Neuronal extracellular vesicles and associated microRNAs induce circuit connectivity downstream BDNF.

Extracellular vesicles (EVs) have emerged as mediators of cellular communication, in part via the delivery of associated microRNAs (miRNAs), small non-coding RNAs that regulate gene expression. We show that brain-derived neurotrophic factor (BDNF) mediates the sorting of miR-132-5p, miR-218-5p, and miR-690 in neuron-derived EVs. BDNF-induced EVs in turn increase excitatory synapse formation in recipient hippocampal neurons, which is dependent on the inter-neuronal delivery of these miRNAs. Transcriptomic analysis further indicates the differential expression of developmental and synaptogenesis-related genes by BDNF-induced EVs, many of which are predicted targets of miR-132-5p, miR-218-5p, and miR-690. Furthermore, BDNF-induced EVs up-regulate synaptic vesicle (SV) clustering in a transmissible manner, thereby increasing synaptic transmission and synchronous neuronal activity. As BDNF and EV-miRNAs miR-218 and miR-132 were previously implicated in neuropsychiatric disorders such as anxiety and depression, our results contribute to a better understanding of disorders characterized by aberrant neural circuit connectivity.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development.

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA-generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain-derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca2+-dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre-miR16, was impaired. Decreased production of miR-16-5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane-targeted TRBP Moreover, miR-16-5p or membrane-targeted TRBP expression blocked BDNF-induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity-dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.

Effect of left ventricular pacing mode and site on hemodynamic, torsional and strain indices.

INTRODUCTION: Several reports have indicated that left ventricular (LV) lead placement at an optimal pacing site is an important determinant of short- and long-term outcome. This study investigated the effect of pacing mode (atrioventricular [AV] or ventricular) and site (LV apical or lateral) outside the ischemic region on the LV hemodynamic, torsional and strain indices in the ischemic myocardium. METHODS: Experiments were conducted in anesthetized open-chest pigs (n = 15) 30 min after LAD ligation to investigate the hemodynamic effects of temporary epicardial AV and ventricular LV pacing at the LV apical (outside the ischemic region) or lateral wall. LV hemodynamic data were recorded (ejection fraction, stroke volume, dP/dtmax, systolic pressure, cardiac output and e/e΄ ratio) and torsional (twist, rotation), as well as deformation (radial and circumferential strain), indices of LV function were assessed using two-dimensional speckle tracking imaging. RESULTS: The LV function was highly dependent on the pacing mode and site. LV dP/dtmax, systolic pressure and twist decreased significantly during LV pacing in comparison to sinus rhythm (p = 0.004, p<0.001, p = 0.002, respectively). Torsion in sinus rhythm decreased significantly during AV-pacing at the lateral wall (0.11±0.04°/mm vs. 0.06±0.02°/mm, p = 0.005) but did not change significantly during AV-pacing at the apex (0.07±0.05°/mm). CONCLUSIONS: LV pacing at the apical or lateral wall, in the ischemic myocardium, leads to a suboptimal response in comparison to sinus rhythm. LV pacing at the apex outside the ischemic area exhibits a better response than pacing at the lateral wall, possibly because pacing from this site leads to a more physiological propagation of electrical conduction.

Non-coding mechanisms of local mRNA translation in neuronal dendrites.

The vast majority of the mammalian genome is transcribed, generating a wealth of transcripts that do not have protein-coding potential. These non-coding RNAs (ncRNAs) have emerged as major mediators of compartmentalized gene expression with many important regulatory functions, and are therefore at the focus of biological research in many cellular systems. The expression of ncRNAs is particularly multifaceted in neurons, as they seem to be expressed in a highly cell-type and activity-dependent manner. Specific subclasses of ncRNAs, especially microRNAs (miRNAs), were implicated in the local regulation of mRNA translation in neuronal dendrites, a process of compartmentalized gene expression that is engaged during synaptic plasticity. Recent discoveries point towards a widespread involvement of ncRNA families in local translation, including less abundant small RNAs (PIWI-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs)) and long ncRNAs (circular RNAs (circRNAs), long intergenic ncRNAs (lincRNAs)). The mechanisms underlying the dendritic transport and the regulatory function of ncRNAs in response to neuronal activity are being elucidated. The emerging picture is an intricate crosstalk between different ncRNA families, mRNAs and RNA-binding proteins (RBPs) that synergistically fine-tune the local dendritic proteome in an activity-dependent manner.

