Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming.

BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

Phenotype Characterization and Course of Patients With Arrhythmogenic Right Ventricular Cardiomyopathy and Biventricular Advanced Heart Failure: A Report of 3 Cases.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) may be complicated by heart failure. Management of advanced heart failure in this context is challenging. METHODS: We reviewed our center's experience with advanced heart failure therapies in patients with ARVC. Three rapidly deteriorating patients with ARVC with biventricular heart failure were found. Their management and outcomes are presented. Data on ventricular fibrosis were available in 2 of them and are also included. RESULTS: The first patient underwent initially successful paracorporeal pulsatile biventricular assist device (BiVAD) implantation. However, a large ischemic stroke occurred 2 weeks later, and the patient died after 2 months. The second patient underwent urgent BiVAD implantation after extracorporeal membrane oxygenation support because of cardiogenic shock, but his course was complicated by multiorgan failure due to systemic infection and the patient died. The last patient, being at Interagency Registry for Mechanically Assisted Circulatory Support 3-4 profile, underwent heart transplant with uneventful recovery. Extensive fibrosis was present in both ventricles of 2 patients undergoing pathology examination. CONCLUSIONS: Patients with ARVC and advanced biventricular heart failure are characterized by extensive ventricular fibrosis and considerable risk, but data on their management are limited. Biventricular circulatory support is associated with suboptimal outcomes, and prioritization for heart transplant seems preferable.

AGO2 localizes to cytokinetic protrusions in a p38-dependent manner and is needed for accurate cell division.

Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole-indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

Left ventricular deformation mechanics over time in patients with thalassemia major with and without iron overload.

BACKGROUND: Myocardial iron overload in patients with thalassemia major (TM) is one of the most important complications. The purpose of the study was to identify advanced echocardiography parameters for early identification of myocardial dysfunction during follow-up of patients with TM. METHODS: Forty TM patients who were 41 ± 5 years old were included in the study and divided into two groups according to cardiac magnetic resonance T2* results (Group 1: Τ2* > 25 ms, Group 2: Τ2* ≤ 25 ms). Liver T2* parameters were also measured. Conventional and deformational echocardiographic parameters were measured at baseline and approximately 2 years later. RESULTS: Thirty-two patients had Τ2* = 34 ± 4 ms (Group 1), and 8 had Τ2* = 17 ± 9 ms (Group 2). Blood consumption was 185 ± 60 and 199 ± 37 ml/kg/yr (p = 0.64), and liver T2* was 4 ± 5 and 17 ± 21 ms (p = 0.01) in Groups 1 and 2, respectively. At baseline, Group 1 had better left ventricular global longitudinal strain (GLS) (- 22 ± 3 vs. - 18 ± 5, p = 0.01) and similar left ventricular ejection fraction (LVEF) (62 ± 5% vs. 58 ± 10%, p = 0.086) than Group 2. At the 28 ± 11-month follow-up, LVEF, GLS, and T2* values in Group 1 (63 ± 3%, - 21 ± 3%, 34 ± 4 ms) and Group 2 (56 ± 11%, - 17 ± 4%, 17 ± 9 ms) did not change significantly compared to their corresponding baseline values. In 8 patients from Group 1, a worsening (> 15%) in LS (p = 0.001) was detected during follow-up, with a marginal reduction in LVEF. CONCLUSIONS: GLS seems to be an efficient echocardiographic parameter for detecting hemochromatosis-related cardiac dysfunction earlier than LVEF. It also seems to be affected by other factors (free radical oxygen, immunogenetic mechanisms or viral infections) in a minority of patients, underscoring the multifactorial etiology of cardiomyopathy.

