Chemosensitizing effect of pentoxifylline in sensitive and multidrug-resistant non-small cell lung cancer cells.

Aim: Multidrug resistance (MDR) is frequent in non-small cell lung cancer (NSCLC) patients, which can be due to its fibrotic stroma. This work explores the combination of pentoxifylline, an anti-fibrotic and chitinase 3-like-1 (CHI3L1) inhibitor drug, with conventional chemotherapy to improve NSCLC treatment. Methods: The effect of pentoxifylline in the expression levels of P-glycoprotein (P-gp), CHI3L1 and its main downstream proteins, as well as on cell death, cell cycle profile, and P-gp activity was studied in two pairs of sensitive and MDR counterpart NSCLC cell lines (NCI-H460/NCI-H460/R and A549/A549-CDR2). Association studies between CHI3L1 gene expression and NSCLC patients' survival were performed using The Cancer Genome Atlas (TCGA) analysis. The sensitizing effect of pentoxifylline to different drug regimens was evaluated in both sensitive and MDR NSCLC cell lines. The cytotoxicity of the drug combinations was assessed in MCF10A non-tumorigenic cells. Results: Pentoxifylline slightly decreased the expression levels of CHI3L1, ß-catenin and signal transducer and activator of transcription 3 (STAT3), and caused a significant increase in the G1 phase of the cell cycle in both pairs of NSCLC cell lines. A significant increase in the % of cell death was observed in the sensitive NCI-H460 cell line. TCGA analysis revealed that high levels of CHI3L1 are associated with low overall survival (OS) in NSCLC patients treated with vinorelbine. Moreover, pentoxifylline sensitized both pairs of sensitive and MDR NSCLC cell lines to the different drug regimens, without causing significant toxicity to non-tumorigenic cells. Conclusion: This study suggests the possibility of combining pentoxifylline with chemotherapy to increase NSCLC therapeutic response, even in cases of MDR.

Minimizing false positives for CTC identification.

BACKGROUND: Cancer is a leading cause of death worldwide, with metastasis playing a significant role. Circulating Tumour Cells (CTCs) can provide important real-time insights into tumour heterogeneity and clonal evolution, making them an important tool for early diagnosis and patient monitoring. Isolated CTCs are typically identified by immunocytochemistry using positive biomarkers (cytokeratin) and exclusion biomarkers (CD45). However, some white blood cell (WBC) populations can express low levels of CD45 and stain non-specifically for cytokeratin, increasing their risk of misclassification as CTCs. There is a clear need to improve CTC detection and enumeration criteria to unequivocally eliminate interfering WBC populations. RESULTS: This study showed that, indeed, some granulocyte subpopulations expressed low levels of CD45 and stained non-specifically for cytokeratin, misidentifying them as CTCs. These same cells, however, strongly expressed CD15, allowing them to be identified as WBCs and excluded from CTC classification. Flow cytometry confirmed the specificity of the CD15 antibody for the granulocyte subpopulation. False positives were considerably reduced from 25 % to 0.2 % by double exclusion, combining a CD15 antibody with a highly specific CD45 antibody. Furthermore, complete elimination of potential false positives was achieved using double exclusion in combination with improved selection of cytokeratin antibody. The study emphasises the importance of a robust exclusion criteria and high antibody specificity in CTC immuno-assays for accurate identification of CTC candidates and thorough exclusion of interfering WBC subpopulations. SIGNIFICANCE: This study demonstrated how misidentifying a granulocyte subpopulation can lead to inaccurate CTC evaluation. However, sensitivity and specificity of CTC identification may be improved by using high-performing antibodies and by including a second exclusion biomarker, in turn, allowing for a more comprehensive clinical application of CTCs.

Novel family of [RuCp(N,N)(P)]+ compounds with simultaneous anticancer and antibacterial activity: Biological evaluation and solution chemistry studies.