Effect of pacing mode and pacing site on torsional and strain parameters and on coronary flow.

BACKGROUND: Right ventricular apical pacing may induce detrimental effects on left ventricular function and coronary flow. In this study, the effects of pacing site and mode on cardiac mechanics and coronary blood flow were evaluated. METHODS: This prospective study included 25 patients who received dual-chamber pacemakers with the ventricular lead placed in the right ventricular apex and presented in sinus rhythm (SR) at their regularly scheduled visits at the pacemaker clinic. Patients underwent complete transthoracic echocardiographic examinations while in SR, followed by noninvasive Doppler assessment of coronary flow in the left anterior descending coronary artery (LAD) and speckle-tracking echocardiography of short-axis planes in SR, atrial pacing (AAI-P), atrioventricular (dual-chamber) pacing (DDD-P), and ventricular pacing (VVI-P). RESULTS: Rotation of the base was significantly decreased with VVI-P compared with AAI-P. Left ventricular twist decreased significantly with DDD-P compared with AAI-P. Circumferential strain of the base significantly decreased with DDD-P and VVI-P compared with SR. The velocity-time integral of diastolic flow in the LAD decreased significantly with DDD-P compared with SR (10.7 ± 2.2 vs 10.2 ± 2.2 vs 8.9 ± 1.6 vs 8.7 ± 2.6 cm in SR and with AAI-P, DDD-P, and VVI-P, respectively, P = .003). Basal rotation and time from onset of the QRS complex to peak basal rotation as a percentage of systole were independently associated with the velocity-time integral of diastolic flow in the LAD during SR and the three pacing modes. CONCLUSIONS: Acute right ventricular apical pacing showed a detrimental effect on left ventricular twist and basal mechanics, with the latter being independently associated with decreased LAD diastolic flow velocity parameters.

MiR-134-dependent regulation of Pumilio-2 is necessary for homeostatic synaptic depression.

Neurons employ a set of homeostatic plasticity mechanisms to counterbalance altered levels of network activity. The molecular mechanisms underlying homeostatic plasticity in response to increased network excitability are still poorly understood. Here, we describe a sequential homeostatic synaptic depression mechanism in primary hippocampal neurons involving miRNA-dependent translational regulation. This mechanism consists of an initial phase of synapse elimination followed by a reinforcing phase of synaptic downscaling. The activity-regulated microRNA miR-134 is necessary for both synapse elimination and the structural rearrangements leading to synaptic downscaling. Results from miR-134 inhibition further uncover a differential requirement for GluA1/2 subunits for the functional expression of homeostatic synaptic depression. Downregulation of the miR-134 target Pumilio-2 in response to chronic activity, which selectively occurs in the synapto-dendritic compartment, is required for miR-134-mediated homeostatic synaptic depression. We further identified polo-like kinase 2 (Plk2) as a novel target of Pumilio-2 involved in the control of GluA2 surface expression. In summary, we have described a novel pathway of homeostatic plasticity that stabilizes neuronal circuits in response to increased network activity.

PICK1 links Argonaute 2 to endosomes in neuronal dendrites and regulates miRNA activity.

MicroRNAs fine-tune gene expression by inhibiting the translation of mRNA targets. Argonaute (Ago) proteins are critical mediators of microRNA-induced post-transcriptional silencing and have been shown to associate with endosomal compartments, but the molecular mechanisms that underlie this process are unclear, especially in neurons. Here, we report a novel interaction between Ago2 and the BAR-domain protein, PICK1. We show that PICK1 promotes Ago2 localization at endosomal compartments in neuronal dendrites and inhibits Ago2 function in translational repression following neuronal stimulation. We propose that PICK1 provides a link between activity-dependent endosomal trafficking and local regulation of translation in neurons.

Atrioventricular left ventricular apical pacing improves haemodynamic, rotational, and deformation variables in comparison to pacing at the lateral wall in intact myocardium: experimental study.

