Proteomic profiling of cisplatin-resistant and cisplatin-sensitive germ cell tumour cell lines using quantitative mass spectrometry.

PURPOSE: Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs. METHODS: Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the 'The Cancer Genome Atlas'. RESULTS: A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs (24 proteins for NTERA-2, 60 proteins for NCCIT, 75 proteins for 2102EP). Western blotting confirmed regulation of key resistance-associated proteins (CBS, ANXA1, LDHA, CTH, FDXR). GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines. CONCLUSION: High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.

Extracellular Vesicles Derived from Neural Progenitor Cells--a Preclinical Evaluation for Stroke Treatment in Mice.

Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell-induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression.

Food impaction: etiology over 35 years and association with eosinophilic esophagitis.

With the emergence of eosinophilic esophagitis (EoE) as a common cause of food impaction (FI) and a presumed increase in incidence of EoE in the population, the effect on the incidence of FI has not been well described. The aim of this study is to describe the incidence of FI and endoscopic findings in these patients and the association with EoE. A population-based retrospective chart review of the Rochester Epidemiology Project database was performed to identify all patients within Olmsted County that presented with FI from 1976 to 2012. A review of all endoscopic findings, biopsy results, and demographic data was performed. 497 patients were identified with FI from 1976 to 2012. The overall incidence of FI has changed from 1976 to 2012 (Fig. 1) (P < 0.001). The peak incidence of 17.12 per 100,000 people occurred in the time period 1995 to 2000. Both the incidence of comorbid gastroesophageal reflux disease (GERD) and proton pump inhibitor (PPI) use increased over the time period of the study (P < 0.001 for both). Of these patients, 188 (46.7%) had no abnormalities on their endoscopy. The most common endoscopic finding was stricture in 71 (17.6%) patients followed closely by Schatzki's ring in 68 (16.9%) patients. 139 patients had biopsies performed within 2 years of FI and 50 (36.0%) of those were diagnosed with EoE. We present for the first time the changing incidence of FI over the last 35 years in a population-based setting. We also demonstrate the rise of EoE as an important clinical consideration in patients with FI.

Microcystin and pyriproxyfen are toxic to early stages of development in Rhamdia quelen: An experimental and modelling study.

The recent increase of freshwater eutrophication has favored cyanobacteria blooms and consequently the increase of toxins such as microcystin-LR in aquatic environments, but few is know about the associated effect of toxin and other compounds. Pyriproxyfen is an insecticide indicated by WHO (World Health Organization) to control Aedes aegypti mosquito (vector of Dengue, Chikungunya and Zika diseases), however, the effects are not well described to non-target species, such as fish. The early life stages (ELS) of fish are more sensitive to chemical stress due to higher metabolic rate, immature immune system and high superficial area/volume ratio. In the current study, ELS of R. quelen a Neotropical fish were exposed to environmentally realistic concentrations of microcystin (1, 10 and 100 µg L-1; M1, M2 and M3 groups, respectively) from an algal extract, pyriproxyfen (1 and 10 µg L-1, P1 and P2) and their association (co-exposure). The hatching, survival and larvae deformities were analyzed, and applied a mathematical model to evaluate the effects on the population size along further generations. Both compounds were toxic to embryos/larvae of fish, but the effects were more pronounced in M2, P1M2 and P2M1 for hatching and M2, P1M2, P2M1 and P1 for survival. Deformities prevailed in groups exposed to the chemicals at 48 hpf (hours post-fertilization) were suggestions of toxicological interaction in P1M2, P2M1 and P2M2 at 48 and 72 hpf. In 96 hpf, the levels of deformities were lower than in previous times. Model predicted population density over 100 years decreased to lower than 0.5 (50%) in all groups, except for P1M1, indicating risk of extinction. P1M2 had the worse results, followed by M2, P1M3 and P2M1. Cyanobacterial blooms can lead to microcystin-LR levels higher than M2 (10 µg L-1), and the suggestion of toxicological interaction with pyriproxyfen is relevant because both compounds may potentially coexist in aquatic environments. Finally, mathematical models may provide an ecological interpretation of the risk of exposure of fish.

Acute LSD effects on response inhibition neural networks.

