Novel nanostructured lipid carriers loading Apigenin for anterior segment ocular pathologies.

Dry eye disease (DED) is a chronic multifactorial disorder of the ocular surface caused by tear film dysfunction and constitutes one of the most common ocular conditions worldwide. However, its treatment remains unsatisfactory. While artificial tears are commonly used to moisturize the ocular surface, they do not address the underlying causes of DED. Apigenin (APG) is a natural product with anti-inflammatory properties, but its low solubility and bioavailability limit its efficacy. Therefore, a novel formulation of APG loaded into biodegradable and biocompatible nanoparticles (APG-NLC) was developed to overcome the restricted APG stability, improve its therapeutic efficacy, and prolong its retention time on the ocular surface by extending its release. APG-NLC optimization, characterization, biopharmaceutical properties and therapeutic efficacy were evaluated. The optimized APG-NLC exhibited an average particle size below 200 nm, a positive surface charge, and an encapsulation efficiency over 99 %. APG-NLC exhibited sustained release of APG, and stability studies demonstrated that the formulation retained its integrity for over 25 months. In vitro and in vivo ocular tolerance studies indicated that APG-NLC did not cause any irritation, rendering them suitable for ocular topical administration. Furthermore, APG-NLC showed non-toxicity in an epithelial corneal cell line and exhibited fast cell internalization. Therapeutic benefits were demonstrated using an in vivo model of DED, where APG-NLC effectively reversed DED by reducing ocular surface cellular damage and increasing tear volume. Anti-inflammatory assays in vivo also showcased its potential to treat and prevent ocular inflammation, particularly relevant in DED patients. Hence, APG-NLC represent a promising system for the treatment and prevention of DED and its associated inflammation.

Brain event-related potentials predict individual differences in inhibitory control.

Stop-signal reaction time (SSRT), the time needed to cancel an already-initiated motor response, quantifies individual differences in inhibitory control. Electrophysiological correlates of SSRT have primarily focused on late event-related potential (ERP) components over midline scalp regions from successfully inhibited stop trials. SSRT is robustly associated with the P300, there is mixed evidence for N200 involvement, and there is little information on the role of early ERP components. Here, machine learning was first used to interrogate ERPs during both successful and failed stop trials from 64 scalp electrodes at 4 ms resolution (n = 148). The most predictive model included data from both successful and failed stop trials, with a cross-validated Pearson's r of 0.32 between measured and predicted SSRT, significantly higher than null models. From successful stop trials, spatio-temporal features overlapping the N200 in right frontal areas and the P300 in frontocentral areas predicted SSRT, as did early ERP activity (<200 ms). As a demonstration of the reproducibility of these findings, the application of this model to a separate dataset of 97 participants was also significant (r = 0.29). These results show that ERPs during failed stops are relevant to SSRT, and that both early and late ERP activity contribute to individual differences in SSRT. Notably, the right lateralized N200, which predicted SSRT here, is not often observed in neurotypical adults. Both the ascending slope and peak of the P300 component predicted SSRT. These results were replicable, both within the training sample and when applied to ERPs from a separate dataset.

Re-emergence of rabies virus maintained by canid populations in Paraguay.

Paraguay has registered no human cases of rabies since 2004, and the last case in dogs, reported in 2009, was due to a variant maintained in the common vampire bat "Desmodus rotundus". In 2014, a dog was diagnosed as positive for rabies with aggression towards a boy and all required measures of control were successfully adopted. Epidemiological investigation revealed that the dog was not vaccinated and had been attacked by a crab-eating fox, "zorro" (Cerdocyon thous). The sample was diagnosed by the Official Veterinary Service of the Country and sent to the Center on Rabies Research from the University of São Paulo, Brazil, for antigenic and genetic characterization. A second sample from a dog positive for rabies in the same region in 2015 and 11 samples from a rabies outbreak from Asuncion in 1996 were also characterized. The antigenic profile of the samples, AgV2, was compatible with one of the variants maintained by dogs in Latin America. In genetic characterization, the samples segregated in the canine (domestic and wild species)-related group in an independent subgroup that also included samples from Argentina. These results and the epidemiology of the case indicate that even with the control of rabies in domestic animals, the virus can still circulate in wildlife and may be transmitted to domestic animals and humans, demonstrating the importance of continuous and improved surveillance and control of rabies, including in wild species, to prevent outbreaks in controlled areas.