Increased autophagy/mitophagy levels in primary tumours of patients with pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS: We assessed the levels of autophagy and mitophagy, that are linked to cancer development and drug resistance, in well differentiated pancreatic neuroendocrine neoplasms (PanNENs) and correlated them with clinico-pathological parameters. METHODS: Fluorescent immunostaining for the autophagy markers LC3Β and p62/or LAMP1 was performed on 22 PanNENs and 11 controls of normal pancreatic tissues and validated through Western blotting. Autophagy quantitative scoring was generated for LC3B-positive puncta and analysed in relation to clinico-pathological parameters. TOMM20/LC3B qualitative assessment of mitophagy levels was undertaken by fluorescent immunostaining. The presence of autophagy/mitophagy was validated by transmission electron microscopy. RESULTS: Autophagy levels (LC3B-positive puncta/cell) were discriminative for normal vs. NEN pancreatic tissue (p = 0.007). A significant association was observed between autophagy levels and tumour grade (Ki67 < 3% vs. Ki67 ≥ 3%; p = 0.021), but not functionality (p = 0.266) size (cut-off of 20 mm; p = 0.808), local invasion (p = 0.481), lymph node- (p = 0.849) and distant metastases (p = 0.699). Qualitative assessment of TOMM20/LC3B demonstrated strong mitophagy levels in PanNENs by fluorescent immunostaining as compared with normal tissue. Transmission electron microscopy revealed enhanced autophagy and mitophagy in PanNEN tissue. Response to molecular targeted therapies in metastatic cases (n = 4) did not reveal any patterns of association to autophagy levels. CONCLUSIONS: Increased autophagy levels are present in primary tumours of patients with PanNENs and are partially attributed to upregulated mitophagy. Grade was the only clinico-pathological parameter associated with autophagy scores.

Ultrasound guidance for volume management in patients with heart failure.

Congestion is one of the most prominent characteristics of patients presented with decompensated heart failure and it implies unfavorable prognosis for the heart failure patient. Neurohumoral and immuno-inflammatory activation secondary to cardiac dysfunction constitute the pivotal mechanisms driving the heart failure syndrome that results in progressive fluid accumulation. In addition, fluid redistribution between different vascular compartments in human body guided from sympathetic activity constitutes another mechanism for heart failure decompensation. Ultrasound applied in the form of echocardiography provides invaluable data for the assessment of intracardiac filling pressures. The type of renal venous flow can provide the degree of renal congestion and probably insight into the pathophysiology of the decompensation of heart failure. Assessment of lung congestion in the patient with heart failure can be accomplished by lung ultrasonography. Additionally, clinical studies on the role of ultrasound in the management and prognosis of the congested patient are reviewed. Special heart failure population supported with left ventricular assist devices and extracorporeal membrane oxygenation support constitute an area where ultrasound guidance of fluid management has gained important role.

The evolving field of nonpharmacological therapies to improve functional capacity in chronic heart failure.

Clinical investigations in chronic heart failure (HF) have been increasingly including endpoints related to functional and exercise capacity. Noninvasive ventilation support (NIVS) constitutes a therapeutic option that could improve several markers of cardiovascular performance and functional capacity along with improvements in HF symptoms such as dyspnea. NIVS with bilevel positive airway pressure (BiPAP) is a therapeutic option that can be better tolerated by patients than continuous positive airway pressure (cPAP) and potentially improve the adherence of patients with HF to NIVS. Future investigations should improve our understanding as to how to select patients with HF most amenable to respond favorably, elucidate the frequency and duration of NIVS session required to maintain the observed short-term beneficial effects for a long period of time, and shed additional light on the mechanisms associated with these benefits. In summary, NIVS appears to be a promising nonpharmacological therapy to improve exercise capacity and quality of life in chronic HF.

Cardiac Rotational Mechanics As a Predictor of Myocardial Recovery in Heart Failure Patients Undergoing Chronic Mechanical Circulatory Support: A Pilot Study.

