A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 µM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection. We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Dog hepatocytes are key effector cells in the liver innate immune response to Leishmania infantum.

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

The efficacy of HRAS and CDK4/6 inhibitors in anaplastic thyroid cancer cell lines.

PURPOSE: Anaplastic thyroid carcinomas (ATCs) are non-responsive to multimodal therapy, representing one of the major challenges in thyroid cancer. Previously, our group has shown that genes involved in cell cycle are deregulated in ATCs, and the most common mutations in these tumours occurred in cell proliferation and cell cycle related genes, namely TP53, RAS, CDKN2A and CDKN2B, making these genes potential targets for ATCs treatment. Here, we investigated the inhibition of HRAS by tipifarnib (TIP) and cyclin D-cyclin-dependent kinase 4/6 (CDK4/6) by palbociclib (PD), in ATC cells. METHODS: ATC cell lines, mutated or wild type for HRAS, CDKN2A and CDKN2B genes, were used and the cytotoxic effects of PD and TIP in each cell line were evaluated. Half maximal inhibitory concentration (IC50) values were determined for these drugs and its effects on cell cycle, cell death and cell proliferation were subsequently analysed. RESULTS: Cell culture studies demonstrated that 0.1 µM TIP induced cell cycle arrest in the G2/M phase (50%, p < 0.01), cell death, and inhibition of cell viability (p < 0.001), only in the HRAS mutated cell line. PD lowest concentration (0.1 µM) increased significantly cell cycle arrest in the G0/G1 phase (80%, p < 0.05), but only in ATC cell lines with alterations in CDKN2A/CDKN2B genes; additionally, 0.5 µM PD induced cell death. The inhibition of cell viability by PD was more pronounced in cells with alterations in CDKN2A/CDKN2B genes (p < 0.05) and/or cyclin D1 overexpression. CONCLUSIONS: This study suggests that TIP and PD, which are currently in clinical trials for other types of cancer, may play a relevant role in ATC treatment, depending on the specific tumour molecular profile.

Enhanced dissolution of ibuprofen using ionic liquids as catanionic hydrotropes.

The therapeutic effectiveness of a drug largely depends on its bioavailability, and thus ultimately on its aqueous solubility. Hydrotropes are compounds able to enhance the solubility of hydrophobic substances in aqueous media and therefore are extensively used in the formulation of drugs and personal care products. Recently, some ionic liquids were shown to display a strong ability to enhance the solubility of biomolecules through hydrotropy. In this work, the impact of the ionic liquid chemical structures and their concentration on the solubility of ibuprofen was evaluated and compared with the performance of conventional hydrotropes. The results obtained clearly evidence the exceptional capacity of ionic liquids to enhance the solubility of ibuprofen. [C4C1im][SCN] and [C4C1im][N(CN)2] seem to be the most promising ionic liquids for ibuprofen solubilisation, where an increase in the solubility of 60- and 120-fold was observed with ionic liquid concentrations of circa 1 mol kg-1, respectively. Dynamic light scattering and molecular dynamics simulations were used to investigate the mechanism of the IL-mediated drug solubility and the results obtained indicate that the structure of aqueous solutions of ionic liquids and the role it plays in the formation of ionic liquid-drug aggregates is the mechanism driving the hydrotropic dissolution.

Skin hydration analysis by experiment and computer simulations and its implications for diapered skin.

BACKGROUND: Experimental work on skin hydration is technologically challenging, and mostly limited to observations where environmental conditions are constant. In some cases, like diapered baby skin, such work is practically unfeasible, yet it is important to understand potential effects of diapering on skin condition. To overcome this challenge, in part, we developed a computer simulation model of reversible transient skin hydration effects. METHODS: Skin hydration model by Li et al. (Chem Eng Sci, 138, 2015, 164) was further developed to simulate transient exposure conditions where relative humidity (RH), wind velocity, air, and skin temperature can be any function of time. Computer simulations of evaporative water loss (EWL) decay after different occlusion times were compared with experimental data to calibrate the model. Next, we used the model to investigate EWL and SC thickness in different diapering scenarios. RESULTS: Key results from the experimental work were: (1) For occlusions by RH=100% and free water longer than 30 minutes the absorbed amount of water is almost the same; (2) Longer occlusion times result in higher water absorption by the SC. The EWL decay and skin water content predictions were in agreement with experimental data. Simulations also revealed that skin under occlusion hydrates mainly because the outflux is blocked, not because it absorbs water from the environment. Further, simulations demonstrated that hydration level is sensitive to time, RH and/or free water on skin. In simulated diapering scenarios, skin maintained hydration content very close to the baseline conditions without a diaper for the entire duration of a 24 hours period. CONCLUSION: Different diapers/diaper technologies are known to have different profiles in terms of their ability to provide wetness protection, which can result in consumer-noticeable differences in wetness. Simulation results based on published literature using data from a number of different diapers suggest that diapered skin hydrates within ranges considered reversible.