BACKGROUND: Recent evidence shows that the serotonin 2A receptor (5-hydroxytryptamine2A receptor, 5-HT2AR) is critically involved in the formation of visual hallucinations and cognitive impairments in lysergic acid diethylamide (LSD)-induced states and neuropsychiatric diseases. However, the interaction between 5-HT2AR activation, cognitive impairments and visual hallucinations is still poorly understood. This study explored the effect of 5-HT2AR activation on response inhibition neural networks in healthy subjects by using LSD and further tested whether brain activation during response inhibition under LSD exposure was related to LSD-induced visual hallucinations. METHODS: In a double-blind, randomized, placebo-controlled, cross-over study, LSD (100 µg) and placebo were administered to 18 healthy subjects. Response inhibition was assessed using a functional magnetic resonance imaging Go/No-Go task. LSD-induced visual hallucinations were measured using the 5 Dimensions of Altered States of Consciousness (5D-ASC) questionnaire. RESULTS: Relative to placebo, LSD administration impaired inhibitory performance and reduced brain activation in the right middle temporal gyrus, superior/middle/inferior frontal gyrus and anterior cingulate cortex and in the left superior frontal and postcentral gyrus and cerebellum. Parahippocampal activation during response inhibition was differently related to inhibitory performance after placebo and LSD administration. Finally, activation in the left superior frontal gyrus under LSD exposure was negatively related to LSD-induced cognitive impairments and visual imagery. CONCLUSION: Our findings show that 5-HT2AR activation by LSD leads to a hippocampal-prefrontal cortex-mediated breakdown of inhibitory processing, which might subsequently promote the formation of LSD-induced visual imageries. These findings help to better understand the neuropsychopharmacological mechanisms of visual hallucinations in LSD-induced states and neuropsychiatric disorders.

Increased thalamic resting-state connectivity as a core driver of LSD-induced hallucinations.

OBJECTIVE: It has been proposed that the thalamocortical system is an important site of action of hallucinogenic drugs and an essential component of the neural correlates of consciousness. Hallucinogenic drugs such as LSD can be used to induce profoundly altered states of consciousness, and it is thus of interest to test the effects of these drugs on this system. METHOD: 100 µg LSD was administrated orally to 20 healthy participants prior to fMRI assessment. Whole brain thalamic functional connectivity was measured using ROI-to-ROI and ROI-to-voxel approaches. Correlation analyses were used to explore relationships between thalamic connectivity to regions involved in auditory and visual hallucinations and subjective ratings on auditory and visual drug effects. RESULTS: LSD caused significant alterations in all dimensions of the 5D-ASC scale and significantly increased thalamic functional connectivity to various cortical regions. Furthermore, LSD-induced functional connectivity measures between the thalamus and the right fusiform gyrus and insula correlated significantly with subjective auditory and visual drug effects. CONCLUSION: Hallucinogenic drug effects might be provoked by facilitations of cortical excitability via thalamocortical interactions. Our findings have implications for the understanding of the mechanism of action of hallucinogenic drugs and provide further insight into the role of the 5-HT2A -receptor in altered states of consciousness.

Acute effects of LSD on amygdala activity during processing of fearful stimuli in healthy subjects.

Lysergic acid diethylamide (LSD) induces profound changes in various mental domains, including perception, self-awareness and emotions. We used functional magnetic resonance imaging (fMRI) to investigate the acute effects of LSD on the neural substrate of emotional processing in humans. Using a double-blind, randomised, cross-over study design, placebo or 100 µg LSD were orally administered to 20 healthy subjects before the fMRI scan, taking into account the subjective and pharmacological peak effects of LSD. The plasma levels of LSD were determined immediately before and after the scan. The study (including the a priori-defined study end point) was registered at ClinicalTrials.gov before study start (NCT02308969). The administration of LSD reduced reactivity of the left amygdala and the right medial prefrontal cortex relative to placebo during the presentation of fearful faces (P<0.05, family-wise error). Notably, there was a significant negative correlation between LSD-induced amygdala response to fearful stimuli and the LSD-induced subjective drug effects (P<0.05). These data suggest that acute administration of LSD modulates the engagement of brain regions that mediate emotional processing.

Hypoxia-stimulated membrane trafficking requires T-plastin.

