Role of Dipolar Interactions on the Determination of the Effective Magnetic Anisotropy in Iron Oxide Nanoparticles.

Challenging magnetic hyperthermia (MH) applications of immobilized magnetic nanoparticles require detailed knowledge of the effective anisotropy constant (Keff ) to maximize heat release. Designing optimal MH experiments entails the precise determination of magnetic properties, which are, however, affected by the unavoidable concurrence of magnetic interactions in common experimental conditions. In this work, a mean-field energy barrier model (ΔE), accounting for anisotropy (EA ) and magnetic dipolar (ED ) energy, is proposed and used in combination with AC measurements to a specifically developed model system of spherical magnetic nanoparticles with well-controlled silica shells, acting as a spacer between the magnetic cores. This approach makes it possible to experimentally demonstrate the mean field dipolar interaction energy prediction with the interparticle distance, dij , ED ≈ 1/dij 3 and obtain the EA as the asymptotic limit for very large dij . In doing so, Keff uncoupled from interaction contributions is obtained for the model system (iron oxide cores with average sizes of 8.1, 10.2, and 15.3 nm) revealing to be 48, 23, and 11 kJ m-3 , respectively, close to bulk magnetite/maghemite values and independent from the specific spacing shell thicknesses selected for the study.

Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3.

Peroxiredoxin 3 (PRDX3) encodes a mitochondrial antioxidant protein, which is essential for the control of reactive oxygen species homeostasis. So far, PRDX3 mutations are involved in mild-to-moderate progressive juvenile onset cerebellar ataxia. We aimed to unravel the molecular bases underlying the disease in an infant suffering from cerebellar ataxia that started at 19 months old and presented severe cerebellar atrophy and peripheral neuropathy early in the course of disease. By whole exome sequencing, we identified a novel homozygous mutation, PRDX3 p.D163E, which impaired the mitochondrial ROS defense system. In mouse primary cortical neurons, the exogenous expression of PRDX3 p.D163E was reduced and triggered alterations in neurite morphology and in mitochondria. Mitochondrial computational parameters showed that p.D163E led to serious mitochondrial alterations. In transfected HeLa cells expressing the mutation, mitochondria accumulation was detected by correlative light electron microscopy. Mitochondrial morphology showed severe changes, including extremely damaged outer and inner membranes with a notable cristae disorganization. Moreover, spherical structures compatible with lipid droplets were identified, which can be associated with a generalized response to stress and can be involved in the removal of unfolded proteins. In the patient's fibroblasts, PRDX3 expression was nearly absent. The biochemical analysis suggested that the mutation p.D163E would result in an unstable structure tending to form aggregates that trigger unfolded protein responses via mitochondria and endoplasmic reticulum. Altogether, our findings broaden the clinical spectrum of the recently described PRDX3-associated neurodegeneration and provide new insight into the pathological mechanisms underlying this new form of cerebellar ataxia.

Significant Surface Spin Effects and Exchange Bias in Iron Oxide-Based Hollow Magnetic Nanoparticles.

Exchange bias (EB) properties have become especially important in hollow magnetic nanoparticles (MNPs) due to the versatility and reduced size of these materials. In this work, we present the synthesis and study of the EB properties of iron-oxide-based hollow MNPs and their precursors Fe/iron oxide MNPs with core/void/shell structure. The two mechanisms involved in EB generation were investigated: the frozen spins present in the nanograins that form the nanoparticles and the surface spins. The effect of external parameters on the coercivity (HC), remanence (MR), exchange bias field (HEB) and frozen spins, such as cooling field (HFC) and temperature, was investigated. Both HC and HEB present a maximum threshold above which their values begin to decrease with HFC, showing a new trend of HEB with HFC and allowing modulation on demand. The existence of surface spins, present on the outer and inner surfaces, was demonstrated, and an intrinsic EB phenomenon (HEB = 444 Oe for hollow iron oxide-based MNPs of 13.1 nm) with significant magnetization (MS~50 emu/g) was obtained. Finally, core/void/shell MNPs of 11.9 nm prior to the formation of the hollow MNPs showed a similar behavior, with non-negligible HEB, highlighting the importance of surface spins in EB generation.