A family of ten novel ruthenium(II)-cyclopentadienyl organometallics of general formula [Ru(η5-C5H5)(N,N)(PPh2(C6H4COOR)][CF3SO3] (1-10) in which (N,N) = 4,4'-R'-2,2'-bipyridyl (R = -H or -CH2CH2OH; R' = -H, -CH3, -OCH3, -CH2OH, and -CH2-biotin) was prepared from [Ru(η5-C5H5)(PPh2(C6H4COOH))2Cl]. All compounds were fully characterized by means of several spectroscopic and analytical techniques, and the molecular structures of [Ru(η5-C5H5)(PPh2(C6H4COOH))2Cl], 1, 3 and 4 have been additionally studied by single-crystal X-ray diffraction. The anticancer activity of all compounds was evaluated in sensitive and multidrug-resistant counterpart cell lines from human colorectal cancer (Colo 205 and Colo 320) and non-small cell lung cancer NSCLC (A549, NCI-H460 versus NCI-H460/R) as well. Notably, compounds 6 and 7 (R CH2CH2OH and (N,N) = bipy or Me2bipy, respectively) showed antiproliferative effect against both cell lines with high intrinsic selectivity towards cancer cells. The antibacterial activity of all compounds was also evaluated against both Gram negative and Gram positive strains, and some compounds in the series showed potent antibacterial activity against Staphylococcus aureus strains, including the methicillin-resistant MRSA strains. Solution speciation studies revealed that the complexes bearing the PPh2(C6H4COO-) ligand are neutral at physiological pH (7.4) in contrast with their ethylene glycol derivatives that have a permanent positive charge. While all compounds are lipophilic, the difference in the distribution coefficient for neutral and charged complexes is around one order of magnitude. Complexes 6 and 7 exhibited excellent biological activity and were selected for further studies. Spectrofluorometric methods were used to investigate their interaction with biomolecules such as human serum albumin (HSA) and calf thymus DNA (ct-DNA). For these complexes, binding site II of HSA is a possible binding pocket through non-covalent interactions. The release of ethidium from the DNA adduct by the charged complexes proves their interaction with DNA in contrast to the neutral ones. In conclusion, Ru(II)-cyclopentadienyl complexes with 2,2'-bipyridyl-derivatives and an ethylene glycol moiety tethered to the phenylphosphane co-ligand are very promising from a therapeutic perspective, in particular complexes 6 and 7 that display remarkable antibacterial activity with a high anti-proliferative effect against colon and non-small cell lung cancers, both clinically challenging neoplasias in need of effective solutions.

Progressive multifocal leukoencephalopathy triggered by COVID-19 in a previously asymptomatic person living with undiagnosed HIV infection.

This report presents the case of a 47-year-old male patient who worked as a mathematics teacher and experienced the sudden onset of disorientation, aphasia, and acalculia during an online class. The current study reveals the first documented case of HIV and progressive multifocal leukoencephalopathy with the detection of SARS-CoV-2 and human polyomavirus 2 (previously known as John Cunningham virus) in the cerebrospinal fluid. Furthermore, serum analysis revealed elevated concentrations of interleukin (IL)-6, IL-17, and IL-8, which are potential factors known to reduce the expression of tight junctions and adhesion molecules in the extracellular matrix, thereby affecting the permeability of the blood-brain barrier. Finally, the study discusses whether SARS-CoV-2 triggers or exacerbates progressive multifocal leukoencephalopathy.

Acesso a medicamentos anti-hipertensivos pelos usuários de uma unidade ambulatorial / Antihypertensive drugs access by users of ambulatory unit

Objetivo: caracterizar o acesso aos medicamentos anti-hipertensivos pelas pessoas com hipertensão arterial atendidas em uma unidade ambulatorial. Metodologia: estudo descritivo, quantitativo, desenvolvido com 103 pessoas com hipertensão arterial em uso de anti-hipertensivos. Os dados foram coletados por meio de questionário com perguntas sociodemográficas, sobre tratamento e acesso aos medicamentos anti-hipertensivos. Utilizou-se a estatística descritiva e teste qui-quadrado de Pearson ou exato de Fisher para análise dos dados. Resultados: Predominou a faixa etária de 50-69 (68,9%), sexo feminino (85,4%) e raça/cor autodeclarada preta (46,6%). Quanto ao acesso aos anti-hipertensivos, 70,9% relataram ter acesso gratuito, 60,2% os obtêm nas unidades de saúde, 65,7% não referiram dificuldades na aquisição e 86,4% que tinham acesso total. Todos os participantes que tinham dificuldade econômica também tinham dificuldade de acesso aos anti-hipertensivos. Verificou-se associação significativa entre a dificuldade de acesso aos anti-hipertensivos com forma de acesso (total ou parcial), quantidade de drogas e disponibilidade do medicamento nas farmácias (p<0,005). Conclusão: observou-se que, embora a maioria dos participantes do estudo não encontre dificuldades para obtenção dos anti-hipertensivos nas farmácias das unidades básicas de saúde, ainda assim, existe uma parcela da população sem acesso total aos anti-hipertensivos de forma gratuita, sendo essencial melhorias dos programas de fornecimento de medicamentos.