Cytotoxic and genotoxic potential of geraniol in peripheral blood mononuclear cells and human hepatoma cell line (HepG2).

Geraniol is an acyclic monoterpene alcohol present in the essential oil of many aromatic plants and is one of the most frequently used molecules by the flavor and fragrance industries. The literature also reports its therapeutic potential, highlighting itself especially as a likely molecule for the development of drugs against cancer. In view of these considerations, this study was designed to evaluate the cytotoxic and genotoxic potential of geraniol, in an in vitro protocol, using two types of human cells: one without the ability to metabolize (peripheral blood mononuclear cells - PBMC), and the other with this capability (human hepatoma cell line - HepG2) through the comet assay and the micronucleus test. Four concentrations (10, 25, 50, and 100 µg/mL) were selected for the genotoxic assessment for PBMC and three (1.25, 2.5, and 5 µg/mL) for HepG2 cells based on cytotoxicity tests (MTT assay). Results showed that geraniol did not present genotoxic or clastogenic/aneugenic effects on both cell types under the conditions studied. However, caution is advised in the use of this substance by humans, since a significant reduction in viability of HepG2 and a marked decrease in cell viability on normal PBMC were verified.

Dynamics of glycerine and water transport across human skin from binary mixtures.

OBJECTIVE: Skin transport properties of glycerine and water from binary mixtures contacting human skin were determined to better understand the mechanism of skin moisturization by aqueous glycerine formulations. METHODS: Steady-state permeation for 3 H2 O and 14 C-glycerine across split-thickness human skin in vitro and desorption dynamics of the same permeants in isolated human stratum corneum (HSC) were experimentally determined under near equilibrium conditions. These data were compared to a priori values developed in the context of a thermodynamic model for binary mixtures of glycerine and water and a previously determined water sorption isotherm for HSC. This allowed the estimation of diffusion and partition coefficients for each permeant in the HSC, as well as HSC thickness, as a function of composition of the contacting solution. These data may be used to estimate water retention and associated HSC swelling related to the absorption and slow release of glycerine from the skin. RESULTS: It took 6+ days for glycerine to completely desorb from HSC immersed in glycerine/water binary solutions. Desorption of both 3 H2 O and 14 C-glycerine from HSC was slower in pure water than from binary mixtures, a result that is largely explained by the greater swelling of HSC in water. Parametric relationships were developed for water and glycerine intradiffusivities in HSC as functions of HSC water content, and a mutual diffusion coefficient was estimated by analogy with glycerine/water binary solutions. The intradiffusivity of 14 C-glycerine in HSC as inferred from sorption/desorption experiments was shown to be approximately 10-fold less than that inferred from permeation experiments, whereas the corresponding values for 3 H2 O were comparable. CONCLUSION: These studies confirm that glycerine enters HSC in substantial quantities and has a long residence time therein. The coupling between bulk water and glycerine transport projected from binary solution data suggests the net effect of glycerine is to slow water loss from the skin. The data support the concept of glycerine as a humectant with an excellent balance of skin penetration and retention characteristics; however, they do not rule out the possibility of an additional biological effect on skin barrier homoeostasis.

Prognostic significance of miR-34a in Ewing sarcoma is associated with cyclin D1 and ki-67 expression.