BACKGROUND: Impaired qualitative and quantitative left ventricular (LV) rotational mechanics predict cardiac remodeling progression and prognosis after myocardial infarction. We investigated whether cardiac rotational mechanics can predict cardiac recovery in chronic advanced cardiomyopathy patients. METHODS AND RESULTS: Sixty-three patients with advanced and chronic dilated cardiomyopathy undergoing implantation of LV assist device (LVAD) were prospectively investigated using speckle tracking echocardiography. Acute heart failure patients were prospectively excluded. We evaluated LV rotational mechanics (apical and basal LV twist, LV torsion) and deformational mechanics (circumferential and longitudinal strain) before LVAD implantation. Cardiac recovery post-LVAD implantation was defined as (1) final resulting LV ejection fraction ≥40%, (2) relative LV ejection fraction increase ≥50%, (iii) relative LV end-systolic volume decrease ≥50% (all 3 required). Twelve patients fulfilled the criteria for cardiac recovery (Rec Group). The Rec Group had significantly less impaired pre-LVAD peak LV torsion compared with the Non-Rec Group. Notably, both groups had similarly reduced pre-LVAD LV ejection fraction. By receiver operating characteristic curve analysis, pre-LVAD peak LV torsion of 0.35 degrees/cm had a 92% sensitivity and a 73% specificity in predicting cardiac recovery. Peak LV torsion before LVAD implantation was found to be an independent predictor of cardiac recovery after LVAD implantation (odds ratio, 0.65 per 0.1 degrees/cm [0.49-0.87]; P=0.014). CONCLUSIONS: LV rotational mechanics seem to be useful in selecting patients prone to cardiac recovery after mechanical unloading induced by LVADs. Future studies should investigate the utility of these markers in predicting durable cardiac recovery after the explantation of the cardiac assist device.

Biologic treatment for rheumatic disease: real-world big data analysis from the Greek country-wide prescription database.

OBJECTIVES: To directly assess the prevalence of inflammatory rheumatic disease under treatment with biologic disease modifying anti-rheumatic drugs (b-DMARDs) and compare treatment patterns between rheumatoid arthritis (RA) and spondyloarthropathy (SpA), including psoriatic arthritis. METHODS: The obligatory country-wide prescription electronic database covering 10.223.000 Greek citizens (95.1% of the population, 99.5% Caucasian), all of whom with fully reinbursed access to b-DMARDs, was used to retrospectively capture all patients under b-DMARDs for RA/SpA between June 2014-May 2015. Age, gender and medications for RA/SpA and co-morbid classical cardiovascular risk factors (hypertension, dyslipidaemia, diabetes) were retrieved and analysed. RESULTS: A total of 9.824 RA (61.2±14.0 years, 79% women) and 9.279 SpA patients (51.4±13.1 years, 41% women) using pharmacy-dispensed prescriptions for b-DMARDs were identified (overall prevalence 0.19%). Tumour necrosis factor inhibitors were used in 73% and 99% of RA and SpA patients, respectively. B-DMARD monotherapy (RA: 18.71%, SpA: 52.11%), b-DMARD switching during 12 months (RA: 7.73%, SpA: 6.26%), and use of methotrexate (RA: 50.25%, SpA: 27.35%) and corticosteroids (RA: 55.8%, SpA: 23.63%) differed between the two patient subgroups. In both subgroups, women received more often than men methotrexate, leflunomide, hydroxychloroquine and corticosteroids, and less often b-DMARD monotherapy. After adjustments for age, gender and concomitant drugs, the probability for anti-hypertensive and lipid-lowering drug prescription was higher in SpA than RA [OR=1.41 (95%CI: 1.29-1.54) and 1.24 (1.14-1.36), respectively, p<0.001], whereas for anti-diabetics it was similar. CONCLUSIONS: In the first country-wide study that examines the characteristics of rheumatic disease patients under b-DMARD we show that their exact prevalence is 0.19%, with RA patients being older by 10 years, only slightly more numerous, and less likely to receive treatment for hypertension and dyslipidaemia than their demographically matched SpA counterparts. Longitudinal studies should assess the implications of these novel findings on the differential financial burden of rheumatic diseases, as well as on cardiovascular morbidity and mortality of these patients.

Pesticide inputs from the sewage treatment plant of Agrinio to River Acheloos, western Greece: occurrence and removal.

This work reports the occurrence and the removal of a wide spectrum of pesticides in municipal wastewaters from the Agrinio region, Greece. Analytical determination was carried out by means of solid-phase extraction followed by gas chromatography equipped with flame thermionic and mass spectrometric detection. The herbicides atrazine, alachlor, isoproturon, and s-metolachlor and the insecticides diazinon, methidathion, fenthion and chlorpyriphos, were more frequently detected in influent and effluent samples while they were also detected in river samples. Isoproturon and diazinon showed the highest concentrations in influent samples that reached concentrations up to 2,328 and 1,486 ng L(-1), respectively. Secondary treatment and cumulative removal rates for herbicides and insecticides ranged between 23-91%, 21->99% and 36->99%, 38->99%, respectively. The data demonstrate that there are significant levels of pesticides entering river waterways.