Aim. To assess the effect on left ventricular (LV) function of atrioventricular (AV) and ventricular pacing at the LV apical or lateral wall and to compare the normal torsional and deformation pattern of the intact LV myocardium with those created by the aforementioned LV pacing modes and sites. Methods. Experiments were conducted in pigs (n = 21) with normal LV function to investigate the acute hemodynamic effects of epicardial AV and ventricular LV pacing at the LV apical or lateral wall. Torsional and deformation indices of LV function were assessed using speckle tracking echocardiography. Results. AV pacing at the apex revealed a significant reduction in the radial strain of the base (P < 0.03), without affecting significantly the ejection fraction and the LV torsion or twist. In contrast, AV pacing at the lateral wall produced, in addition to the reduction of the radial strain of the base (P < 0.01), significant reduction of the circumferential and the radial strain of the apex (both P < 0.01) as well as of the ejection fraction (P < 0.002) and twist (P < 0.05). Conclusions. In pig hearts with intact myocardium, LV function is maintained at sinus rhythm level when AV pacing is performed at the LV apex.

Clinical profile and therapeutic management of patients with atrial fibrillation in Greece: results from the Registry of Atrial Fibrillation to Investigate New Guidelines (RAFTING).

INTRODUCTION: Atrial fibrillation (AF) is the most common arrhythmia with significant morbidity, including a 5-fold increase in stroke risk. The management of AF, including antithrombotic therapy (AT), varies considerably among countries. Representative data concerning AF features and management in Greece are generally lacking. METHODS: The Registry of Atrial Fibrillation To Investigate New Guidelines (RAFTING) is a country-wide prospective observational study of AF in Greece that enrolled consecutive patients with a diagnosis of AF in emergency departments of 31 hospitals of different types according to the population's geographical distribution. RESULTS: RAFTING enrolled 1127 patients, 51% females, aged 71 ± 12 years. Paroxysmal AF was present in 54% of patients and newly diagnosed AF in 28%; 68% of patients with a previous AF history had undergone a median of 4 cardioversions. A high rate of comorbidities was present, including arterial hypertension in 75% and heart failure in 40%. The median CHADS2 and CHA2DS2VASc scores were 2 and 3, respectively; AT had been prescribed in 87% of non-newly diagnosed patients, with warfarin being prescribed in 56% of them. Among all patients on warfarin, INR values were within therapeutic range in 34% of cases during inhospital measurement. Hospital admission occurred in 82% of cases, with in-hospital mortality 0.8%. CONCLUSIONS: RAFTING provides updated insights into the current features and management of AF in Greece. The majority of patients have a sufficiently high risk to warrant oral anticoagulation and further attempts to comply with the existing guidelines are warranted.

The small GTPase Arf1 modulates Arp2/3-mediated actin polymerization via PICK1 to regulate synaptic plasticity.

Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.

Kinetically controlled formation of formamide trimer from first principles.

The formation of formamide trimers was simulated using Car-Parrinello molecular dynamics. A variety of different initial setups were compared to study the effects of spatial arrangement, concentration, and temperature on the trimer product distribution. A total of nine different trimer species were obtained by simulation. Although a triangular initial arrangement of the three monomers is found to favour a less energetically stable chain-like product at high concentration, the more compact global minimum structure is expected to be the most abundant species overall in experiment. This is because there is evidence of a low activation barrier for conversion of the chain-like trimer to the lowest-energy structure. For one, this process is observed upon increasing the length of the trajectories. Furthermore, a slight rise in temperature drastically reduces the number of chain-like trimers. With regard to the intermolecular forces driving the aggregation dynamics, dispersion corrections to the underlying density functional theory description have a strong effect on the product distribution, again favouring the global minimum species. Certain local minimum structures are significantly destabilised relative to the global minimum by dispersion correction while the relative energies of the majority of species are practically unchanged.

Longterm followup after tapering mycophenolate mofetil during maintenance treatment for proliferative lupus nephritis.

OBJECTIVE: To determine the timing for safe reduction of mycophenolate mofetil (MMF) dose during remission-maintenance therapy of proliferative lupus nephritis. METHODS: The study population consisted of 44 patients evaluated retrospectively; MMF dose was empirically tapered in 18/44 patients until the latest observation. RESULTS: Patients reducing MMF ≤ 18 months after remission/complete remission had a 6.8-fold/6.3-fold higher risk of relapse compared to those taking a stable dose (p = 0.001, p = 0.011, respectively). Reducing MMF later than 18 months was not associated with increased relapse rates. CONCLUSION: Reducing MMF > 1.5 years after remission/complete remission seems to warrant drug tapering without increased risk of disease flare in proliferative lupus nephritis.

Pacing within the ischemic area significantly decreases the left ventricular ejection fraction during experimental acute myocardial infarction.