BACKGROUND: At diagnosis, identification of reliable biological indicators of prognosis to allow stratification of patients according to different risks is an important but still unresolved aspect in the treatment of Ewing sarcoma (EWS) patients. This study aimed to explore the role of miR-34A expression on prognosis of EWS patients. PATIENTS AND METHODS: Specimens from 109 patients with non-metastatic EWS treated at the Rizzoli Institute with neoadjuvant chemotherapy (protocols ISG/SSGIII, EW-1, EW-2, EW-REN2, EW-REN3, EW-PILOT) and 17 metastases were studied. Sixty-eight patients (62%) remained disease-free and 41 (38%) relapsed (median follow-up: 67 months, range 9-241 months). Expression of miR-34a and of some of its targets (cyclin D1, bcl-2, SIRT1 and YY1) was evaluated by qRT-PCR using TaqMan MicroRNA Assays and/or by immunohistochemistry on tissue microarrays from the same patients. RESULTS: High expression of miR-34a in localized tumors was significantly related to better event-free and overall survival (P = 0.004). Relevance of miR-34a was confirmed by using different calibrators (normal mesenchymal stem cells and different normal tissues). By multivariate Cox regression analysis, low miR-34a expression as well as nontotal necrosis and high levels of lactate dehydrogenase were all confirmed as independent risk factors associated with poor outcome. Expression of miR-34a was lower in metastases than in primary tumors. It inversely correlated with expression of cyclin D1 and Ki-67. CONCLUSIONS: By demonstrating its relationship with clinical outcome, we propose evaluation of miR-34a at diagnosis of EWS patients to allow early risk stratification. Validation of these results would nonetheless ultimately need a prospective assessment.

Vitreous rebleeding rate after vitrectomy or phacovitrectomy in patients with proliferative diabetic retinopathy: A retrospective study.

BACKGROUND: The most common complication after vitrectomy is the rebleeding in vitreous cavity. It is important to detect the different factors that can increase the vitreous rebleeding rate in these patients. OBJECTIVE: To carry out a retrospective review of the rate of vitreous rebleeding after vitrectomy or phacovitrectomy. METHOD: Retrospective, descriptive and comparative study of patients with a diagnosis of proliferative diabetic retinopathy with phacovitrectomy or vitrectomy procedure. Personal background data, type of surgical intervention and grade of the surgeon who carried out the procedure were obtained. RESULTS: 1227 files of diabetic patients with vitrectomy or phacovitrectomy were reviewed. 65% presented systemic arterial hypertension. The average glomerular filtration rate was 63.50 (±32.36) ml/min/1.73 m2 and glycosylated hemoglobin (HBA1C) of 8% (4.6 to 15%). In the comparison of variables, a significant difference in the rate of vitreous rebleeding was obtained comparing phacovitrectomy with vitrectomy. (p = 0.003), in the relationship between vitrectomy with vitreous rebleeding, an odds ratio of 1.44 was obtained. CONCLUSION: The results obtained show a lower rate of rebleeding in patients undergoing phacovitrectomy in patients with proliferative diabetic retinopathy.

A contrast set mining based approach for cancer subtype analysis.

The task of detecting common and unique characteristics among different cancer subtypes is an important focus of research that aims to improve personalized therapies. Unlike current approaches mainly based on predictive techniques, our study aims to improve the knowledge about the molecular mechanisms that descriptively led to cancer, thus not requiring previous knowledge to be validated. Here, we propose an approach based on contrast set mining to capture high-order relationships in cancer transcriptomic data. In this way, we were able to extract valuable insights from several cancer subtypes in the form of highly specific genetic relationships related to functional pathways affected by the disease. To this end, we have divided several cancer gene expression databases by the subtype associated with each sample to detect which gene groups are related to each cancer subtype. To demonstrate the potential and usefulness of the proposed approach we have extensively analysed RNA-Seq gene expression data from breast, kidney, and colon cancer subtypes. The possible role of the obtained genetic relationships was further evaluated through extensive literature research, while its prognosis was assessed via survival analysis, finding gene expression patterns related to survival in various cancer subtypes. Some gene associations were described in the literature as potential cancer biomarkers while other results have been not described yet and could be a starting point for future research.

Immune response in breast cancer surgery. A comparative and prospective study of different anesthetic techniques.

INTRODUCTION: The main reason for high mortality in breast cancer is local recurrence and metastasis, despite surgery as the first therapeutic option. The anesthesia used in the operation room can determine the immune response. METHODS: A prospective, comparative and non- randomised study in patients undergoing breast cancer surgery was conducted in our hospital after obtaining approval from the Hospital's Institutional Review Board. Patients were divided in two groups: Group A received general anesthesia with propofol and opioids. Group B, in addition to general anesthesia, three interfascial blocks (Pec I, Pec II and BRILMA) were performed in all patients. Three blood samples were taken 1) previous anesthetic induction; 2) two hours after the end of the surgery and 3) 24-48 h after surgery. Leukocytes, CD3, CD4, CD8 and Natural Killer cells were determined at each time. RESULTS: 103 patients were included. 59 (group A) received general anesthesia and 54 (group B) general anesthesia and interfascial blocks. Regarding baseline characteristics, age was significantly higher in the group that received general anesthesia and mastectomy was more frequent in the group that received interfascial blocks. We observed after surgery an increase in leukocytes level that returns close to baseline levels. On the other hand, a reduction in the immune response was observed that also returns to the previous level 48 h after surgery. Group A and B get similar results and also subgroups of hormonal receptors (HER+, PR and/or ER+). CONCLUSIONS: Interfascial blocks in chest wall added to general anesthesia in breast cancer surgery has not shown a significant difference in the inflammatory response or immunological depression compared to general anesthesia as the only anesthetic technique. It seems to trend less immunological depression in the interfascial block group.