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Nrg1 haploinsufficiency alters inhibitory cortical circuits.

Neuregulin 1 (NRG1) and its receptor ERBB4 are schizophrenia (SZ) risk genes that control the development of both excitatory and inhibitory cortical circuits. Most studies focused on the characterization ErbB4 deficient mice. However, ErbB4 deletion concurrently perturbs the signaling of Nrg1 and Neuregulin 3 (Nrg3), another ligand expressed in the cortex. In addition, NRG1 polymorphisms linked to SZ locate mainly in non-coding regions and they may partially reduce Nrg1 expression. Here, to study the relevance of Nrg1 partial loss-of-function in cortical circuits we characterized a recently developed haploinsufficient mouse model of Nrg1 (Nrg1tm1Lex). These mice display SZ-like behavioral deficits. The cellular and molecular underpinnings of the behavioral deficits in Nrg1tm1Lex mice remain to be established. With multiple approaches including Magnetic Resonance Spectroscopy (MRS), electrophysiology, quantitative imaging and molecular analysis we found that Nrg1 haploinsufficiency impairs the inhibitory cortical circuits. We observed changes in the expression of molecules involved in GABAergic neurotransmission, decreased density of Vglut1 excitatory buttons onto Parvalbumin interneurons and decreased frequency of spontaneous inhibitory postsynaptic currents. Moreover, we found a decreased number of Parvalbumin positive interneurons in the cortex and altered expression of Calretinin. Interestingly, we failed to detect other alterations in excitatory neurons that were previously reported in ErbB4 null mice suggesting that the Nrg1 haploinsufficiency does not entirely phenocopies ErbB4 deletions. Altogether, this study suggests that Nrg1 haploinsufficiency primarily affects the cortical inhibitory circuits in the cortex and provides new insights into the structural and molecular synaptic impairment caused by NRG1 hypofunction in a preclinical model of SZ.

Axial Ligation in Ytterbium(III) DOTAM Complexes Rationalized with Multireference and Ligand-Field ab Initio Calculations.

The nature of the axial ligand coordinated to the Yb3+ ion in [Yb(DOTAM)]3+ has profound consequences in the magnetic anisotropy and optical properties of the complex, as evidenced by 1H NMR and UV-vis spectroscopies. The pseudocontact shifts of 1H nuclei and the 2F5/2 ← 2F7/2 absorption band were found to be very sensitive to the nature of the axial ligand (MeOH, H2O, MeOH, or F-). The energy levels of the 2F5/2 and 2F7/2 manifolds in [Yb(DOTAM)(X)]3+ (X = MeOH, H2O, or dimethyl sulfoxide (DMSO)) and [Yb(DOTAM)F]2+ complexes were assigned from the analysis of the optical spectra and ab initio calculations based on CASSCF wave functions that considered dynamic correlation through perturbation theory (NEVPT2) and spin-orbit coupling effects. The magnetic anisotropies obtained with ab initio calculations are in good agreement with the experimental values derived from 1H NMR spectral data, though for the [Yb(DOTAM)(H2O)]3+ and [Yb(DOTAM)F]2+ complexes, the explicit inclusion of a few second-sphere water molecules is required to improve the calculated data. Crystal-field calculations show that the observed pseudocontact shifts do not correlate well with the crystal-field parameter B20, as predicted by Bleaney's theory. The change in the sign of the magnetic anisotropy from prolate (X = MeOH, H2O, or DMSO) to oblate in [Yb(DOTAM)F]2+ is related to the relative energies of the 4fz3 orbital and the 4fx3/4fy3 pair, which are affected by the coordination ability of the axial ligand.

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

Polyunsaturated Fatty acids modify the gating of kv channels.

Polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which are attributed to their capability to modulate ion channels. This PUFAs ability has been reported to be due to their effects on the gating properties of ion channels. In the present review, we will focus on the role of PUFAs on the gating of two Kv channels, Kv1.5 and Kv11.1. Kv1.5 channels are blocked by n-3 PUFAs of marine [docosahexaenoic acid (DHA) and eicosapentaenoic acid] and plant origin (alpha-linolenic acid, ALA) at physiological concentrations. The blockade of Kv1.5 channels by PUFAs steeply increased in the range of membrane potentials coinciding with those of Kv1.5 channel activation, suggesting that PUFAs-channel binding may derive a significant fraction of its voltage sensitivity through the coupling to channel gating. A similar shift in the activation voltage was noted for the effects of n-6 arachidonic acid (AA) and DHA on Kv1.1, Kv1.2, and Kv11.1 channels. PUFAs-Kv1.5 channel interaction is time-dependent, producing a fast decay of the current upon depolarization. Thus, Kv1.5 channel opening is a prerequisite for the PUFA-channel interaction. Similar to the Kv1.5 channels, the blockade of Kv11.1 channels by AA and DHA steeply increased in the range of membrane potentials that coincided with the range of Kv11.1 channel activation, suggesting that the PUFAs-Kv channel interactions are also coupled to channel gating. Furthermore, AA regulates the inactivation process in other Kv channels, introducing a fast voltage-dependent inactivation in non-inactivating Kv channels. These results have been explained within the framework that AA closes voltage-dependent potassium channels by inducing conformational changes in the selectivity filter, suggesting that Kv channel gating is lipid dependent.

Effects of n-3 Polyunsaturated Fatty Acids on Cardiac Ion Channels.

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, and these effects have been attributed to their capability to modulate ion channels. In the present review, we will focus on the effects of PUFAs on a cardiac sodium channel (Na(v)1.5) and two potassium channels involved in cardiac atrial and ventricular repolarization (K(v)) (K(v)1.5 and K(v)11.1). n-3 PUFAs of marine (docosahexaenoic, DHA and eicosapentaenoic acid, EPA) and plant origin (alpha-linolenic acid, ALA) block K(v)1.5 and K(v)11.1 channels at physiological concentrations. Moreover, DHA and EPA decrease the expression levels of K(v)1.5, whereas ALA does not. DHA and EPA also decrease the magnitude of the currents elicited by the activation of Na(v)1.5 and calcium channels. These effects on sodium and calcium channels should theoretically shorten the cardiac action potential duration (APD), whereas the blocking actions of n-3 PUFAs on K(v) channels would be expected to produce a lengthening of cardiac action potential. Indeed, the effects of n-3 PUFAs on the cardiac APD and, therefore, on cardiac arrhythmias vary depending on the method of application, the animal model, and the underlying cardiac pathology.

Stereoselective interactions between local anesthetics and ion channels.

Local anesthetics are useful probes of ion channel function and structure. Stereoselective interactions are especially interesting because they can reveal three-dimensional relationships between drugs and channels with otherwise identical biophysical and physicochemical properties. Furthermore, stereoselectivity suggests direct and specific receptor-mediated action, and identification of such stereospecific interactions may have important clinical consequences. The fact that drug targets are able to discriminate between the enantiomers present in a racemic drug is the consequence of the ordered asymmetric macromolecular units that form living cells. However, almost 25% of the drugs used in the clinical practice are racemic mixtures, and their individual enantiomers frequently differ in both their pharmacodynamic and pharmacokinetic profiles. Moreover, their effects can be similar to or different from the pharmacological effect of the drug and may contribute to the undesired effects of the drug. In other cases, the pharmacological effects induced by the two enantiomers on the molecular target are opposite. In the present manuscript, we will review the stereoselective effects of bupivacaine-like local anesthetics on cardiac sodium and potassium channels.

Protein kinase C (PKC) activity regulates functional effects of Kvß1.3 subunit on KV1.5 channels: identification of a cardiac Kv1.5 channelosome.

K(v)1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (I(Kur)). The regulatory K(v)ß1.3 subunit converts K(v)1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by K(v)ß1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack K(v)ß subunits) transiently cotransfected with K(v)1.5+K(v)ß1.3 and also rat ventricular and atrial tissue to study native α-ß subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that K(v)1.5 and K(v)ß1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, K(v)ß1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed K(v)ß1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of K(v)1.5 and K(v)ß1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between K(v)1.5, K(v)ß1.3, the receptor for activated C kinase (RACK1), PKCßI, PKCßII, and PKCθ in HEK293 cells. A very similar K(v)1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.