Coordinative task difficulty and behavioural errors are associated with increased long-range beta band synchronization.

The neural network and the task-dependence of (local) activity changes involved in bimanual coordination are well documented. However, much less is known about the functional connectivity within this neural network and its modulation according to manipulations of task complexity. Here, we assessed neural activity via high-density electroencephalography, focussing on changes of activity in the beta frequency band (~15-30Hz) across the motor network in 26 young adult participants (19-29 years old). We investigated how network connectivity was modulated with task difficulty and errors of performance during a bimanual visuomotor movement consisting of dial rotation according to three different ratios of speed: an isofrequency movement (1:1), a non-isofrequency movement with the right hand keeping the fast pace (1:3), and the converse ratio with the left hand keeping the fast pace (3:1). To quantify functional coupling, we determined neural synchronization which might be key for the timing of the activity within brain regions during task execution. Individual source activity with realistic head models was reconstructed at seven regions of interest including frontal and parietal areas, among which we estimated phase-based connectivity. Partial least squares analysis revealed a significant modulation of connectivity with task difficulty, and significant correlations between connectivity and errors in performance, in particular between sensorimotor cortices. Our findings suggest that modulation of long-range synchronization is instrumental for coping with increasing task demands in bimanual coordination.

Understanding bimanual coordination across small time scales from an electrophysiological perspective.

Bimanual movement involves a variety of coordinated functions, ranging from elementary patterns that are performed automatically to complex patterns that require practice to be performed skillfully. The neural dynamics accompanying these coordination patterns are complex and rapid. By means of electro- and magneto-encephalographic approaches, it has been possible to examine these dynamics during bimanual coordination with excellent temporal resolution, which complements other neuroimaging modalities with superb spatial resolution. This review focuses on EEG/MEG studies that unravel the processes involved in movement planning and execution, motor learning, and executive functions involved in task switching and dual tasking. Evidence is presented for a spatio-temporal reorganization of the neural networks within and between hemispheres to meet increased task difficulty demands, induced or spontaneous switches in coordination mode, or training-induced neuroplastic modulation in coordination dynamics. Future theoretical developments will benefit from the integration of research techniques unraveling neural activity at different time scales. Ultimately this work will contribute to a better understanding of how the human brain orchestrates complex behavior via the implementation of inter- and intra-hemispheric coordination networks.

A 1.3-mb 7q11.23 atypical deletion identified in a cohort of patients with williams-beuren syndrome.

Williams-Beuren syndrome is a rare multisystem neurodevelopmental disorder caused by a 1.55-1.84-Mb hemizygous deletion on chromosome 7q11.23. The classical phenotype consists of characteristic facial features, supravalvular aortic stenosis, intellectual disability, overfriendliness, and visuospatial impairment. So far, 26-28 genes have been shown to contribute to the multisystem phenotype associated with Williams-Beuren syndrome. Among them, haploinsufficiency of the ELN gene has been shown to cause the cardiovascular anomalies. Identification of patients with atypical deletions has provided valuable information for genotype-phenotype correlation, in which other genes such as LIMK1,CLIP2, GTF2IRD1, or GTF2I have been correlated with specific cognitive profiles or craniofacial features. Here, we report the clinical and molecular characteristics of a patient with an atypical deletion that does not include the GTF2I gene and only partially includes the GTF2IRD1 gene.

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain.

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aß42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

[Behavioral factors and sensorial identificatory deficits as predictors for dementia of the Alzheimer's type]. / Factores de comportamiento y déficit sensoriales identificatorios como predictores de la demencia tipo Alzheimer.