Using Ant Programming Guided by Grammar for Building Rule-Based Classifiers.

The extraction of comprehensible knowledge is one of the major challenges in many domains. In this paper, an ant programming (AP) framework, which is capable of mining classification rules easily comprehensible by humans, and, therefore, capable of supporting expert-domain decisions, is presented. The algorithm proposed, called grammar based ant programming (GBAP), is the first AP algorithm developed for the extraction of classification rules, and it is guided by a context-free grammar that ensures the creation of new valid individuals. To compute the transition probability of each available movement, this new model introduces the use of two complementary heuristic functions, instead of just one, as typical ant-based algorithms do. The selection of a consequent for each rule mined and the selection of the rules that make up the classifier are based on the use of a niching approach. The performance of GBAP is compared against other classification techniques on 18 varied data sets. Experimental results show that our approach produces comprehensible rules and competitive or better accuracy values than those achieved by the other classification algorithms compared with it.

Using statistical deformable models to reconstruct vocal tract shape from magnetic resonance images.

The mechanisms involved in speech production are complex and have thus been subject to growing attention by the scientific community. It has been demonstrated that magnetic resonance imaging (MRI) is a powerful means in the understanding of the morphology of the vocal tract. Over the last few years, statistical deformable models have been successfully used to identify and characterize bones and organs in medical images and point distribution models (PDMs) have gained particular relevance. In this work, the suitability of these models has been studied to characterize and further reconstruct the shape of the vocal tract in the articulation of Portuguese European (EP) speech sounds, one of the most spoken languages worldwide, with the aid of MR images. Therefore, a PDM has been built from a set of MR images acquired during the artificially sustained articulation of 25 EP speech sounds. Following this, the capacity of this statistical model to characterize the shape deformation of the vocal tract during the production of sounds was analysed. Next, the model was used to reconstruct five EP oral vowels and the EP fricative consonants. As far as a study on speech production is concerned, this study is considered to be the first approach to characterize and reconstruct the vocal tract shape from MR images by using PDMs. In addition, the findings achieved permit one to conclude that this modelling technique compels an enhanced understanding of the dynamic speech events involved in sustained articulations based on MRI, which are of particular interest for speech rehabilitation and simulation.

[E-health tools to overcome the gap in epilepsy care before, during and after COVID-19 pandemics]. / Herramientas de salud digital para superar la brecha de atención en epilepsia antes, durante y después de la pandemia de COVID-19.

INTRODUCTION: Epilepsy is a common chronic neurological disorder that affects around 50 million worldwide and there is an abundance of literature on the health care gap for this sector of the population. This gap will increase with the current pandemic due to COVID-19. AIM: To evaluate the current availability of digital health tools for the care of people with epilepsy according to the world medical literature and their use during said pandemic. DEVELOPMENT: We reviewed the publications in scientific journals in the last decade that had as their main topic the use of digital health tools or telemedicine focused on the care of patients with epilepsy, including 4 months after the national quarantines due to the appearance of the virus SARS-CoV-2. Seventeen publications were found on the use of telemedicine focused on epilepsy. The most widely used tools internationally are online platforms, followed by mobile applications, videoconferences, epileptic seizure capture systems, checklists, algorithms for understanding medical data, phone calls, tele-encephalography and text messages. None was published during the COVID-19 current pandemic. CONCLUSIONS: Although there is little literature on the use of digital health tools focused on epilepsy, there are several that can be used to fight the attention gap, especially in this global pandemic by COVID-19 that forces quarantines of people and communities for long periods. It is necessary to remove barriers and facilitate patient access to these new information technologies.