Objective: to characterize access to antihypertensive drugs by patients with arterial hypertension treated at an ambulatory unit. Methodology: descriptive, quantitative study, developed with a group of 103 people with arterial hypertension currently using antihypertensive drugs. The data were collected through a questionnaire with sociodemographic questions, with respect to treatment and access to antihypertensive drugs. Descriptive statistics and Pearson's chi-square test or Fisher's exact test were used for data analysis. Results: the age group 50-69 (68.9%), female (85.4%) and black self-declared race (46.6%) predominated. Regarding access to antihypertensive drugs, 70.9% reported having free access, 60.2% obtained them at health units, 65.7% did not mention difficulties in acquiring them and 86.4% that had full access. All participants who had economic difficulties also had difficulty accessing antihypertensive drugs. It was identified a significant association between difficulty in accessing antihypertensive drugs and the means of access (total or partial), quantity of drugs and availability of the drug in pharmacies (p<0.005). Conclusion: it was observed that, that most of the study participants did not find it difficult to obtain antihypertensive drugs in the pharmacies of basic health units, notwithstanding, there is a portion of the population without full access to antihypertensive drugs free of charge, improvements in drug supply programs are essential.

Economic evaluation of meningococcal vaccination with vaccine for serogroups A, C, W e Y: A systematic revision / Avaliação económica da vacinação meningocócica com a vacina para os serogrupos A, C, W e Y: Uma revisão sistemática

Introduction: Meningococcal disease, caused by infection due to Neisseria meningitidis, have a high burden of disease and entails excess costs for countries. The purpose of this paper is to analyse meningococcal disease, the preventive strategies adopted, and to analyse economic evaluations of the meningococcal vaccine for serogroups A, C, W and Y (MenACWY). Methods: A narrative literature review and a systematic literature review were conducted, in which these databases were used: B-ON, PUBMED, EBSCO, Cochrane, NHS EED, Science Direct, Scopus and Web of Science. The inclusion criteria were based on the PICO methodology, the articles included were full economic evaluations and excluded partial. Results: Meningococcal disease still presents a high incidence in the World and in Portugal. The systematic review identified that in seven of the thirteen articles, MenACWY was not cost-effective considering the incidence of disease and the price of the vaccine. The strategies considered cost-effective had high cost-effectiveness thresholds and some even considered cost-effective despite being above. Conclusion: Incidence of this disease in Portugal is higher than in the European Union, due to serogroups B, C, W, and Y. The cost-effectiveness of the MenACWY is still dubious, so the incidence and the price are important to the introduction in the Portuguese National Vaccination Programme.

Introdução: A doença meningocócica, causada por infeção por Neisseria meningitidis, constitui uma elevada carga de doença e acarreta elevados custos aos países. Os objetivos gerais deste artigo são: analisar a doença meningocócica, as estratégias de prevenção adotadas, e analisar as avaliações económicas na utilização da vacina meningocócica para os serogrupos A, C, W e Y (MenACWY). Métodos: Foram realizadas uma pesquisa bibliográfica e uma revisão sistemática da literatura, na qual foram utilizadas as bases de dados B-ON, PUBMED, EBSCO, Cochrane, NHS EED, Science Direct, Scopus e Web of Science. Os critérios de inclusão foram construídos com metodologia PICO e incluíam avaliações económicas completas, tendo sido excluídas as parciais. Resultados: A doença meningocócica apresenta uma elevada incidência no Mundo e em Portugal. A revisão sistemática identificou em sete dos treze artigos, que a MenACWY não era custo-efetiva considerando a incidência no país e o preço da vacina. Nas estratégias que consideraram custo-efetiva, os limiares de custo-efetividade eram muito elevados e em alguns foi considerada custo-efetiva apesar de o ultrapassar. Conclusão: A incidência desta em Portugal está acima da média da União Europeia devido aos serogrupos B, C, W, e Y. A relação custo-efetividade da MenACWY é ainda dúbia, pelo que incidência e preço são importantes para a introdução no Programa Nacional de Vacinação Português.