Occurrence and removal of fungicides in municipal sewage treatment plant.

This work reports the occurrence and the removal of widely used fungicides, in municipal sewage treatment plant receiving combined sewage discharges and operating with primary (mechanical), secondary (activated sludge) and tertiary (sand filtration and chlorination) treatments that are commonly applied in Greece. Target analytes included compounds belonging to triazoles and anilino-pyrimidines. Analytical determination in wastewaters and river water samples was carried out by means of solid-phase extraction followed by gas chromatography equipped with flame thermionic and mass spectrometric detection. The fungicides tebuconazole and cyproconazole were more frequently detected in influent and effluent samples at concentrations up to 1893 and 1735 ng L(-1), respectively. Effluent concentrations were below 691.1 ng L(-1) recorded for tebuconazole. With the exception of triadimefon, all the azole fungicides and pyrimethanil showed relatively low removal efficiencies after secondary and tertiary treatments. Mean removal rates of the fungicides after secondary treatment ranged between 31% for pyrimethanil and 65% for triadimefon. Mean overall removal efficiencies after tertiary treatment ranged between 46% for pyrimethanil and 93% for triadimefon. The findings demonstrate that significant levels of fungicides enter river waterways and that only some of these compounds are being reduced in low levels by municipal wastewater treatment processes.

Silicon scaffolds promoting three-dimensional neuronal web of cytoplasmic processes.

Primary neurons were grown on structured silicon (Si) substrates, in the absence of chemotropic factors or synthetic extracellular matrix. The Si substrates used for the study comprise hierarchical structures in the micro- and nanolength scales. The substrates were structured via femtosecond laser irradiation of the Si wafer, in a reactive SF(6) environment. Electron microscopy revealed that the neurons formed an elaborate web of cytoplasmic processes in the absence of glial elements. The neuronal cytoplasm autografted the depth of the spikes, and the neurite sprouting took place over the spikes surface. Here we demonstrate how microfabrication of a Si surface provides an excellent platform for multifaceted studies of neuronal specimens.

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival.

Cardiac contractility is regulated through the activity of various key Ca(2+)-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca(2+) transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca(2+) by SR membranes during relaxation. Recently, the antiapoptotic HS-1-associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575-594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca(2+) levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca(2+) stores.

Genetic divergence among marine and lagoon Atherina boyeri populations in Greece using mtDNA analysis.

Genetic differentiation and phylogenetic relationships among 15 Atherina boyeri populations from several marine and lagoon or lake sites in Greece were investigated using mtDNA analysis. PCR-RFLP analysis of 12s, 16s rRNA genes and D-loop revealed 23 haplotypes. All the lake or lagoon populations, as well as the Kymi and Kalymnos populations that originated from sites with lagoonlike environmental conditions, showed haplotypes 1-6, clearly distinguishable from the marine populations, which exhibited types 7-23. The genetic divergence values estimated between the lagoon and the marine populations ranged from 5.55 to 10.45%. The high genetic differentiation observed between these two types of populations is also highlighted by the dendrograms obtained using UPGMA and maximum parsimony methods.

Molecular phylogeny of grey mullets (Teleostei: Mugilidae) in Greece: evidence from sequence analysis of mtDNA segments.

Mitochondrial DNA sequence analysis has been used to explore genetic differentiation and phylogenetic relationships among five species of the Mugilidae family, Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens. DNA was isolated from samples originating from the Messolongi Lagoon in Greece. Three mtDNA segments (12s rRNA, 16s rRNA, and CO I) were PCR amplified and sequenced. Sequencing analysis revealed that the greatest genetic differentiation was observed between M. cephalus and all the other species studied, while C. labrosus and L. aurata were the closest taxa. Dendrograms obtained by the neighbor-joining method and Bayesian inference analysis exhibited the same topology. According to this topology, M. cephalus is the most distinct species and the remaining taxa are clustered together, with C. labrosus and L. aurata forming a single group. The latter result brings into question the monophyletic origin of the genus Liza.