TITLE: Herramientas de salud digital para superar la brecha de atención en epilepsia antes, durante y después de la pandemia de COVID-19.Introducción. La epilepsia es un trastorno neurológico crónico común que afecta alrededor de 50 millones de personas en el mundo y abunda la bibliografía sobre la brecha de atención en salud a este sector de la población. Dicha brecha aumentará con la pandemia actual de COVID-19. Objetivo. Evaluar la disponibilidad actual de herramientas de salud digital para la atención a personas con epilepsia según la literatura médica mundial y su uso durante dicha pandemia. Desarrollo. Se hizo una revisión de las publicaciones en revistas científicas en la última década que tuvieran como tema principal el uso de herramientas de salud digital o telemedicina enfocada a la atención de los pacientes con epilepsia, incluyendo cuatro meses después de las cuarentenas nacionales por la aparición del virus SARS-CoV-2. Se encontraron 17 publicaciones sobre el uso de telemedicina enfocada a la epilepsia. Las herramientas más utilizadas internacionalmente son las plataformas en línea, seguidas de las aplicaciones móviles, videoconferencias, sistemas de captación de crisis epilépticas, listas de verificación, algoritmos de comprensión de datos médicos, llamadas telefónicas, telelectroencefalografía y mensajes de texto. Ninguna se publicó durante la presente pandemia. Conclusiones. Hay poca bibliografía sobre herramientas de salud digital enfocadas a epilepsia, pero existen varias que pueden emplearse para luchar contra la brecha de atención, especialmente en esta pandemia mundial de COVID-19 que obliga a las personas y comunidades a mantenerse en cuarentena por la emergencia sanitaria. Es necesario eliminar barreras y facilitar el pronto acceso de los pacientes a estas nuevas tecnologías de información.

Cytotoxicity profiling of deep eutectic solvents to human skin cells.

The tailor-made character of deep eutectic solvents (DES) turns them very attractive to be used in several applications, including in health-related areas such as pharmaceutical, nutraceutical, and cosmetic industries. However, although DES has been touted as "green" solvents, several works proved that their potential toxicity should not be neglected. Using the premise of DES applicability in the cosmetic and pharmaceutical sectors, we chose two cell lines to work as a skin model (keratinocytes HaCaT and tumor melanocytes MNT-1), to assess DES cytotoxicity. The effect of three different hydrogen bond acceptors (HBA) ([Chol]Cl, [N1111]Cl and [N4444]Cl) and three different hydrogen bond donors (HBD) (hexanoic and butanoic acid, ethylene glycol, 1-propanol and urea) were evaluated through a common viability assay (MTT assay). Results were promising since [Chol]Cl and [N1111]Cl- based DES showed good biocompatibility for the tested cells. [N4444]Cl-based DES, however, showed cytotoxicity for both cell lines, with the HBA being the driver of the toxicity. Interestingly, some compounds increased cell viability in the HaCaT cell line, namely [Chol]Cl, ethylene glycol, hexanoic acid, urea, and all [Chol]Cl and [N1111]Cl-based DES and should be considered as targets for future studies. These results highlight their possible use in cosmetic or pharmaceutical formulations.

The alpha1A-adrenoceptor gene is required for the alpha1L-adrenoceptor-mediated response in isolated preparations of the mouse prostate.

BACKGROUND AND PURPOSE: This study investigated whether deletion of the alpha1A-adrenoceptor gene influences contractile responses of mouse prostate to noradrenaline. Responses of mouse prostate to noradrenaline are known to be mediated by alpha1L-adrenoceptors, which are thought to be a functional phenotype of alpha1A-adrenoceptor. EXPERIMENTAL APPROACH: Prostate tissues from alpha1A-adrenoceptor knockout mice which were homozygous (alpha1A -/-) and heterozygous (alpha1A +/-) for the disrupted alpha1A-adrenoceptor gene, as well as wild-type (alpha1A +/+) littermates were mounted in glass-isolated organ baths. Electrical field stimulation of nerves and exogenous application of noradrenaline were used to investigate the effects of alpha1A-adrenoceptor disruption on prostate contractility. KEY RESULTS: Frequency-response curves to electrical field stimulation (0.5 ms pulse duration, 60 V, 0.1-20 Hz) yielded frequency-dependent contractions. At frequencies of 10 and 20 Hz, prostates from alpha1A -/- mice elicited an approximately 30% decreased response compared with prostates from alpha(1A)+/+ mice. Prazosin (0.3 muM) attenuated responses to electrical field stimulation in prostates from alpha1A +/+ and alpha1A +/- mice but not from alpha1A -/- mice. Increasing concentrations of exogenously administered noradrenaline (10 nM-1 mM) produced mean concentration-response curves in prostates from alpha1A +/+ and alpha1A +/- mice, which were not different. Maximum responses to noradrenaline were decreased by approximately 80% in prostates from alpha1A -/- mice compared with alpha1A +/+ mice. Prazosin attenuated responses to noradrenaline in all genotypes. CONCLUSIONS AND IMPLICATIONS: alpha1L-Adrenoceptor-mediated responses in mouse prostate are abolished in alpha1A -/- mice, demonstrating that the alpha1A-adrenoceptor gene is essential to the manifestation of the prostatic alpha1L-adrenoceptor phenotype. This implies that alpha1L-adrenoceptors are indeed a functional phenotype of alpha1A-adrenoceptor.