BACKGROUND: The aim of this study was to examine the effects on left ventricular (LV) function of LV apical or/and lateral wall pacing during an experimental acute myocardial infarction. METHODS: In 12 anesthetized pigs, epicardial LV pacing at the apex or lateral wall, or at both sites simultaneously, was performed before and after left anterior descending (LAD) ligation. Data concerning LV function were obtained by two-dimensional echo during spontaneous sinus rhythm (SR) and during pacing before and 15, 45, 60, and 90 minutes after LAD ligation. RESULTS: Before ligation of the LAD, pacing at the lateral wall (48.04 ± 6.25%) or both sites (45.71 ± 6.31%) reduced the LV ejection fraction (EF) significantly (P < 0.01) in comparison to SR (55.44 ± 4.10%). However, during pacing at the apex (50.19 ± 6.50%), the reduction was not significant. After LAD ligation, the EF during lateral pacing (43.02 ± 7.71%) was significantly higher than during apical pacing (38.78 ± 8.26%, P < 0.04) but was not significantly different from that during dual-site pacing (41.65 ± 8.69%). CONCLUSIONS: Pacing within the ischemic LV apical zone after LAD ligation impairs left ventricular ejection fraction, as compared with pacing the nonischemic LV lateral wall, and should therefore be avoided in clinical settings where the LV pacing site may be chosen.

Cognitive deficits in patients with antiphospholipid syndrome: association with clinical, laboratory, and brain magnetic resonance imaging findings.

BACKGROUND: Antiphospholipid syndrome (APS) is a multisystem disorder characterized by arterial and venous thromboses, pregnancy morbidity, and various neuropsychiatric manifestations. Cognitive dysfunction in APS has been poorly recognized. We examined for the first time, to our knowledge, the presence of cognitive dysfunction in patients with APS and its association with clinical, laboratory, and cerebral magnetic resonance imaging characteristics. METHODS: Sixty patients (39 with primary APS and 21 with systemic lupus erythematosus-related APS) and 60 healthy individuals matched for age, sex, and education were examined by means of a comprehensive 3-hour battery of neuropsychological tests. Twenty-three patients had a history of central nervous system involvement. Fifty-nine of 60 patients underwent brain magnetic resonance imaging at the time of neuropsychological assessment. A disease control group not fulfilling criteria for APS (15 patients with systemic lupus erythematosus and 10 with rheumatoid arthritis) was also included. The demographic, clinical, and laboratory characteristics of patients were recorded. RESULTS: Twenty-five (42%) of the 60 patients with APS had cognitive deficits compared with 11 (18%) healthy control subjects (P = .005). No patient was diagnosed as having dementia. The most commonly involved cognitive domains were complex attention and verbal fluency. No difference was found in cognitive performance between patients with primary APS and those with systemic lupus erythematosus-related APS. No relationship was detected between cognitive dysfunction and prior central nervous system disease. We noted a significant association between cognitive dysfunction and livedo reticularis (P = .004) as well as between cognitive dysfunction and the presence of white matter lesions on the findings of brain magnetic resonance imaging (P=.01). No difference was detected in cognitive performance between the disease control group and healthy individuals (P=.86). CONCLUSIONS: Cognitive deficits may often be found among patients with APS, independent of any history of central nervous system involvement. Livedo reticularis and the presence of white matter lesions on brain magnetic resonance imaging are associated with an increased risk for cognitive dysfunction in APS.

Highly active antiretroviral therapy interruption: predictors and virological and immunologic consequences.

OBJECTIVE: To characterize the magnitude and the predictors of highly active antiretroviral therapy (HAART) interruption (TI) and to investigate its immunologic and virological consequences. METHODS: Using Concerted Action on Seroconversion to AIDS and Death in Europe data from 8,300 persons with well-documented seroconversion dates, we identified subjects with stable first HAART (for at least 90 days) not initiated during primary infection. A TI was defined as an interruption of all antiretroviral therapy drugs for at least 14 days. RESULTS: Of 1,551 subjects starting HAART, 299 (19.3%) interrupted treatment. Median (interquartile range) duration of the TI was 189 (101-382) days. The cumulative probability (95% confidence interval) of TI at 2 years was 15.9% (14.0%-18.1%). Women were more likely to have a TI than men in the same exposure group (35.8% vs 24.2% among drug users, 22.1% vs 13.3% among heterosexuals; P < 0.05). Higher baseline viremia and poor immunologic response to HAART were associated with higher probabilities of TI. Median (interquartile range) individual CD4 cell loss during TI was 94 (1-220) cells/microL. Older age at HAART (>40 yr), lower pre-HAART nadir (<200 cells/microL), and lower CD4 at start of TI (<350 cells/microL) were significantly associated with greater relative CD4 loss during TI. CONCLUSIONS: We estimate that almost 1 in 6 subjects on HAART interrupts treatment by 2 years. Further research is needed to investigate the reasons why TI is higher in women. We have identified characteristics of subjects with the greatest risk for CD4 loss in whom TI may have greater risks.