Stromal Vascular Fraction Obtained From Subcutaneous Adipose Tissue: Ex-Obese and Older Population as Main Clinical Targets.

INTRODUCTION: Human adipose tissue contains a heterogeneous and synergistic mixture of cells called stromal vascular fraction (SVF) with highly proliferative and angiogenic properties, conferring promising applicability in the field of regenerative medicine. This study aims to investigate if age, body mass index (BMI), history of obesity and massive weight loss, and harvest site are related to SVF cell marker expression. METHODS: A total of 26 samples of subcutaneous adipose tissue were harvested from patients admitted to the Plastic and Reconstructive department in University Hospital Center of São João, Porto, Portugal, for body contouring surgery. The percentage of cells expressing CD31, CD34, CD45, CD73, CD90, and CD105 was assessed and compared with patient's age, BMI, history of obesity and massive weight loss (ex-obese group), and harvest site. RESULTS: In the ex-obese group, a significantly higher number of cells expressing CD90 (P = 0.002) was found. BMI, harvest site, and age appear to have no association with SVF subpopulations. CONCLUSIONS: This study suggests that ex-obese patients have a higher percentage of SVF cells expressing CD90, which correlates with higher proliferative and angiogenic rates. The effect of former obesity and massive weight loss on the expression of CD90 is a new and relevant finding because it makes this population a suitable candidate for reconstructive and aesthetic surgery and other fields of regenerative medicine. The use of SVF appears also promising in older patients because no negative correlation between increasing age and different cell markers expression was found.

Interaction of Polycyclic Aromatic Hydrocarbon compounds in fish primary hepatocytes: From molecular mechanisms to genotoxic effects.

Polycyclic Aromatic Hydrocarbons (PAHs) are persistent pollutants normally found in the environment as complex mixtures. Although several individual PAHs are classified as mutagenic and carcinogenic pollutants, the interaction effects between compounds in a mixture may trigger different toxicological mechanisms and, consequently, yield different effects to organisms which are not accounted for in risk assessment guidelines. Given the ubiquity of PAHs, understanding the mechanistic features of their mixtures is a pressing research need. Therefore, the present work aimed to disclose the interaction effects of three PAHs with different carcinogenic potential and chemical structure, in primary hepatocyte cells of gilt-headed seabreams (Sparus aurata). Hepatocytes were exposed to Phenanthrene (Phe), Benzo[a]pyrene (B[a]P) and Benzo[b]fluoranthene (B[b]F) and their mixtures at different proportions and several cellular responses were analyzed: cellular viability, CYP1A1 activity (EROD assay) and protein expression level (Western blot); transcript (mRNA) levels of CYP1A1, EPXH1 and GST-3 (qRT-PCR); genotoxic effects (DNA strand breakage) by the Comet assay. Results show that B[a]P induced CYP1A1 gene and protein expression increasing its activity and, therefore, increasing the production of metabolites that trigger genotoxic DNA damage (%). Most importantly, mixtures containing Phe and B[a]P increased even further CYP1A1 mRNA levels and DNA damage (up to 70 %) which suggests that, although Phe is considered a non-carcinogenic PAH, it potentiates CYP1A1 synthesis induced by B[a]P, increasing its genotoxicity. These findings indicate that the upregulation of CYP1A1 by carcinogenic PAHs will not weaken even when in mixtures with non-carcinogenic PAHs. On contrary, non-carcinogenic PAHs may potentiate the genotoxic effect of carcinogenic PAH and therefore mixture composition should be taken in account when assessing PAH toxicity. In fact, our results point to the need of redefining Environmental Risk Assessment protocols for mixtures of carcinogenic pollutants.

Immunophenotypic Analysis of T Lymphocytes and Cytokine Production in Elderly Practicing Physical Activities and Its Relationship with Quality of Life and Depression.