Relationship between IL-6/ERK and NF-κB: a study in normal and pathological human prostate gland.

BACKGROUND: There is growing evidence that inflammation is a causal factor in cancer, where pro-inflammatory cytokines such as IL-6, IL-1 or TNF-α could induce cellular proliferation by activation of NF-κB. This study focuses on the IL-6/ERK transduction pathway, its relationship with NF-κB, and the consequences of dysregulation in the development of prostate pathologies such as benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate cancer (PC). METHODS: Immunohistochemical and Western blot analyses for IL-6, gp-130, Raf-1, MEK-1, ERK-1, p-MEK, ERK-2, p-ERK, NF-κB/p-50 and NF-κB/p-65 were carried out in 20 samples of normal prostate glands, 35 samples of BPH, 27 samples with a diagnosis of PIN (low-grade PIN or high-grade PIN), and 95 samples of PC (23 with low, 51 with medium and 21 with high Gleason scores). RESULTS: Immunoreaction to IL-6, gp-130, ERK-1, ERK-2, p-ERK and NF-κB/p50 was found in the cytoplasm of epithelial cells in normal prostate samples; p-MEK was found in the nucleus of epithelial cells; but not expression to Raf-1, MEK-1 and NF-κB/p65. In BPH, all of these proteins were immunoexpressed, while there was increased immunoexpression of IL-6, gp-130, p-MEK, ERK-1, ERK-2 and NF-κB/p50 (cytoplasm). In PC, immunoexpression of IL-6 and gp-130 were similar to that found in BPH; while immunoexpression of Raf-1, MEK-1, p-MEK, ERK-1, ERK-2, p-ERK, NF-κB/p50 (nucleus and cytoplasm), and NF-κB/p65 (nucleus and cytoplasm) was higher than in BPH. CONCLUSION: Translocation of NF-κB to the nucleus in PC and high-grade PIN could be stimulated by the IL-6/ERK transduction pathway, but might also be stimulated by other transduction pathways, such as TNF-α/NIK, TNF/p38, IL-1/NIK or IL-1/p38. Activation of NF-κB in PC could regulate IL-6 expression. These transduction pathways are also related to activation of other transcription factors such as Elk-1, ATF-2 or c-myc (also involved in cell proliferation and survival). PC is a heterogeneous disease, where multiple transduction pathways might alter the apoptosis/proliferation balance. Significant attention should be give to the combination of novel agents directed towards inactivation of pro-inflammatory cytokines than can disrupt tumour cell growth.

Características del mal de ojo en buenavista, estudio antropológico y descriptivo: Bogotá abril a noviembre de 1989

Las enfermedades culturales son un grupo de fenómenos relativamente frecuentes en muchas de nuestras comunidades y sin embargo muy poco investigadas por la medicina. El presente trabajo estudia al Mal de Ojo como una de ellas, analizando sus características generales (origen, etiología, sintomatología,características del ojeador, diagnóstico y tratamiento), e igualmente desarrolla una discusión teórica sobre la influencia de la cultura en el enfermar. Para investigar las características del fenomeno en una población concreta, se seleccionó una muestra de 175 madres de familia de un barrio de los Cerros del Norte de Bogotá (Buenavista, y en ella se aplicó un formulario que evaluaba las características mencionadas sobre el Mal de Ojo. Asi mismo se incluyeron preguntas sobre edad, escolaridad, procedencia, ocupación, tiempo de residencia en las ciudades, y además sobre el concepto general de enfermedad para ver su relación con la creencia o no en el fenómeno estudiado. Con relación al concepto de enfermedad, encontramos una fusión de elementos "miasmáticos", etiopatológicos (derivados del coantacto con las instituciones de salud y en general del fenómeno de aculturación por el cambio a la vida urbana) y de otros mágico-empiricos (que se destacan como los pricipales), lo cual justifica en gran parte el hallazgo de una prevalencia importante de dicho fenómeno en nuestro estudio: el 28 por ciento