Loss of Direct Vascular Contact to Astrocytes in the Hippocampus as an Initial Event in Alzheimer's Disease. Evidence from Patients, In Vivo and In Vitro Experimental Models.

Alzheimer's disease (AD) is characterized by the accumulation of aggregated amyloid peptides in the brain parenchyma and within the walls of cerebral vessels. The hippocampus-a complex brain structure with a pivotal role in learning and memory-is implicated in this disease. However, there is limited data on vascular changes during AD pathological degeneration in this susceptible structure, which has distinctive vascular traits. Our aim was to evaluate vascular alterations in the hippocampus of AD patients and PDAPP-J20 mice-a model of AD-and to determine the impact of Aß40 and Aß42 on endothelial cell activation. We found a loss of physical astrocyte-endothelium interaction in the hippocampus of individuals with AD as compared to non-AD donors, along with reduced vascular density. Astrocyte-endothelial interactions and levels of the tight junction protein occludin were altered early in PDAPP-J20 mice, preceding any signs of morphological changes or disruption of the blood-brain barrier in these mice. At later stages, PDAPP-J20 mice exhibited decreased vascular density in the hippocampus and leakage of fluorescent tracers, indicating dysfunction of the vasculature and the BBB. In vitro studies showed that soluble Aß40 exposure in human brain microvascular endothelial cells (HBMEC) was sufficient to induce NFκB translocation to the nucleus, which may be linked with an observed reduction in occludin levels. The inhibition of the membrane receptor for advanced glycation end products (RAGE) prevented these changes in HBMEC. Additional results suggest that Aß42 indirectly affects the endothelium by inducing astrocytic factors. Furthermore, our results from human and mouse brain samples provide evidence for the crucial involvement of the hippocampal vasculature in Alzheimer's disease.

An intra-operative feature-based classification of microelectrode recordings to support the subthalamic nucleus functional identification during deep brain stimulation surgery.

Objective. The subthalamic nucleus (STN) is the most selected target for the placement of the Deep Brain Stimulation (DBS) electrode to treat Parkinson's disease. Its identification is a delicate and challenging task which is based on the interpretation of the STN functional activity acquired through microelectrode recordings (MERs). Aim of this work is to explore the potentiality of a set of 25 features to build a classification model for the discrimination of MER signals belonging to the STN.Approach.We explored the use of different sets of spike-dependent and spike-independent features in combination with an ensemble trees classification algorithm on a dataset composed of 13 patients receiving bilateral DBS. We compared results from six subsets of features and two dataset conditions (with and without standardization) using performance metrics on a leave-one-patient-out validation schema.Main results.We obtained statistically better results (i.e. higher accuracyp-value = 0.003) on the RAW dataset than on the standardized one, where the selection of seven features using a minimum redundancy maximum relevance algorithm provided a mean accuracy of 94.1%, comparable with the use of the full set of features. In the same conditions, the spike-dependent features provided the lowest accuracy (86.8%), while a power density-based index was shown to be a good indicator of STN activity (92.3%).Significance.Results suggest that a small and simple set of features can be used for an efficient classification of MERs to implement an intraoperative support for clinical decision during DBS surgery.

Characterization of Microelectrode Recordings for the Subthalamic Nucleus identification in Parkinson's disease.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective treatment for Parkinson's disease, when the pharmacological approach has no more effect. DBS efficacy strongly depends on the accurate localization of the STN and the adequate positioning of the stimulation electrode during DBS stereotactic surgery. During this procedure, the analysis of microelectrode recordings (MER) is fundamental to assess the correct localization. Therefore, in this work, we explore different signal feature types for the characterization of the MER signals associated to STN from NON-STN structures. We extracted a set of spike-dependent (action potential domain) and spike-independent features in the time and frequency domain to evaluate their usefulness in distinguishing the STN from other structures. We discuss the results from a physiological and methodological point of view, showing the superiority of features having a direct electrophysiological interpretation.Clinical Relevance- The identification of a simple, clinically interpretable, and powerful set of features for the STN localization would support the clinical positioning of the DBS electrode, improving the treatment outcome.

Hydrocephalus: an underrated long-term complication of microvascular decompression for trigeminal neuralgia. A single institute experience.