Aging is a complex process often associated with a chronic inflammatory profile that alters several biological functions, including the immune system and cognitive and physical capacity. The practice of physical activity is increasingly gaining popularity as a method of preventing infections, depression, and other disorders that affect the quality of life of the elderly. Thus, this work analyzes the profile of cytokines and molecular markers expressed in immune cells of elderly people who practice physical activities or not, evaluating their impacts on the immune system and quality of life. For this, 48 individuals were recruited, and peripheral blood samples were collected for hemogram analysis, cytokine determination, and immunophenotyping. Elderly people were separated into two groups: practitioners with low-intensity physical activity and non-practitioners. Quality of life was assessed using the Whoqol-Old instrument, and depression was assessed using the Beck II Depression Inventory. When comparing the scores of the Whoqol-Old and Beck questionnaires, we observed a significant negative correlation between these two factors. The perception of a higher quality of life was present in the elderly who exercised and was related to greater autonomy and sensory abilities, whereas the presence of depression was lower. In the hemogram, we observed higher basophil and segmented counts in the sedentary elderly, whereas lymphocytes and monocytes had lower counts. Elderly practitioners of physical activities had higher levels of IFN-γ, IL-4, and IL-10; increased expression of CD69, PD1, and TIM-3 in CD4+ T lymphocytes and increased CD14+CD80+ and CD14+CD86+ monocytes. Elderly people with an increased perception of quality of life had higher levels of IFN-γ, higher expression of CD14+CD80+CD86+, and decreased levels of TRAIL. An increase in TRAIL was observed in individuals with depression, in addition to an increased expression of CD14+CD86+. These results show a clear correlation between the quality of life, level of depression, physical activity, and immune system function. Although some cytokines with a typical proinflammatory profile (IFN-γ) were observed, the results point to a protective state with benefits reflected in the general well-being of the elderly who exercise.

Obesity phenotypes are, in part, associated with physical activity in diabetic hemodialysis patients.

PURPOSE: To investigate the prevalence of obesity phenotypes and their association with physical activity levels among diabetic hemodialysis patients. METHODS: This is a cross-sectional study with 84 diabetic hemodialysis patients (63.5 ± 9.4 years, 54.8% of men). Obesity was diagnosed as high body fat (≥ 40% for male and ≥ 30% for female). Sarcopenic obesity was considered if low skeletal muscle mass (< 20.0 kg for males and < 15.0 kg for females) and obesity were combined. Dynapenic obesity was defined in the presence of low handgrip strength (< 27 kg for males and < 16 kg for females) and obesity. Muscle failure obesity was confirmed in the concomitant presence of obesity, sarcopenia, and dynapenia. Physical activity level was assessed by the Baecke questionnaire and patients were classified as low physical activity according to the first tertile for each of and total domains. RESULTS: Fifty-four patients (64%) presented obesity. From these, 5 (6%), 19 (23%) and 8 (10%) were classified as sarcopenic obese, dynapenic obese, and muscle failure obese, respectively, and 22 (26%) were only obese. Patients with sarcopenic obesity and muscle failure obesity had lower leisure and locomotion physical activity scores than non-obese, whereas the total domain score did not differ across the groups. Muscle failure obesity was independently associated with low leisure physical activity (OR 10.8, 95% CI 1.3-88.1). Only sarcopenic obesity was independently associated with the locomotion and total physical activity domains (OR 15.4, 95% CI 1.4-90.2 and OR 17.0, 95% CI 1.5-95.4, respectively). CONCLUSION: Our study found a lower prevalence of sarcopenic obesity compared to dynapenic obesity and muscle failure obesity among diabetic hemodialysis patients. Moreover, sarcopenic obesity and muscle failure obesity, but not dynapenic obesity, were associated with low physical activity levels.

Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion.

Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.

Synthesis of glutathione as a central aspect of PAH toxicity in liver cells: A comparison between phenanthrene, Benzo[b]Fluoranthene and their mixtures.