AIM: Traffic between the plasma membrane and the endomembrane compartments is an essential feature of eukaryotic cells. The secretory pathway sends cargoes from biosynthetic compartments to the plasma membrane. This is counterbalanced by a retrograde endocytic route and is essential for cell homoeostasis. Cells need to adapt rapidly to environmental challenges such as the reduction of pO2 which, however, has not been analysed in relation to membrane trafficking in detail. Therefore, we determined changes in the plasma membrane trafficking in normoxia, hypoxia, and after reoxygenation. METHODS: Membrane trafficking was analysed by using the bulk membrane endocytosis marker FM 1-43, the newly developed membrane probe mCLING, wheat germ agglutinin as well as fluorescently labelled cholera toxin subunit B. Additionally, the uptake of specific membrane proteins was determined. In parallel, a non-biased SILAC screen was performed to analyse the abundance of membrane proteins in normoxia and hypoxia. RESULTS: Membrane trafficking was increased in hypoxia and quickly reversed upon reoxygenation. This effect was independent of the hypoxia-inducible factor (HIF) system. Using SILAC technology, we identified that the actin-bundling protein T-plastin is recruited to the plasma membrane in hypoxia. By the use of T-plastin knockdown cells, we could show that T-plastin mediates the hypoxia-induced membrane trafficking, which was associated with an increased actin density in the cells as determined by electron microscopy. CONCLUSION: Membrane trafficking is highly dynamic upon hypoxia. This phenotype is quickly reversible upon reoxygenation, which suggests that this mechanism participates in the cellular adaptation to hypoxia.

Impact of polygenic schizophrenia-related risk and hippocampal volumes on the onset of psychosis.

Alterations in hippocampal volume are a known marker for first-episode psychosis (FEP) as well as for the clinical high-risk state. The Polygenic Schizophrenia-related Risk Score (PSRS), derived from a large case-control study, indicates the polygenic predisposition for schizophrenia in our clinical sample. A total of 65 at-risk mental state (ARMS) and FEP patients underwent structural magnetic resonance imaging. We used automatic segmentation of hippocampal volumes using the FSL-FIRST software and an odds-ratio-weighted PSRS based on the publicly available top single-nucleotide polymorphisms from the Psychiatric Genomics Consortium genome-wide association study (GWAS). We observed a negative association between the PSRS and hippocampal volumes (ß=-0.42, P=0.01, 95% confidence interval (CI)=(-0.72 to -0.12)) across FEP and ARMS patients. Moreover, a higher PSRS was significantly associated with a higher probability of an individual being assigned to the FEP group relative to the ARMS group (ß=0.64, P=0.03, 95% CI=(0.08-1.29)). These findings provide evidence that a subset of schizophrenia risk variants is negatively associated with hippocampal volumes, and higher values of this PSRS are significantly associated with FEP compared with the ARMS. This implies that FEP patients have a higher genetic risk for schizophrenia than the total cohort of ARMS patients. The identification of associations between genetic risk variants and structural brain alterations will increase our understanding of the neurobiology underlying the transition to psychosis.

Neuroimaging in moderate MDMA use: A systematic review.

MDMA ("ecstasy") is widely used as a recreational drug, although there has been some debate about its neurotoxic effects in humans. However, most studies have investigated subjects with heavy use patterns, and the effects of transient MDMA use are unclear. In this review, we therefore focus on subjects with moderate use patterns, in order to assess the evidence for harmful effects. We searched for studies applying neuroimaging techniques in man. Studies were included if they provided at least one group with an average of <50 lifetime episodes of ecstasy use or an average lifetime consumption of <100 ecstasy tablets. All studies published before July 2015 were included. Of the 250 studies identified in the database search, 19 were included. There is no convincing evidence that moderate MDMA use is associated with structural or functional brain alterations in neuroimaging measures. The lack of significant results was associated with high methodological heterogeneity in terms of dosages and co-consumption of other drugs, low quality of studies and small sample sizes.