BACKGROUND: Hydrocephalus is a common complication of posterior fossa surgery, but its real incidence after microvascular decompression (MVD) for idiopathic trigeminal neuralgia (TN) still remains unclear. The aim of this study was to focus on the potential association between MVD and hydrocephalus as a surgery-related complication. METHODS: All patients who underwent MVD procedure for idiopathic TN at our institute between 2009 and 2014 were reviewed to search for early or late postoperative hydrocephalus. RESULTS: There were 259 consecutive patients affected by idiopathic TN who underwent MVD procedure at our institution between 2009 and 2014 (113 men, 146 women; mean age 59 years, range 30-87 years; mean follow-up 40.92 months, range 8-48 months). Nine patients (3.47 %) developed communicating hydrocephalus after hospital discharge and underwent standard ventriculo-peritoneal shunt. No cases of acute hydrocephalus were noticed. CONCLUSIONS: Our study suggests that late communicating hydrocephalus may be an underrated potential long-term complication of MVD surgery.

Transition to superdiffusive behavior in intracellular actin-based transport mediated by molecular motors.

Intracellular transport of large cargoes, such as organelles, vesicles, or large proteins, is a complex dynamical process that involves the interplay of adenosine triphosphate-consuming molecular motors, cytoskeleton filaments, and the viscoelastic cytoplasm. In this work we investigate the motion of pigment organelles (melanosomes) driven by myosin-V motors in Xenopus laevis melanocytes using a high-spatio-temporal resolution tracking technique. By analyzing the obtained trajectories, we show that the melanosomes mean-square displacement undergoes a transition from a subdiffusive to a superdiffusive behavior. A stochastic theoretical model, which explicitly considers the collective action of the molecular motors, is introduced to generalize the interpretation of our data. Starting from a generalized Langevin equation, we derive an analytical expression for the mean square displacement, which also takes into account the experimental noise. By fitting theoretical expressions to experimental data we were able to discriminate the exponents that characterize the passive and active contributions to the dynamics and to estimate the "global" motor forces correctly. Then, our model gives a quantitative description of active transport in living cells with a reduced number of parameters.

Global spatiotemporal order and induced stochastic resonance due to a locally applied signal.

We study the phenomenon of spatiotemporal stochastic resonance (STSR) in a chain of diffusively coupled bistable oscillators. In particular, we examine the situation in which the global STSR response is controlled by a locally applied signal and reveal a wave-front propagation. In order to deepen the understanding of the system dynamics, we introduce, on the time scale of STSR, the study of the effective statistical renormalization of a generic lattice system. Using this technique we provide a criterion for STSR, and predict and observe numerically a bifurcationlike behavior that reflects the difference between the most probable value of the local quasiequilibrium density and its mean value. Our results, tested with a chain of nonlinear oscillators, appear to possess some universal qualities and may stimulate a deeper search for more generic phenomena.

Scanning FCS, a novel method for three-dimensional particle tracking.

We describe a novel method to track fluorescent particles in three dimensions with nanometre precision and millisecond time resolution. In this method, we use our two-photon excitation microscope. The galvomotor-driven x-y scanning mirrors allow the laser beam to move repetitively in a circular path with a radius of half the width of the point spread function of the laser. When the fluorescent particle is located within the scanning radius of the laser, the precise position of the particle in the x-x plane can be determined by its fluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective was used to change the laser focus at two planes (half width of the point spread function apart). The difference of the fluorescence intensity in the two planes is used to calculate the z-position of the fluorescent particle. The laser beam is allowed to scan multiple circular orbits before it is moved to the other plane, thus improving the signal to noise ratio. With a fast feedback mechanism, the position of the laser beam is directed to the centre of the fluorescent particle, thus allowing us to track a particle in three dimensions. In this contribution we describe some calibration experiments performed to test the three-dimensional tracking capability of our system over a large range.

Implication of SAR of male medfly attractants in insect olfaction.

Medfly (Ceratitis capitata) males are strongly attracted by different compounds, not described as pheromones. The best attractants reported are (+)-alpha-copaene, a sesquiterpene of natural source and (-)-ceralure-B1, a non-natural iodinated cyclohexane ester. Although their origin, atomic composition, chemical and physical properties are rather different, they show similar attraction to medflies. The question of why these compounds, act behaviorally in the same way, has been never addressed in research papers. We show here for the first time that these compounds have quite similar stereochemistry, water accessible surfaces, certain local dipole moments and, to some extent, similar octanol/water partition coefficient (log P). When seven carbons, one oxygen and one iodine belonging to (-)-ceralure-B1 are selectively chosen based on topological homology with (+)-alpha-copaene and are overlaid with nine corresponding carbons of (+)-alpha-copaene, the RMS is 0.367 A. This represents a high degree of steric resemblance. Local dipole moments and charges are similar in those regions where the molecules show topological homologies. Thus, we hypothesize that these two molecules could interact with the same male medfly's odorant receptor(s). The implications of this result in future research in insect olfaction is discussed.