Association of clinical progression in classic Kaposi's sarcoma with reduction of peripheral B lymphocytes and partial increase in serum immune activation markers.

OBJECTIVE: To evaluate various immunologic markers in the peripheral blood of patients with early and advanced classic Kaposi's sarcoma (CKS). DESIGN: Cross-sectional study. SETTING: A major referral center for skin and venereal diseases. PATIENTS: Sixty-eight patients with histologically confirmed CKS staged according to a modified version of the Mitsuyasu-Groopman classification in stage I-II (cutaneous involvement only) and stage IV (skin and systemic involvement). MAIN OUTCOME MEASURES: Concentrations of neopterin and beta2-microglobulin, titer of anti-human herpesvirus 8 antibodies, number of natural killer cells, and numbers of total lymphocytes, B lymphocytes, T lymphocytes, and their subsets in peripheral blood. RESULTS: The median values of beta2-microglobulin and neopterin were elevated in patients with CKS in stage IV (median, 3.679 microg/mL [312.72 nmol/L] and 14.0 nmol/L, respectively) compared with patients in stage I-II (median, 2.406 microg/mL [204.51 nmol/L] and 6.5 nmol/L, respectively). A statistically significant reduction in total lymphocyte and B-lymphocyte counts was observed in patients with advanced-stage CKS (1679/microL and 79/microL, respectively) compared with patients in earlier stages of the disease (2142/microL and 224/microL, respectively). The human herpesvirus 8 antibody titer, determined by latent immunofluorescent assay, decreased from stage I-II to stage IV, although not at a statistically significant level (P = .14). CONCLUSION: The evolution of CKS from the early stages of the disease to the more advanced may be associated with a partial activation of the immune system and a gradual decrease in the number of total and B lymphocytes.

Registration of antithrombotic policy for the management of acute coronary syndromes in the Greek population.

INTRODUCTION: In this study an attempt was made for the first time in the Greek population to register the antithrombotic medication given to patients with acute coronary syndromes (ACS), both during their hospitalisation and on discharge. METHODS: The study was designed to include all patients with ACS in a total of 22 hospitals in Athens and other parts of Greece. An analysis was made of differences in the administration of antithrombotic agents in relation to region, sex and age. RESULTS: From the data recorded it emerged that patients in Athenian hospitals more often receive aspirin and heparin than do those in other regions. Also, women with acute myocardial infarction are given aspirin and platelet glycoprotein IIb/IIIa inhibitors less frequently than men. In addition, elderly patients with non-Q infarction and unstable angina are treated less often with clopidogrel than are younger patients. CONCLUSIONS: A large number of patients with ACS do not receive antithrombotic medication in accordance with the guidelines. Furthermore, it appears that population groups who are considered to have higher risk and poorer prognosis, such as the elderly and women, are undertreated.

Attitudes and behavior towards contraception among Greek women during reproductive age: a country-wide survey.

OBJECTIVES: The study objectives were to explore current contraceptive behavior of Greek women during reproductive age. STUDY DESIGN: Data were drawn from a country-wide survey, conducted with the use of a self-administered questionnaire. The sample, numbering 797 women of ages 16-45 years, was representative of the Greek female population of reproductive age. RESULTS: The most common contraceptive method reported was the male condom (MC) (33.9%) followed by coitus interruptus (CI) (28.8%), oral pill (4.8%), and coil (3.6%). Other methods counted for a 5% and no use of contraception reached a 23.8%. Attitudes over responsibility of using contraception were also explored. The majority of respondents (52%) stated that contraception use is the responsibility of men. The probability of reporting that women should be responsible in using contraception was higher in women aged 25-34 years, in those with higher level of knowledge over contraception issues and in those with an experience of abortion. CONCLUSIONS: The need for sexual education and easy access to counseling services is apparent in order to promote optimal contraception decision-making. The role of women in taking active responsibility over contraception use should be of great importance in reproductive health promotion projects.