Polycyclic Aromatic Hydrocarbons (PAH) are a class of organic pollutants normally found as mixtures with effects often hard to predict, which poses a major challenge for risk assessment. In this study, we address the effects of Phenanthrene (Phe), benzo[b]fluoranthene (B[b]F) and their mixtures (2 Phe:1 B[b]F; 1 Phe: 1 B[b]F; 1 Phe: 2 B[b]F) over glutathione (GSH) synthesis and function in HepG2 cells. We analyzed the effects on cellular viability, ROS production, glutathione (GSH) levels, protein-S-glutathionylation (PSSG), the activity of glutathione peroxidase (GPx), glutathione-S-transferases (GST) and glutathione reductase (GR). Transcript (mRNA) levels of glutathione synthesis enzymes - glutathione cysteine ligase catalytical (GCLC) and modifying (GCLM) sub-units and glutathione synthetase (GS) - and Nrf2 translocation to the nucleus were analyzed. Phe showed a higher cytotoxicity (IC50 = 130 µM after 24 h) than B[b]F related to a higher ROS production (up-to 50% for Phe). In agreement, GSH levels were significantly increased (up-to 3-fold) by B[b]F and were accompanied by an increase in the levels of PSSG, which is a mechanism that protect proteins from oxidative damage. The upregulation of GSH was the consequence of Nrf2 signaling activation and increased levels of GCLC, GCLM and GS mRNA observed after exposure to B[b]F, but not during exposure to Phe. Most interestingly, all mixtures showed higher cytotoxicity than individual compounds, but intriguingly it was the 1 Phe: 1B[b]F mixture showing the highest cytotoxicity and ROS production. GSH levels were not significantly upregulated not even in the mixture enriched in B[b]F. These results point to the role of GSH as a central modulator of PAH toxicity and demonstrate the idiosyncratic behavior of PAH mixtures even when considering only two compounds in varying ratios.

Ozonoterapia en el tratamiento de la Gingivo estomatitis herpética aguda. Presentación de caso / Ozone therapy in the treatment of acute herpetic gingivo stomatitis. Case presentation

El ozono presenta actividad antiparasitaria por su potente acción bactericida. En el presente estudio experimental se evaluó la efectividad del ozono en el tratamiento de la gingivoestomatitis herpética aguda (GEHA) en una paciente diagnosticada con esta enfermedad. La paciente recibió dos curas de OLEOZON® oral diaria, en toda la cavidad oral y en sus labios donde se extendía la enfermedad. Los resultados muestran que la aplicación de oleozón oral logró la sanación completa de la zona donde apareció la lesión sin dejar huellas en un periodo de 3-7 días, el dolor alivió aproximadamente a los tres minutos de aplicado el medicamento y desapareció a las 24 horas., mejorando las condiciones de la cavidad bucal y facilitando su alimentación. Se pudo comprobar el efecto antiparasitario, fungicida y virucida del Ozono ante la presencia de las lesiones herpéticas, así como cicatrizante y estimulante de la circulación mejorando la llegada de elementos nutritivos y oxígeno a la mucosa bucal y encía, contribuyendo a la mejoría de la paciente ya que el dolor se alivió aproximadamente a los tres minutos de aplicado el medicamento y desapareció a las 24 horas.

Binding of Flavylium Ions to Sulfonatocalix[4]arene and Implication in the Photorelease of Biologically Relevant Guests in Water.

The formation of host-guest complexes between seven flavylium cations and water-soluble p-sulfonatocalix[4]arene (SC4) was investigated by UV/vis absorption, fluorescence, and NMR spectroscopies. The results show the cationic guests form complexes with affinities in the submillimolar range. A representative chalcone/flavylium photoswitch was investigated in more detail regarding its pH- and light-triggered interconversion between the two forms. The dramatic affinity differentiation of the SC4 binding of the two switchable species (40 M-1 for the trans-chalcone versus 3.5 × 104 M-1 for the flavylium cation) enables the pH-gated photocontrol of the complexation process. These responsive properties were explored to demonstrate the competitive and selective release of biologically relevant guests from their supramolecular complexes with p-sulfonatocalix[4]arene (SC4), following the principle of AND logic. The guest release can be reverted by the thermally activated reaction of the flavylium ion back to the trans-chalcone.

Toxicity Evaluation of Quantum Dots (ZnS and CdS) Singly and Combined in Zebrafish (Danio rerio).

The exponential growth of nanotechnology has led to the production of large quantities of nanomaterials for numerous industrial, technological, agricultural, environmental, food and many other applications. However, this huge production has raised growing concerns about the adverse effects that the release of these nanomaterials may have on the environment and on living organisms. Regarding the effects of QDs on aquatic organisms, existing data is scarce and often contradictory. Thus, more information is needed to understand the mechanisms associated with the potential toxicity of these nanomaterials in the aquatic environment. The toxicity of QDs (ZnS and CdS) was evaluated in the freshwater fish Danio rerio. The fishes were exposed for seven days to different concentrations of QDs (10, 100 and 1000 µg/L) individually and combined. Oxidative stress enzymes (catalase, superoxide dismutase and glutathione S-transferase), lipid peroxidation, HSP70 and total ubiquitin were assessed. In general, results suggest low to moderate toxicity as shown by the increase in catalase activity and lipid peroxidation levels. The QDs (ZnS and CdS) appear to cause more adverse effects singly than when tested combined. However, LPO results suggest that exposure to CdS singly caused more oxidative stress in zebrafish than ZnS or when the two QDs were tested combined. Levels of Zn and Cd measured in fish tissues indicate that both elements were bioaccumulated by fish and the concentrations increased in tissues according to the concentrations tested. The increase in HSP70 measured in fish exposed to 100 µg ZnS-QDs/L may be associated with high levels of Zn determined in fish tissues. No significant changes were detected for total ubiquitin. More experiments should be performed to fully understand the effects of QDs exposure to aquatic biota.

