In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, Pathological, Toxicological, and Pharmacological Studies on Epididymal Function.

Contraction of cauda epididymal duct (CE) smooth muscle is one of the very first events of the seminal emission phase of ejaculation. The contraction of CE smooth muscle is governed by a complex interaction of hormones, autacoids, and by the neurotransmitters released from the epididymal intramural nerve endings, and any impairment in the CE smooth muscle contraction has the potential to impair male fertility. Apart the obvious pathophysiological and toxicological importance of CE smooth muscle contraction, modulation of CE contraction has pharmaceutical interest offering a druggable target to development of drugs to improve/impair male fertility. The in vitro contraction experiments constitute a valuable approach to an in-depth evaluation of functional and molecular changes resulting from pathologies or drug exposure. Therefore, this chapter consists in a description of in vitro pharmacological reactivity contractility of the epididymal duct in a controlled medium, maintained at 30 °C of temperature and continuously bubbled with 95% O2 and 5% CO2 to obtain cumulative concentration-response curves that has been fundamental to some of our investigations on epididymal physiology, toxicology, and pharmacology.

Biomimetic cardiovascular platforms for in vitro disease modeling and therapeutic validation.

Bioengineered tissues have become increasingly more sophisticated owing to recent advancements in the fields of biomaterials, microfabrication, microfluidics, genetic engineering, and stem cell and developmental biology. In the coming years, the ability to engineer artificial constructs that accurately mimic the compositional, architectural, and functional properties of human tissues, will profoundly impact the therapeutic and diagnostic aspects of the healthcare industry. In this regard, bioengineered cardiac tissues are of particular importance due to the extremely limited ability of the myocardium to self-regenerate, as well as the remarkably high mortality associated with cardiovascular diseases worldwide. As novel microphysiological systems make the transition from bench to bedside, their implementation in high throughput drug screening, personalized diagnostics, disease modeling, and targeted therapy validation will bring forth a paradigm shift in the clinical management of cardiovascular diseases. Here, we will review the current state of the art in experimental in vitro platforms for next generation diagnostics and therapy validation. We will describe recent advancements in the development of smart biomaterials, biofabrication techniques, and stem cell engineering, aimed at recapitulating cardiovascular function at the tissue- and organ levels. In addition, integrative and multidisciplinary approaches to engineer biomimetic cardiovascular constructs with unprecedented human and clinical relevance will be discussed. We will comment on the implementation of these platforms in high throughput drug screening, in vitro disease modeling and therapy validation. Lastly, future perspectives will be provided on how these biomimetic platforms will aid in the transition towards patient centered diagnostics, and the development of personalized targeted therapeutics.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

In vitro assessment of commercial sunscreens available in Latin America.

In Latin America, people have largely abandoned the practice of wearing hats and traditional clothing that provided skin protection. Sunscreen application has therefore become essential to protect against the increased sun exposure. The physician-prescribed medical-grade sunscreens provide sufficient sun protection but the requirement for regular use puts a financial burden on the patient that is often not sustainable. An appropriate sunscreen should provide a high and broad ultraviolet (UV) protection against UVB and UVA. Several over-the-counter (OTC) sunscreens have been developed for sale at affordable prices and are available for purchase in convenient locations, such as local grocery stores. The aim of this study was to assess the in vitro UV protection of 34 popular OTC sunscreens found in the Latin American market. UV absorbance/transmittance was quantified by diffusion transmission spectroscopy using coarse silica plaques. Photostability was tested by irradiating them with simulated solar light and calculating the sun protection factor (SPF), critical length of absorption (C lambda ), UVA/UVB ratio, and the spectral uniformity index (SUI). The results indicated that the in vitro SPFs were significantly lower than the value declared on the labels, particularly for those claiming high SPF values; however, the majority of these sunscreens offered high levels of UV protection. Considering the advantages of low cost and ample accessibility, we concluded that this sample of OTC sunscreens can be beneficial to the general public by providing some level of skin protection from solar radiation, and may be promoted to improve compliance with recommended photoprotection behavior.

In vitro assessment of commercial sunscreens available in Latin America / Valoración in vitro de protectores solares comerciales existentes en Latinoamérica

In Latin America, people have largely abandoned the practice of wearing hats and traditional clothing that provided skin protection. Sunscreen application has therefore become essential to protect against the increased sun exposure. The physician-prescribed medical-grade sunscreens provide sufficient sun protection but the requirement for regular use puts a financial burden on the patient that is often not sustainable. An appropriate sunscreen should provide a high and broad ultraviolet (UV) protection against UVB and UVA. Several over-the-counter (OTC) sunscreens have been developed for sale at affordable prices and are available for purchase in convenient locations, such as local grocery stores. The aim of this study was to assess the in vitro UV protection of 34 popular OTC sunscreens found in the Latin American market. UV absorbance/transmittance was quantified by diffusion transmission spectroscopy using coarse silica plaques. Photostability was tested by irradiating them with simulated solar light and calculating the sun protection factor (SPF), critical length of absorption (C λ ), UVA/UVB ratio, and the spectral uniformity index (SUI). The results indicated that the in vitro SPFs were significantly lower than the value declared on the labels, particularly for those claiming high SPF values; however, the majority of these sunscreens offered high levels of UV protection. Considering the advantages of low cost and ample accessibility, we concluded that this sample of OTC sunscreens can be beneficial to the general public by providing some level of skin protection from solar radiation, and may be promoted to improve compliance with recommended photoprotection behavior.