Quantitation of plasma membrane calcium pump phosphorylated intermediates by electrophoresis.

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and (32)P-labeled EP by digital analysis of both the stained gel and its autoradiogram, respectively. The principal advantages of this method over typical procedures (filtration and centrifugation) are the low amount of enzyme required and the substantial decrease in the blank values and data scattering produced by unspecific phosphorylation and nonquantitative recovering of the enzyme. Application of this new method to a purified preparation of the plasma membrane calcium ATPase (PMCA) results in overcoming the difficulties of measuring EP at high ATP concentrations. A biphasic behavior of the substrate curve for EP was observed when the study was extended to ATP levels within the physiological range. Since, in principle, the method does not require the use of highly purified preparations, it could be helpful for the study of phosphorylated intermediates especially under conditions in which small amounts of protein are available, e.g., mutated variants of P-ATPases.

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability.

Ca(2+) pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions.

Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions.

Thermal stability of plasma membrane Ca(2+) pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca(2+) pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K(LD)) showing that those phospholipids with higher affinity provide greater stability to the Ca(2+) pump. We developed a method for directly measure K(LD) by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K(LD) values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.

Analysis of recombinant human platelet-derived growth factor by reversed-charge capillary zone electrophoresis.

Reversed-charge capillary zone electrophoresis (RC-CZE) has been developed as a clipping (proteolysis) assay for homodimeric protein recombinant human platelet-derived growth factor (rhPDGF-BB), a major serum mitogenic factor involved in subcutaneous wound healing. When expressed in yeast, the protein is excreted as a fully folded homodimeric protein consisting of two antiparallel B chains held together by two interchain disulfide bonds. During fermentation, internal proteolysis (clipping between residues Arg32 and Thr33) and C-terminal truncation (Arg32 and Thr109) may occur. Internal proteolysis yields three potential forms of rhPDGF-BB: intact (both B chains are intact), single-clipped (one B chain is clipped), and double-clipped (both B chains are clipped). Clipping also creates new C-terminal sites for further C-terminal truncations and leads to a very complex mixture of isoforms. Routine baseline resolution of these three forms by various modes of HPLC proved unsuccessful. When the disulfide bonds of antiparallel chains are reduced, the complex peptide mixture can be analyzed by RP-HPLC; however, only the level of total clipping is identified. Since RC-CZE separation relies upon differences in molecular charge/size ratio, it can resolve the three rhPDGF-BB forms differing in the additional exposed residues. The choice of reversed-charge CZE columns (amine-coated column) allows proteins of high pI such as rhPDGF-BB (pI > 10) to be readily analyzed while minimizing protein loss from column adsorption. To simplify the electropherogram of clipped forms, the sample is treated first with carboxypeptidase B to reduce the charge microheterogeneity of partial Arg32 truncation. Analysis of rhPDGF-BB by RC-CZE yields a baseline separation between the three forms, intact and single- and double-clipped rhPDGF-BB.

Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection.

This paper describes the development and validation of a new off-line approach to quantitate both covalent and noncovalent, in-solution aggregates present in protein formulations and compares the new assay to established HPLC methods. This off-line analysis is well suited for use in QC release testing, formulation development and stability indicating applications. An inexpensive, continuous source HPLC fluorometer has been adapted with the addition of second order filters for use as a sensitive right-angle scatterometer which can determine the molecular weight of protein aggregates in solution. When used as an HPLC detector, right-angle light scattering is a sensitive method which can determine the molecular weight of peaks separable by HPLC, thus discriminating between monomers of different conformations and aggregates. The weight-averaged molecular weight of aggregate peaks can be calculated with system calibration, yielding the average number of monomers per aggregate. If the protein concentration is high enough for an adequate signal, the off-line technique of right-angle light scattering of protein formulations has advantages of convenience and speed over the HPLC approach. Samples are placed in standard fluorometer cuvettes and toluene is used as a calibrator. Data are presented which show the off-line (static) method to be extremely rapid, rugged and precise. The accuracy of this approach is demonstrated through cross-validation to traditional GPC analysis of protein aggregate distributions. This non-invasive light scattering approach is particularly useful when non-covalent protein aggregation is reversible and readily altered by chromatographic separations typically used for characterizing aggregates.