Pattern and pathways for mercury lifespan bioaccumulation in Carcinus maenas.

Carcinus maenas is an important and exploited natural resource in temperate estuaries, being consumed by local populations and also used as fish bait. Through bioaccumulation and bioamplification processes occurring in polluted estuarine ecosystems, this species may directly or indirectly represent a major pathway for Human contamination. No information is available on the overall lifespan bioaccumulation pattern of mercury for this species, which would be an invaluable tool for assessing the risks associated with C. maenas harvest in contaminated areas. Taking that in mind, the main objectives of this work were to clarify the lifespan bioaccumulation pattern of mercury for this species. A consistent lifespan accumulation pattern was found for this species, both for total and organic mercury, and coherent differences were found between genders, suggesting different physiological responses to contamination. From our results two scenarios emerge: (a) for low mercury contamination areas where the diet is the major pathway for metal uptake, with higher levels in muscle and hepatopancreas (twice as high as in gills) and higher organic mercury fractions (as high as 80-90% in muscle), crabs bioaccumulate with age and (b) on high contaminated areas, environmental exposure is predominant, with higher levels found in gills (reaching 0.8 mg kg(-1)), lower organic fractions and no mercury increment with age. Detoxification strategies, dietary differences and species mobility may account for these two accumulation patterns.

Stimulation of epithelial CB1 receptors inhibits contractions of the rat prostate gland.

BACKGROUND AND PURPOSE: This study investigated whether stimulation of cannabinoid receptors influences smooth muscle contractility in the rat prostate gland. EXPERIMENTAL APPROACH: Immunohistochemistry was used to characterize and localize cannabinoid receptors in the rat prostate gland. Isolated organ bath experiments were carried out to investigate the effects of cannabinoids on prostate contractility. KEY RESULTS: Immunohistochemical studies of the rat prostate yielded positive immunoreactivity for the CB(1) receptor, but not the CB(2) receptor. Double labelling revealed that CB(1) receptors were not colocalized with alpha-actin in the smooth muscle layer but were primarily expressed within the epithelial lining of the prostatic acini. The cannabinoid receptor agonist WIN 55,212-2 (10 nM - 10 microM) inhibited contractile responses to electrical-field stimulation (10 Hz, 0.5 ms, 60 V for 2 s per minute) in a concentration-dependent manner. The CB(1) selective antagonists, SR141716 (1 microM) and LY 320135 (1 microM), reversed the WIN 55,212-2-mediated inhibition but the CB(2) selective antagonist, SR144528 (1 microM), did not. Furthermore, the cyclooxygenase inhibitor indomethacin (0.1 microM) caused significant reversal of the WIN 55,212-2 mediated inhibition of contractile responses, whereas the nitric oxide synthase inhibitor N (omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mM) did not. Prostaglandin E(2) (10 nM - 10 microM), produced a similar concentration-dependent inhibition to WIN 55,212-2. CONCLUSIONS AND IMPLICATIONS: WIN 55,212-2, an agonist at cannabinoid receptors, causes inhibition of smooth muscle contraction in the rat prostate by activating epithelial CB(1) receptors. This inhibition is mediated via the cyclooxygenase pathway.