En Latinoamérica, la población ha abandonado la costumbre de usar sombrero y ropa tradicional para protegerse del sol. En consecuencia, es básico el uso de protectores solares si se realizan actividades bajo sol. Los protectores solares que se usan en la práctica médica son adecuados, pero su uso frecuente condiciona una carga económica que muchos pacientes no pueden solventar debido a sus costos considerables. Un protector apropiado contiene una amplia y elevada protección ultravioleta (UV) A y B. En las tiendas de conveniencia, existen numerosos protectores solares a precios más accesibles. El objetivo del estudio fue determinar la protección UV in vitro de 34 protectores solares con amplia presencia comercial (de venta sin prescripción médica) en el mercado latinoamericano. La absorbancia/transmitancia de la radiación UV se cuantificó mediante espectroscopía de transmisión difusa. Placas de sílice esmerilado fueron recubiertas con el producto y expuestas a radiación solar simulada para conocer su fotoestabilidad. Se calcularon índices como el factor de protección solar (SPF), longitud crítica de absorción (C λ), relación UVA/UVB y el índice de uniformidad espectral (SUI). Se encontró que el SPF in vitro fue inferior al establecido en las etiquetas, especialmente en aquellos con valores altos. No obstante, la mayoría de los protectores incluidos ofrecen niveles de protección UV elevados. Considerando su amplia accesibilidad y menor costo, concluimos que esta muestra comercial de protectores solares podría utilizarse en el entorno clínico para favorecer su apego junto a las otras medidas de fotoprotección sugeridas.

Glutathione-s-transferase modified electrodes for detecting anticancer drugs.

With the fast growth of cancer research, new analytical methods are needed to measure anticancer drugs. This is usually accomplished by using sophisticated analytical instruments. Biosensors are attractive candidates for measuring anticancer drugs, but currently few biosensors can achieve this goal. In particular, it is challenging to have a general method to monitor various types of anticancer drugs with different structures. In this work, a biosensor was developed to detect anticancer drugs by modifying carbon paste electrodes with glutathione-s-transferase (GST) enzymes. GST is widely studied in the metabolism of xenobiotics and is a major contributing factor in resistance to anticancer drugs. The measurement of anticancer drugs is based on competition between 1-chloro-2,4-dinitrobenzene (CDNB) and the drugs for the GST enzyme in the electrochemical potential at 0.1V vs. Ag/AgCl by square wave voltammetry (SWV) or using a colorimetric method. The sensor shows a detection limit of 8.8µM cisplatin and exhibits relatively long life time in daily measurements.

Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 µmol L (-1) and 164 ± 13.4 µmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

The footfault test as a screening tool in the 6-hydroxydopamine rat model of Parkinson's disease.

The administration of 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway is a rat model of Parkinson's disease (PD). The footfault test is a behavioural task in which rodents have their motor functions assessed. Here, we observed that unilaterally 6-OHDA-lesioned animals show a context-induced ipsilateral rotational behaviour when placed on the footfault apparatus for 3 min and this may be used as index to detect lesioned animals. Our results showed a sensitivity and specificity of 100% for lesions higher than 94% and 64%, respectively (ROC curve: AUC=0.988). A binary logistic regression model showed an expB=1.116 (95% CI, 1.007-1.236) and C=-9.081+/-4.554 (p=0.046) using the nigral tyrosine hidroxylase immunocontent as standard (each unit represents a 10%-lesion extension). Additionally, the footfault test was more sensitive than apomorphine challenging at 1mg/kg when these tests were carried out days apart and it was less sensitive than methylphenidate at 40 mg/kg (sign test, p<0.05). Therefore, the footfault test may be very useful in the PD animal model for screening animals since it is fast and simple and it does not require a drug to induce rotational activity.

Investigations of the antioxidant properties of plant extracts using a DNA-electrochemical biosensor.

In this work, the results of a method based on an electrochemical biosensor to detect DNA damage in vitro for the evaluation of the antioxidant properties of plant extracts are reported. The biosensor consisted of a dsDNA immobilized on a screen-printed electrode surface (SPE). DNA damage was promoted by the generation of the *OH radicals via Fenton-type reaction. The interaction of the radical species with immobilised DNA in the absence and presence of antioxidants was evaluated by means of changes in the guanine oxidation peak obtained by square wave voltammetry. The results demonstrated that the DNA-based biosensor is suitable as a rapid screening test for the evaluation of antioxidant properties of samples.

7A7 MAb: a new tool for the pre-clinical evaluation of EGFR-based therapies.

The epidermal growth factor receptor (EGFR) is highly expressed in many types of epithelial tumors. EGFR overexpression has been associated with an advanced stage of the disease, with resistance to standard therapies, and, for certain tumors, with poor patient prognosis. As a result, EGFR has been considered a meaningful target in anti-tumor strategies. Active and passive immunotherapies blocking EGFR and its ligands have been explored. But for successful pre-clinical evaluation of these approaches, well-established murine tumor models are not available and highly desirable. We described, for the first time, the generation and characterization of an anti-murine EGFR extracellular domain monoclonal antibody (7A7 MAb) (IgG1). 7A7 was generated by immunization of Balb/c mice with the recombinant extracellular domain of murine EGFR (rECD-mEGFR). 7A7 recognized an epitope present in the amino acidic core of the antigen and is cross-reactive with the human EGFR. Interestingly, this MAb was able to specifically bind EGFR at the cell surface, allowing the assessment of its differential expression in a panel of murine cells. Noteworthy, in a preliminary immunohistochemical study with 7A7 MAb, recognition of Balb/c mice skin sections and EGFR-positive tumors were observed. We concluded that 7A7 MAb is a valuable tool for EGFR-based therapeutic pre-clinical studies.