domain and the structural data from this study, suggested several roles for the glycans, including spacing and orienting CD59 on the cell surface and protecting the molecule from proteases. This work completes the initial structural analysis of CD59, providing the most complete view of any cell surface glycoprotein studied to date.

The C5a receptor (C5aR) is a 7 transmembrane G-protein coupled receptor (GPCR) that mediates the powerful pro-inflammatory effect of the complement activation product C5a. Excess C5a generated under pathological conditions has been implicated in a variety of conditions including sepsis, asthma and rheumatoid arthritis, but very little is known about the regulation of expression of the C5aR. The 5′ promoter region and 3′ untranslated region (UTR) of the C5aR mRNA were cloned, generating enhanced green fluorescent protein (EGFP)-reporter plasmids, which were transfected into the monocytic cell line U937. Most of the cloned 2 kb 5′ region was dispensable for the expression of the reporter constructs and the majority of regulatory sequences are in the first 200 bp. Three motifs, a NFκB, a CCAAT and a NFAT site, were identified to be of importance by site directed mutagenesis for basal expression. Analysis of the 3′UTR of the C5aR mRNA showed that it contained two AU-rich elements (AREs), however site directed mutagenesis showed that these had no effect on basal expression. While the phorbol ester PMA and dibutyryl cAMP increased C5aR protein expression, these agents had no effect on the regulation of expression via the promoter or the 3′UTR. This is the first study to investigate the role of both the promoter and 3′UTR in regulating C5aR expression and our results show that regulation of the human C5aR is similar but not identical to that of the mouse C5aR.

Transcription factors are the common convergence points of signal transduction pathways to affect gene transcription. Signal transduction activity results in posttranslational modification (PTM) of transcription factors and the sum of these modifications at any given time point will determine the action of the transcription factor. It has been suggested that these PTMs provide a transcription factor code analogous to the histone code. However, the number and variety of these modifications and the lack of knowledge in general of their dynamics precludes at present a concise view of how combinations of PTMs affect transcription factor function. Also, a single type of PTM such as phosphorylation can have opposing effects on transcription factor activity. Transcription factors of the Forkhead box O (FOXO) class are predominantly regulated through signaling, by phosphoinositide 3-kinase/protein kinase B (also known as AKT) pathway and a reactive oxygen species/c-Jun N-terminal kinase pathway. Both pathways result in increased FOXO phosphorylation yet with opposing result. Whereas PKB-mediated phosphorylation inactivates FOXO, c-Jun N-terminal kinase-mediated phosphorylation results in activation of FOXO. Here we discuss regulation of FOXO transcription factors by phosphorylation as an example for understanding integration of signal transduction at the level of transcription activity.

Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, such as intravascular haemolysis, intravascular coagulation and renal failure. Current therapies are not effective, often not based in scientific research and can be even detrimental. A lack of understanding of the mechanism of action of the venom of the Loxosceles spider had thus far prevented development of effective therapies. In this review we aim to give an overview of our contributions to the understanding of the mechanism of action of the Loxosceles venom and propose targets and therapeutics for medical intervention.

Snake venoms are a complex mixture of components, which have a wide range of actions both on prey and human victims. The genus Bothrops causes the vast majority of snakebites in Central and South America, being responsible for 80% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including oedema, haemorrhage and necrosis, which can lead to permanent disability. Systemic manifestations such as haemorrhage, coagulopathy, shock and acute renal failure may also occur. In the present study we have investigated the action of venoms from 19 species of snakes from the genus Bothrops, occurring in Brazil, on the complement system in in vitro studies. All venoms were able to activate the classical complement pathway, in the absence of sensitizing antibody. This activation was in part associated with the cleavage of C1-Inhibitor by proteases present in these venoms, which disrupts complement activation control. No modification of the membrane bound complement regulators, such as DAF, CR1 and CD59 was detected, after treatment of human erythrocytes with the snake venoms. Some of the Bothrops venoms were also able to activate alternative and lectin pathways, as measured in haemolytic and ELISA assays. C3a, C4a and C5a were generated in sera treated with the venoms, not only through C-activation, but also by the direct cleavage of complement components, as determined using purified C3 and C4. Metallo- and/or serine-protease inhibitors prevented cleavage of C3 and C4. These results suggest that Bothrops venoms can activate the complement system, generating a large amount of anaphylatoxins, which may play an important role in the inflammatory process presented in humans after snake envenomations, and they may also assist, due to their vasodilatory effects, to enhance the spreading of other venom components.