INTRODUCTION: Several behavior factors had been studied as associated to Alzheimer, but not its prediction power. AIM: To examine whether five behavior variables (psychosocial introversion, coping deficit, apathy, demotivation and perception blockade) could predict the beginning of dementia in mild cognitive impairment initially non dementia cases, after a six-year follow-up period. SUBJECTS AND METHODS: 197 mild cognitive impairment non dementia cases (mean: 72.6 years) and an equal number age, gender, schooling and familial income matched normal cases (mean: 73.0 years) were selected to participate from an university data base of older than 60 year people initiated at 1994 for epidemiologic multifactorial studies. Behavior variables were recovery by means of a protocol, applied directly or with the help of relative caregivers, and designed under the hypothetical theory of Alzheimer as a conclusion of a progressive psychosocial capsuletion's process. RESULTS: Introversion, coping deficit, apathy and demotivation appear as frequently and progressive but not necessarily simultaneously or unchained factors, but with possibility of reversion or taking a different way. Perception blockade (three identificatory component of senses impairment at least) seems like the irreversible phase in the process, with near 100% of previous detection for the final diagnostic of Alzheimer's cases. CONCLUSION: Perception blockade, in spite of its great fluctuation and individual variations, appears as more objective, easy, non invasive and firmly as reveille and predictor of this dementia, and we suggest that it appears as the immediate cause of the wide reported histopathological disorders by way of a disintegration of neural net owing to the normal reinforcement deficit.

Factores de comportamiento y déficit sensoriales identificatorios como predictores de la demencia tipo Alzheimer / Behavioral factors and sensorial identificatory deficits as predictors for dementia of the Alzheimer´s type

Introducción. Se sabe que diversos comportamientos se hallan asociados a la demencia tipo Alzheimer, pero no se ha explorado su poder predictor. Objetivo. Determinar si en adultos mayores de 60 años inicialmente con afectaciones cognitivas o ejecutivas sin demencia, cinco factores de comportamiento (introversión psicosocial, déficit de afrontamiento de pérdidas personales, apatía, desmotivación y bloqueo perceptivo) pueden predecir dicha demencia. Sujetos y métodos. 197 personas con déficit cognitivo leve sin demencia (edad media: 72,6 años) e igual número con envejecimiento normal (edad media: 73 años), igualadas en edad, género, escolaridad e ingresos, se seleccionaron de una base de datos iniciada en 1994 para estudios epidemiológicos multifactoriales. Se utilizó un protocolo aplicado bien directamente o a través de familiares cuidadores, y diseñado especialmente bajo la teoría hipotética del Alzheimer como la culminación de un proceso progresivo de aislamiento social. Resultados. La introversión, el déficit de afrontamiento de las pérdidas personales, la apatía y la desmotivación se muestran frecuentes y los dos últimos progresivos pero no siempre simultáneos o encadenados, aunque con la posibilidad de reversión o de tomar un camino diferente al Alzheimer. El bloqueo perceptivo (déficit de al menos tres componentes identificatorios de los sentidos) aparece como la instancia irreversible del proceso, con cerca del 100% de detección previa a los casos diagnosticados como Alzheimer. Conclusiones. El bloqueo sensorial, pese a ser fluctuante y variable individualmente, es el indicador más objetivo, no invasivo, sencillo y firme como diana y predictor del curso hacia esta demencia. Se conjetura que el déficit sensorial identificatorio puede causar la desintegración de las redes neuronales por déficit de reforzamiento y conducir a las alteraciones de las funciones cerebrales típicas del Alzheimer

Introduction. Several behavior factors had been studied as associated to Alzheimer, but not its prediction power. Aim. To examine whether five behavior variables (psychosocial introversion, coping deficit, apathy, demotivation and perception blockade) could predict the beginning of dementia in mild cognitive impairment initially non dementia cases, after a six-year follow-up period. Subjects and methods. 197 mild cognitive impairment non dementia cases (mean: 72.6 years) and an equal number age, gender, schooling and familial income matched normal cases (mean: 73.0 years) were selected to participate from an university data base of older than 60 year people initiated at 1994 for epidemiologic multifactorial studies. Behavior variables were recovery by means of a protocol, applied directly or with the help of relative caregivers, and designed under the hypothetical theory of Alzheimer as a conclusion of a progressive psychosocial capsuletion’s process. Results. Introversion, coping deficit, apathy and demotivation appear as frequently and progressive but not necessarily simultaneously or unchained factors, but with possibility of reversion or taking a different way. Perception blockade (three identificatory component of senses impairment at least) seems like the irreversible phase in the process, with near 100% of previous detection for the final diagnostic of Alzheimer’s cases. Conclusion. Perception blockade, in spite of its great fluctuation and individual variations, appears as more objective, easy, non invasive and firmly as reveille and predictor of this dementia, and we suggest that it appears as the immediate cause of the wide reported histopathological disorders by way of a disintegration of neural net owing to the normal reinforcement deficit

Two isoforms of the thyrotropin-releasing hormone receptor generated by alternative splicing have indistinguishable functional properties.