BDNF Val66Met polymorphism and hippocampal volume in neuropsychiatric disorders: A systematic review and meta-analysis.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a neurotrophin involved in neurogenesis and synaptic plasticity in the central nervous system, especially in the hippocampus, and has been implicated in the pathophysiology of several neuropsychiatric disorders. Its Val66Met polymorphism (refSNP Cluster Report: rs6265) is a functionally relevant single nucleotide polymorphism affecting the secretion of BDNF and is implicated in differences in hippocampal volumes. METHODS: This is a systematic meta-analytical review of findings from imaging genetic studies on the impact of the rs6265 SNP on hippocampal volumes in neuropsychiatric patients with major depressive disorder, anxiety, bipolar disorder or schizophrenia. RESULTS: The overall sample size of 18 independent clinical cohorts comprised 1695 patients. Our results indicated no significant association of left (Hedge's g=0.08, p=0.12), right (g=0.07, p=0.22) or bilateral (g=0.07, p=0.16) hippocampal volumes with BDNF rs6265 in neuropsychiatric patients. There was no evidence for a publication bias or any demographic, clinical, or methodological moderating effects. Both Val/Val homozygotes (g=0.32, p=0.004) and Met-carriers (g=0.20, p=0.004) from the patient sample had significantly smaller hippocampal volumes than the healthy control sample with the same allele. The magnitude of these effects did not differ between the two genotypes. CONCLUSION: This meta-analysis suggests that there is no association between this BDNF polymorphism and hippocampal volumes. For each BDNF genotype, the hippocampal volumes were significantly lower in neuropsychiatric patients than in healthy controls.

Treatment cost of non-small cell lung cancer in three European countries: comparisons across France, Germany, and England using administrative databases.

INTRODUCTION: Lung cancer is a highly prevalent condition with non-small cell lung cancer (NSCLC), representing ∼ 80%. Given its high prevalence and poor survival rates, it is important to understand costs associated with NSCLC treatment. OBJECTIVES: To carry out an incidence-based study in three European countries: France, Germany, and the UK, to estimate the cost of NSCLC treatment. METHODS: Three similar administrative databases were accessed; Hospital Episode Statistics (England), Gesundheitsforen Leipzig (Germany), French Hospital Discharge system (France), using ICD-9/10 codes and treatment/surgery algorithms to identify NSCLC patients. An incidence population of NSCLC patients was obtained using an index year (ranging from 2007-2008), ensuring the absence of prior lung cancer (12-months). Data were extracted on treatment information, patient characteristics, and disease staging. Average NSCLC treatment costs were estimated by age and severity. For England, 20,081 patients were identified, for France, 15,061, and for Germany, 1038. RESULTS: In-patient length of stay was 8.9, 8.7, and 10.1 days for France, England, and Germany, respectively, for the first year. Average total costs for the 2-year follow-up period for France, England, and Germany were €25,063, €17,777, and €32,500, respectively. Sub-group analyses showed higher costs for younger patients and those with metastatic disease. CONCLUSION: Considerable differences in average treatment costs were observed. In-patient costs dominate in the first year of treatment in all countries. The study highlights the costly nature of NSCLC.

Femoroacetabular impingement inducing non-union of a femoral neck fracture: a case report.

INTRODUCTION: We describe a case in which femoroacetabular impingement (FAI) was identified as the cause of non-union of a femoral neck fracture and the subsequent treatment strategy. MATERIALS AND METHODS: Retrospective review of a 35-year-old patient, without any risk factors for non-union, who sustained a femoral neck fracture. Pre-existing FAI was identified as the cause for the non-union of the femoral neck fracture, with successful treatment of the non-union according to established arthroscopic treatment of the hip. RESULTS: After treatment of the FAI, the non-union healed uneventfully within 3 months. CONCLUSIONS: FAI may be a less common but potential cause of delayed union or non-union in the setting of femoral neck fracture in the young.

Integrin genetic variants and stage-specific tumor recurrence in patients with stage II and III colon cancer.

Integrins (ITGs) are key elements in cancer biology, regulating tumor growth, angiogenesis and lymphangiogenesis through interactions of the tumor cells with the microenvironment. Moving from the hypothesis that ITGs could have different effects in stage II and III colon cancer, we tested whether a comprehensive panel of germline single-nucleotide polymorphisms (SNPs) in ITG genes could predict stage-specific time to tumor recurrence (TTR). A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole-blood samples were analyzed for germline SNPs in ITG genes using PCR-restriction fragment length polymorphism or direct DNA sequencing. In the multivariable analysis, stage II colon cancer patients with at least one G allele for ITGB3 rs4642 had higher risk of recurrence (hazard ratio (HR)=4.027, 95% confidence interval (95% CI) 1.556-10.421, P=0.004). This association was also significant in the combined stage II-III cohort (HR=1.975, 95% CI 1.194-3.269, P=0.008). The predominant role of ITGB3 rs4642 in stage II diseases was confirmed using recursive partitioning, showing that ITGB3 rs4642 was the most important factor in stage II diseases. In contrast, in stage III diseases the combined analysis of ITGB1 rs2298141 and ITGA4 rs7562325 allowed to identify three distinct prognostic subgroups (P=0.009). The interaction between stage and the combined ITGB1 rs2298141 and ITGA4 rs7562325 on TTR was significant (P=0.025). This study identifies germline polymorphisms in ITG genes as independent stage-specific prognostic markers for stage II and III colon cancer. These data may help to select subgroups of patients who may benefit from ITG-targeted treatments.