We have investigated the role of the 3'untranslated region (3'UTR) in the expression of decay accelerating factor (DAF), one of the major membrane regulators of Complement activation. We show here that the 3'UTR of DAF contains an adenylate uridine rich element (ARE) AUUUAUUUAUAUUUAUUUA, which belongs to Class II Cluster 4 of the AU-rich element-containing mRNA (ARED) database. Enhanced Green Fluorescent Protein (EGFP) Reporter constructs containing the DAF 3'UTR showed reduced levels of expression when transfected into a variety of cell lines compared to 3'UTR reporter constructs without the ARE sequence. Furthermore, the inhibitor of mRNA transcription Actinomycin D had a much stronger effect on mRNA half-life of the ARE-containing 3'UTR demonstrating that this ARE destabilises the mRNA. Electrophoretic Mobility Shift Assays (EMSA) using biotinylated RNA probes, demonstrated that cytoplasmic Human antigen R (HuR) bound to the DAF ARE. Transfection experiments using HuR specific siRNA increased DAF expression whilst plasmids containing the coding sequence of HuR had the opposite effect, demonstrating that HuR reduced the stability of DAF mRNA and suggesting that it is of importance in regulating the expression of DAF. These data suggest that modulators of HuR could potentially be used to alter DAF expression and therefore increase the susceptibility of malignant cells to immunotherapy.

A study of the components of the fruits of Kigelia pinnata was undertaken to identify compounds with potential growth inhibitory activity against human melanoma cells, since extracts from the fruits of this plant have been described in traditional medicine to have application in the treatment of skin cancer and other skin ailments. A bioactivity-guided fractionation process yielded a number of crude fractions, which demonstrated cytotoxicity in vitro against human melanoma cells. Compounds isolated and identified included the isocoumarins, demethylkigelin (1) and kigelin (2), fatty acids, oleic (3) and heneicosanoic acids (4), the furonaphthoquinone, 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione (5), and ferulic acid (6). A number of structurally related synthetic compounds were also tested using the MTT assay. The most potent series of these compounds, the furonaphthoquinones, also demonstrated a cytotoxic effect in two human breast cancer cell lines tested.

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to the dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1,3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo in a number of human tumor xenograft models.

Background: As nuclear receptors and transcription factors have an important regulatory function in adipocyte differentiation and fat storage, genetic variation in these key regulators and downstream pathways may be involved in the onset of obesity.Objective: To explore associations between single nucleotide polymorphisms ( SNPs) in candidate genes from regulatory pathways that control fatty acid and glucose metabolism, and repeated measurements of body mass index (BMI) and waist circumference in a large Dutch study population.Methods: Data of 327 SNPs across 239 genes were analyzed for 3575 participants of the Doetinchem cohort, who were examined three times during 11 years, using the Illumina Golden Gate assay. Adjusted random coefficient models were used to analyze the relationship between SNPS and obesity phenotypes. False discovery rate q-values were calculated to account for multiple testing. Significance of the associations was defined as a q-valueResults: Two SNPs ( in NR1H4 and SMARCA2 in women only) were significantly associated with both BMI and waist circumference. In addition, two SNPs ( in SIRT1 and SCAP in women only) were associated with BMI alone. A functional SNP, in IL6, was strongly associated with waist.Conclusion: In this explorative study among participants of a large population-based cohort, five SNPs, mainly located in transcription mediator genes, were strongly associated with obesity phenotypes. The results from whole genome and candidate gene studies support the potential role of NR1H4, SIRT1, SMARCA2 and IL6 in obesity. Although replication of our findings and further research on the functionality of these SNPs and underlying mechanism is necessary, our data indirectly suggest a role of GATA transcription factors in weight control. International Journal of Obesity ( 2009) 33, 1143-1152; doi:10.1038/ijo.2009.152; published online 4 August 2009

Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.