Respuesta inmunológica en cirugía del cáncer de mama. Estudio comparativo y prospectivo de diferentes técnicas anestésicas / Immune response in breast cancer surgery. A comparative and prospective study of different anesthetic techniques

Introducción: El principal motivo de la alta mortalidad en el cáncer de mama es la recurrencia local y las metástasis, siendo la cirugía la primera opción terapéutica. La técnica anestésica utilizada en quirófano puede modificar la respuesta inmunológica del paciente. Métodos: Estudio prospectivo, comparativo y no aleatorizado en pacientes intervenidos de cáncer de mama en el Hospital Universitario de Getafe (Madrid) tras la aprobación del Comité Ético del Hospital.Dividimos a los pacientes en dos grupos: grupo A, que recibió anestesia general con propofol y fármacos opiáceos; grupo B, en el que además de la anestesia general, se realizaron tres bloqueos interfasciales (Pec I, Pec II y BRILMA) en todos los pacientes. Se obtuvieron tres muestras sanguíneas: 1) antes de la inducción anestésica; 2) 2h después de finalizar la cirugía y 3) 24-48h posquirúrgicas. En cada muestra, se analizaron el número de leucocitos, células CD3, CD4 y CD8, así como las células natural killer (NK). Resultados: Se incluyeron en el estudio un total de 103 pacientes; 59 (grupo A) recibieron anestesia general y 54 (grupo B) anestesia general y bloqueos interfasciales. Según las características basales, la edad fue significativamente superior en las pacientes que recibieron anestesia general. La mastectomía se realizó con más frecuencia en el grupo que recibió bloqueos interfasciales. Observamos que después de la cirugía hay un aumento en el número de leucocitos pero regresa a los niveles basales a las 48h, comportamiento que se repite a nivel inmunológico: disminuye después de la cirugía pero vuelve a niveles previos a las 48h de la cirugía. Los grupos A y B presentan resultados similares en el resto de parámetros estudiados, al igual que los subgrupos según los receptores hormonales (HER+, PR y/o ER+).(AU)

Introduction: The main reason for high mortality in breast cancer is local recurrence and metastasis, despite surgery as the first therapeutic option. The anesthesia used in the operation room can determine the immune response. Methods: A prospective, comparative and non-randomized study in patients undergoing breast cancer surgery was conducted in our hospital after obtaining approval from the Hospital's Institutional Review Board. Patients were divided in two groups: Group A received general anesthesia with propofol and opioids. Group B, in addition to general anesthesia, three interfascial blocks (Pec I, Pec II and BRILMA) were performed in all patients. Three blood samples were taken 1) previous anesthetic induction; 2) two hours after the end of the surgery and 3) 24-48hours after surgery. Leukocytes, CD3, CD4, CD8 and Natural Killer cells were determined at each time. Results: 103 patients were included. 59 (group A) received general anesthesia and 54 (group B) general anesthesia and interfascial blocks. Regarding baseline characteristics, age was significantly higher in the group that received general anesthesia and mastectomy was more frequent in the group that received interfascial blocks.We observed after surgery an increase in leukocytes level that returns close to baseline levels. On the other hand, a reduction in the immune response was observed that also returns to the previous level 48hours after surgery. Group A and B get similar results and also subgroups of hormonal receptors (HER+, PR and/or ER+). Conclusions: Interfascial blocks in chest wall added to general anesthesia in breast cancer surgery has not shown a significant difference in the inflammatory response or immunological depression compared to general anesthesia as the only anesthetic technique. It seems to trend less immunological depression in the interfascial block group.(AU)

Leishmania infantum exerts immunomodulation in canine Kupffer cells reverted by meglumine antimoniate.

Kupffer cells (KC) are the liver macrophage population that resides in the hepatic sinusoids and efficiently phagocyte pathogens by establishing an intimate contact with circulating blood. KC constitute the liver host cells in Leishmania infection, nevertheless little is described about their role, apart from their notable contribution in granulomatous inflammation. The present study aims to investigate how canine KC sense and react to the presence of Leishmania infantum promastigotes and amastigotes by evaluating the gene expression of specific innate immune cell receptors and cytokines, as well as the induction of nitric oxide and urea production. Complementarily, the impact of a leishmanicidal drug - meglumine antimoniate (MgA) - in infected KC was also explored. KC revealed to be susceptible to both parasite forms and no major differences were found in the immune response generated. L. infantum parasites seem to interact with KC innate immune receptors and induce an anergic state, promoting immune tolerance and parasite survival. The addition of MgA to infected KC breaks the parasite imposed silence and increased gene expression of Toll-like receptors (TLR) 2 and TLR4, possibly activating downstream pathways. Understanding how KC sense and react to parasite presence could bring new insights into the control or even elimination of canine leishmaniasis.