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation.

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-ß family, TGF-beta 1 (TGF-ß1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-ß1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-ß1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-ß1 and its receptor, TßRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1-P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-ß receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-ß1. Treatment of granular cell cultures with TGF-ß1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TßRI activation because addition of a pharmacological inhibitor of TGF-ß, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-ß1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons.

Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides.

Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ' positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 µ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.

Fluorescent and paramagnetic chitosan nanoparticles that exhibit high magnetic resonance relaxivity: synthesis, characterization and in vitro studies.

We report water-in-oil (W/O) microemulsion synthesis of fluorescently bright and paramagnetically strong bimodal chitosan nanoparticles (BCNPs). The W/O microemulsion system provides a confined environment for producing monodispersed BCNPs. Average particle size as estimated by the Transmission Electron Microscopy was 28 nm. The water to surfactant molar ratio of 22 produced small size fairly monodispersed BCNPs. Fluorescein isothiocyanate (FITC, a fluorescent dye) and Gd-DOTA (a paramagnetic Gd ion chelating agent) were covalently attached to chitosan polymer backbone prior to BCNP synthesis. The purpose of the covalent attachment of fluorescent and paramagnetic labels to chitosan is to prevent leakage of these labels from the BCNPs. The BCNPs were cross-linked with tartaric acid using water-soluble carbodiimide coupling chemistry in order to maintain particulate integrity. Zeta potential value of +27.6 mV confirmed positive surface charge of cross-linked BCNPs. Fluorescence excitation and emission spectra of BCNPs were similar to that of bare FITC spectra, showing characteristic 520 nm emission at the 490 excitation. Paramagnetic gadolinium ion (Gd3+) concentration in the BCNPs was determined by inductively coupled plasma (ICP) emission spectroscopy. The longitudinal (T1) and transverse (T2) proton relaxation times were determined as a function of Gd3+ concentration in the BCNPs at 4.7 Tesla. Proton relaxivity (R1 value) of BCNPs was calculated to be 41.1 mM Gd(-1)s(-1). The reported R1 value of Gd-DOTA chelates is however 5.8 mM Gd(-1)s(-1). High proton relaxivity of BCNPs is attributed to hydrated chitosan environment around Gd chelates which additionally contributed to overall water exchange process. To demonstrate in vitro bioimaging capability, J774 macrophage cells were incubated with BCNPs. Confocal images clearly showed BCNP uptake by J774 cells. Internalization of BCNPs was confirmed by co-labeling J774 cells with a red-emitting membrane dye. BCNP green emission was mostly observed from middle of cells and within the red-emitting membrane boundary.

Disclosure model for pediatric patients living with HIV in Puerto Rico: design, implementation, and evaluation.

The American Academy of Pediatrics strongly encourages the disclosure of HIV status to school-age children and further recommends that adolescents know their HIV status. Limited information exists on the impact of disclosure. We designed and implemented a disclosure model hypothesized to be associated with healthy psychological adjustment and improved medication adherence. We report the model's design and implementation and results of a quasi-experimental study of the disclosure's effects on health care professionals (n = 16), caregivers (n = 39), and HIV-infected youth (n = 40) in Puerto Rico. Information was collected from youth, caregivers, and professionals by semistructured interviews and questionnaires. Most youth (70%) had feelings of normalcy 6 months post-disclosure, and most also improved their adherence to therapy after disclosure as reported by both patients (58%) and caregivers (59%). Eighty-five percent of youth and 97% of caregivers considered disclosure a positive event for themselves and their families. Fewer health care professionals reported feelings of fear, discomfort, and insecurity after protocol participation.