10 páginas, 4 figuras -- PAGS nros. 30451-30460, Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca2+ to maintain its native fold and pentameric subunit assembly or by specific Ca2+-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca2+, conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration., Complement (C)2 is the principle effector in the innate host defense against infection and also contributes to tissue damaging inflammatory reactions, processing and clearance of immune complexes and regulation of the immune response (1). Activation is controlled by a group of C regulatory proteins that includes factor H (fH), the plasma protein responsible for regulating the alternative pathway, an amplification loop that is pivotal in physiological and pathological activation of C (2). fH is a single-chain serum glycoprotein of 150 kDa consisting of a string of 20 homologous units, each of about 60 amino acids, called short consensus repeats (SCR). Deficiency of fH causes unregulated consumption of C with secondary deficiency of C3 (3). Although fH is a plasma protein, many of its activities occur on the cell membrane. Indeed, mutations in the carboxyl terminus of fH that affect membrane binding capacity cause defective C regulation at the membrane and are associated with the renal disease, atypical hemolytic uremic syndrome (4). Within the fH molecule there are multiple binding sites for C3b, heparin, and several other ligands (5). An interaction of fH with the plasma pentraxin molecule, C-reactive protein (CRP), the classical major acute phase reactant, was first reported almost 25 years ago. Mold et al. (6) showed that CRP coating of pneumococci had divergent effects, increasing activation of the classical pathway while inhibiting the alternative pathway; this latter effect was fH-dependent and associated with a doubling of fH binding to the CRP-coated pneumococcus. The same group later provided direct evidence of fH binding to plastic-immobilized CRP, although when the assay format was reversed and fluid phase CRP was offered to immobilized fH, less than 1% of added CRP bound (7). In a surface plasmon resonance study, when CRP was covalently immobilized on the chip surface through amine coupling in the absence of Ca2+, calcium-dependent binding of the recombinant fH fragments comprising both SCR 7 and SCRs 8–11 was seen (8)., The seventh SCR of fH is a “hotspot” for ligand interactions with reported binding to heparin, CRP, and streptococcal M-protein (9). When a fH polymorphism, resulting in a single amino acid change (Y402H) in SCR7 was implicated as a powerful risk factor for age-related macular degeneration (AMD) (10–12), there was immediately intense interest in exploring differences in ligand binding between these polymorphic variants that might explain the disease association. Several groups have lately reported that the fH Y402 and H402 variants show differential binding affinity for CRP (13–17). Each used similar methods with CRP directly immobilized on a surface (ELISA plate or surface plasmon resonance chip) and either intact fH variants or recombinant fragments in the fluid phase. All concluded that both fH variants bound CRP and that the H402 variant bound less well than the Y402 variant. C proteins are present in drusen, the hallmark of the retinal pathology of AMD, but there is controversy about CRP, some reporting that it is absent (18), whereas others find it in the retinal layers and drusen deposits and more abundantly in H402 homozygotes than Y402 homozygotes (19). These observations have led to the suggestion that binding of fH to CRP in the retina limits C activation and the H402 variant because it binds less well and permits more C activation, thus, predisposing to disease. This has potentially important therapeutic implications, especially with the recent development of the first specific inhibitor of CRP ligand binding and pathogenic function in vivo (20). As a first step toward evaluation of this suggestion, we have investigated the interaction between CRP and fH under conditions in which CRP retains its native fold and homopentameric assembly of protomers and which are, thus, most likely to be of physiological and pathophysiological relevance. Here we report that there is no interaction between fH and intact native CRP in the fluid phase or with CRP immobilized directly to plastic in the presence of Ca2+ or immobilized via its specific Ca2+-dependent binding to its natural ligands. fH binds CRP only after the CRP has been denatured, either before immobilization or by coating onto plastic in the absence of Ca2+. We conclude that the reported binding of fH to CRP is likely caused by denaturation of CRP, which has long been known to result from its adsorption to plastic surfaces in the non-physiological absence of Ca2+ (21, 22) or by other structural modifications of CRP resulting from its covalent immobilization. Alternative mechanisms for the strong association of the fH H402 variant with AMD should be sought, The work was supported by Wellcome Trust Programme Grant 068590 (to B. P. M.), Wellcome Trust University Award 068823 (to C. L. H.), and Spanish Ministerio de Educacion y Cultura Grant SAF2005-00913 (to S. R. d.-C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Envenomation by spiders belonging to the Loxosceles genus (brown spider) often results in local dermonecrotic lesions. We have previously shown that Loxosceles sphingomyelinase D (SMase D), the venom component responsible for all the pathological effects, induced the expression of matrix metalloproteinases (MMPs) in rabbits and in human keratinocytic cells. We also showed that the SMase D-induced apoptosis and MMP expression of keratinocytes was inhibited by tetracyclines. We have further investigated the ability of tetracyclines to inhibit or prevent the dermonecrotic lesion induced by Loxosceles venom in vivo and in vitro models. Primary cultures of rabbit fibroblasts incubated with increasing concentrations of venom or SMase D showed a decrease in cell viability, which was prevented by tetracyclines. In vivo experiments showed that topical treatments with tetracycline of rabbits, inoculated with crude Loxosceles intermedia venom or recombinant SMase D, significantly reduced the progression of the dermonecrotic lesion. Furthermore, tetracyclines also reduced the expression of MMP-2 and prevented the induction of MMP-9. Our results suggest that tetracycline may be an effective therapeutic agent for the treatment of cutaneous loxoscelism.