Association of common gene variants in the WNT/ß-catenin pathway with colon cancer recurrence.

Wnt/ß-catenin signaling has a central role in the development and progression of most colon cancers (CCs). Germline variants in Wnt/ß-catenin pathway genes may result in altered gene function and/or activity, thereby causing inter-individual differences in relation to tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of Wnt/ß-catenin pathway genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II CC. A total of 234 patients treated with 5-fluorouracil-based chemotherapy were included in this study. Whole-blood samples were analyzed for putative functional germline polymorphisms in SFRP3, SFRP4, DKK2, DKK3, Axin2, APC, TCF7L2, WNT5B, CXXC4, NOTCH2 and GLI1 genes by PCR-based restriction fragment-length polymorphism or direct DNA sequencing. Polymorphisms with statistical significance were validated in an independent study cohort. The minor allele of WNT5B rs2010851 T>G was significantly associated with a shorter TTR (10.7 vs 4.9 years; hazard ratio: 2.48; 95% CI, 0.96-6.38; P=0.04) in high-risk stage II CC patients. This result remained significant in multivariate Cox's regression analysis. This study shows that the WNT5B germline variant rs2010851 was significantly identified as a stage-dependent prognostic marker for CC patients after 5-fluorouracil-based adjuvant therapy.

SS18-SSX2 and the mitochondrial apoptosis pathway in mouse and human synovial sarcomas.

Synovial sarcoma is a deadly malignancy with limited sensitivity to traditional cytotoxic chemotherapy. SS18-SSX fusion oncogene expression characterizes human synovial sarcomas and drives oncogenesis in a mouse model. Elevated expression of BCL2 is considered a consistent feature of the synovial sarcoma expression profile. Our objective was to evaluate the expression of apoptotic pathway members in synovial sarcomas and interrogate the impact of modulating SS18-SSX expression on this pathway. We show in human and murine synovial sarcoma cells that SS18-SSX increases BCL2 expression, but represses other anti-apoptotic genes, including MCL1 and BCL2A1. This repression is achieved by directly suppressing expression via binding through activating transcription factor 2 (ATF2) to the cyclic adenosine monophosphate (AMP) response element (CRE) in the promoters of these genes and recruiting TLE1/Groucho. The suppression of these two anti-apoptotic pathways silences the typical routes by which other tumors evade BH3-domain peptidomimetic pharmacotherapy. We show that mouse and human synovial sarcoma cells are sensitive in vitro to ABT-263, a BH3-peptidomimetic, much more than the other tested cancer cell lines. ABT-263 also enhances the sensitivity of these cells to doxorubicin, a traditional cytotoxic chemotherapy used for synovial sarcoma. We also demonstrate the capacity of ABT-263 to stunt synovial sarcomagenesis in vivo in a genetic mouse model. These data recommend pursuit of BH3-peptidomimetic pharmacotherapy in human synovial sarcomas.

Feasibility of in vivo myelin water imaging using 3D multigradient-echo pulse sequences.

Quantitative myelin water imaging is able to show demyelinating processes and, therefore, provides insight into the pathology of white matter diseases such as multiple sclerosis. So far, mapping of the myelin water fraction most often was performed using single-slice multiecho spin-echo sequences. Recently, a different approach using two-dimensional multigradient-echo pulse sequences was suggested. In this work, a solution to three-dimensional in vivo myelin water fraction imaging is presented that applies multigradient-echo pulse sequences and uses non-negative least squares algorithms to analyze the multicomponent T*(2) decay. The suggested method offers not only whole brain coverage but also clinically practicable acquisition times. The obtained myelin water fraction values are low (6.9% for white matter) but are able to detect demyelination in multiple sclerosis lesions. However, the clinical application of the proposed method remains questionable, because further measurements that clarify the possibility of detecting ongoing processes in lesions are needed.