Capillary electrophoresis of abnormal hemoglobins associated with alpha-thalassemias.

Capillary electrophoresis was evaluated for separation of hemoglobin species associated with alpha-thalassemias, and for identification of hemoglobin variants commonly found in the same human populations. Separation of hemoglobins was achieved using capillary isoelectric focusing with chemical mobilization; visible-wavelength absorbance detection was used to identify hemoglobins against a background of nonheme-containing proteins. This technique could easily differentiate hemoglobins Bart's and H (associated with alpha-thalassemias) from hemoglobin variants. Analysis of globin chains derived from intact hemoglobins was performed by free zone capillary electrophoresis under denaturing conditions. This technique was useful for distinguishing Hb Bart's and Hb H, and for confirming the identity of hemoglobin variants.

Preliminary assessment of multidrug therapy in leprosy patients in Western Samoa.

Western Samoa is an island state in the Polynesian part of the South Pacific Ocean with a total population of around 160,000. In 5 July 1985 multidrug therapy (MDT) as advised by the WHO was started in leprosy patients there. One hundred and eighteen cases (99 males, 19 females) on the active list were included in the MDT programme. Sixty-three (53.3%) were paucibacillary and 55 (46.6%) were multibacillary. Eighteen patients (15.26%, 15 paucibacillary, 3 multibacillary) were below 15 years of age. In this paper we present a preliminary evaluation of WHO/MDT, in leprosy patients in Western Samoa as of 31 December 1987. Assessment was done on clinical, bacteriological and histopathological grounds. During the evaluation period the acceptance of MDT by Samoan patients was found adequate, even though surveillance is still continuing. As of 31 December 1987 there were 40 patients (34 male, 6 female) on the active list. Thirty-five (87.5%) were on multibacillary and 5 (12.5%) on paucibacillary regimen. Only 2 patients (both paucibacillary) were below 15 years of age. The prevalence rate was now estimated to be 0.25/1000. Leprosy cannot be considered to be a serious public health problem in the country. However there is still need for improvement in case-finding, recording and follow up matters.

Improved radioreceptor assay of opiate narcotics in human serum: application to fentanyl and morphine metabolism.

A recently developed radioreceptor assay (RRA) (1) that employs 3H-naloxone and rat brain membrane homogenates was improved two ways. First, the brain membranes were preincubated in the presence of sodium ions, and second, manganase-II ions were added to the sample incubations. These changes enhanced the assay sensitivity and reproducibility with stored membrane preparations and reduced the effects of serum constituents (Na+) on ligand-receptor binding. Patient sera were assayed by radioimmunoassay (RIA) and RRA after fentanyl administration and by high-performance liquid chromatography (HPLC) and RRA after morphine administration. The results with both fentanyl assays were comparable, and no fentanyl metabolites were detectable by RRA after HPLC of serum extracts. In contrast, preliminary results with the HPLC-RRA procedure suggest the presence of an active morphine metabolite of unknown structure in sera obtained from patients on morphine therapy.

[Molecular polarity dependence of the transformed steroid effect on Na,K-ATPase]. / Zavisimost' vliianiia transformirovannykh steroidov na Na,K-ATPazy ot bifil'nosti ikh molekul.

The transformed steroids containing an additional cycle E (delta-lactone or 16.23-pyranone) or 23-carbethoxy side chain (1.10(-5) M) inhibit Na,K-ATPase from pig kidney medulla. The steroid structure has a noticeable effect on ATPase inhibiton varying from 9 to 35%. The data obtained suggest that the position, stereochemistry and the uneven distribution of polar substituents in the steroid molecule are essential for ATPase inhibition by steroid aglycons.

Simultaneous determination of linear and branched residues in poly(ADP-ribose).

Methodology for the routine and simultaneous determination of the linear and branched residues of poly(ADP-ribose) is described. The main features of the procedure consist of the isolation of poly(ADP-ribose) by affinity chromatography; enzymatic digestion of the polymer to the unique nucleosides ribosyladenosine and diribosyladenosine which are derived from linear and branched residues, respectively; formation of fluorescent derivatives of ribosyladenosine and diribosyladenosine; and identification and quantification of these compounds by high-pressure liquid chromatography coupled with fluorescence detection. A variation on the methodology which allows the detection and quantification of ribosyladenosine and diribosyladenosine without formation of their fluorescent derivatives is also presented. Analyses of several cell lines for their capacity to synthesize poly(ADP-ribose) with a branched structure showed that the proportion of branched sites was constant (0.7-0.8%) in each of the cell lines.