Two cDNA clones encoding two different isoforms of the thyrotropin-releasing hormone receptor (TRH-R) from GH3 rat anterior pituitary cells have been isolated. One isoform corresponds to the TRH-R(412) receptor previously described (de la Peña, P., Delgado, L. M., del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899). The second one, named TRH-R(387), contains a 52-base pair deletion, which yields a new variant of the receptor protein 25 amino acid shorter and which contains 12 new residues on its carboxyl terminus. This new isoform is produced by alternative splicing of a retained intron in the primary transcript of a gene represented only once in the rat genome. Furthermore, the perfect colinearity between genomic DNA and TRH-R(412) cDNA demonstrates that no other introns are present within the coding region of the TRH receptor gene. Functional expression in Xenopus laevis oocytes indicates that both cDNAs encode fully functional TRH receptors. Otherwise, indistinguishable electrophysiological responses to TRH are evoked in oocytes expressing both receptor isoforms.

Okadaic acid and calyculin A enhance the effect of thyrotropin-releasing hormone on GH3 rat anterior pituitary cells excitability.

Thyrotropin-releasing hormone (TRH) causes a transient hyperpolarization followed by several minutes of increased action potential frequency in patch-perforated current-clamped GH3 cells. Treatment of cells for 5 min with either 2 or 100 nM of the protein phosphatase inhibitor okadaic acid does not affect electrical activity of the cells, but potentiates the enhancement of action potential frequency elicited by a subsequent addition of TRH. Alternatively, 100 nM (but not 2 nM) of okadaic acid added during the second phase of TRH action, further increases the frequency of firing above that produced by the hormone. Similar effects to those of 2 nM okadaic acid are observed with 20 nM calyculin A. These data suggest that a protein phosphatase plays a major role in regulating the delayed effects of TRH on cell excitability in GH3 cells.

Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells.

Hyperpolarization of patch-perforated GH3 rat anterior pituitary cells in high-K+ Ca(2+)-free medium reveals an inwardly rectifying K+ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs+, Ba2+, Cd2+ and Co2+, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH3 cell inwardly rectifying K+ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.

Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells.

Functional thyrotropin-releasing hormone (TRH) receptors have been expressed in Xenopus laevis oocytes following the microinjection of total and poly(A)+ RNA from GH3 rat anterior pituitary tumour cells. Under voltage-clamp conditions, application of the peptide induced a biphasic Ca(2+)-dependent chloride current. The amplitude of the initial, fast, component of the response was dependent on the concentration of the hormone and on the amount of mRNA injected. Size fractionation of poly(A)+ RNA on a continuous sucrose gradient and Northern blot analysis indicated that the receptor was encoded by an mRNA of approx. 3.5 kb. A 3.28 kbp cDNA encoding the TRH receptor has been cloned and sequenced. Full functionality of the predicted 412-amino-acid receptor protein was demonstrated by functional expression of cell surface receptors in Xenopus oocytes after both cytoplasmic injection of sense RNA transcribed in vitro from this cDNA and nuclear injection of the cDNA under the control of the Herpes simplex virus thymidine kinase promoter. The predicted protein contains seven putative membrane-spanning domains and shows significant sequence identify with some G-protein-coupled receptors. RNA blot analysis indicates that the mRNA for the TRH receptor is exclusively expressed in the pituitary gland. Expression studies performed with clones in which the 3' region of the mRNA has been successively shortened indicate that the 3' terminal region is not an important determinant for efficient functional expression in oocytes.

Effects of hypothalamic peptides on electrical activity and membrane currents of 'patch perforated' clamped GH3 anterior pituitary cells.

'Perforated-patch' recordings of rat anterior pituitary GH3 cells allow long and stable monitoring of electrical activity and membrane currents. Under current clamp conditions, the biphasic effect of thryotropin releasing hormone (TRH) consisting of a transient hyperpolarization followed by a longer phase of increased action potential frequency is fully preserved. Somatostatin suppresses action potential activity and antagonizes the second phase of enhanced spiking caused by TRH. Voltage clamp records of isolated currents indicate that TRH affects calcium-dependent potassium currents, but does not alter either voltage-dependent potassium or calcium currents at times and concentrations at which the electrical activity is increased.