Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells.

BACKGROUND: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. METHODS: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. RESULTS: Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71+/CD235a+ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. CONCLUSION: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.

Annatto Oil Loaded Nanostructured Lipid Carriers: A Potential New Treatment for Cutaneous Leishmaniasis.

Annatto (Bixa orellana L.) is extensively used as food pigment worldwide. Recently, several studies have found it to have healing and antioxidant properties, as well as effective action against leishmaniasis. Therefore, the purpose of this study was to incorporate the oil obtained from annatto seeds into a nanostructured lipid carrier (NLC) and evaluate its physicochemical properties and biological activity against Leishmania major. Nanoparticles were prepared by the fusion-emulsification and ultrasonication method, with the components Synperonic™ PE (PL) as the surfactant, cetyl palmitate (CP) or myristyl myristate (MM) as solid lipids, annatto oil (AO) (2% and 4%, w/w) as liquid lipid and active ingredient, and ultra-pure water. Physicochemical and biological characterizations were carried out to describe the NLCs, including particle size, polydispersity index (PDI), and zeta potential (ZP) by dynamic light scattering (DLS), encapsulation efficiency (EE%), thermal behavior, X-ray diffraction (XRD), transmission electron microscopy (TEM), Electron Paramagnetic Resonance (EPR), cytotoxicity on BALB/c 3T3 fibroblasts and immortalized human keratinocyte cells, and anti-leishmaniasis activity in vitro. Nanoparticles presented an average diameter of ~200 nm (confirmed by TEM results), a PDI of less than 0.30, ZP between -12.6 and -31.2 mV, and more than 50% of AO encapsulated in NLCs. Thermal analyses demonstrated that the systems were stable at high temperatures with a decrease in crystalline structure due to the presence of AOs (confirmed by XRD). In vitro, the anti-leishmania test displayed good activity in encapsulating AO against L. major. The results indicate that the oily fraction of Bixa orellana L. in NLC systems should be evaluated as a potential therapeutic agent against leishmaniasis.

Shrub changes with proximity to anthropogenic disturbance in boreal wetlands determined using bi-temporal airborne lidar in the Oil Sands Region, Alberta Canada.

In this study, we used bi-temporal airborne lidar data to compare changes in vegetation height proximal to anthropogenic disturbances in the Oil Sands Region of Alberta, Canada. We hypothesize that relatively low-impact disturbances such as seismic lines will increase the fragmentation of wetlands, resulting in shrub growth. Bi-temporal lidar data collected circa 2008 and 2018 were used to identify correspondence between the density of anthropogenic disturbances, wetland shape complexity and changes in vegetation height within >1800 wetlands near Fort McKay, Alberta, Canada. We found that up to 50% of wetlands were disturbed by anthropogenic disturbance in some parts of the region, with the highest proportional disturbance occurring within fens. Areas of dense anthropogenic disturbance in bogs resulted in increased growth and expansion of shrubs, while we found the opposite to occur in fens and swamps during the 10-year period. Up to 30% of bogs had increased shrubification, while shrub changes in fens and swamps varied depending on density of disturbance and did not necessarily correspond with shrub growth. As wetland shapes became increasingly elongated, the prevalence of shrubs declined between the two time periods, which may be associated with hydrological drivers (e.g. elongated may indicate surface and ground-water discharge influences). The results of this study indicate that linear disturbances such as seismic lines, considered to have relatively minimal impacts on ecosystems, can impact proximal wetland shape, fragmentation and vegetation community changes, especially in bogs.

Nanocomposite gels of poloxamine and Laponite for ß-Lapachone release in anticancer therapy.

Nano-hybrid systems have been shown to be an attractive platform for drug delivery. Laponite® RD (LAP), a biocompatible synthetic clay, has been exploited for its ability to establish of strong secondary interactions with guest compounds and hybridization with polymers or small molecules that improves, for instance, cell adhesion, proliferation, and differentiation or facilitates drug attachment to their surfaces through charge interaction. In this work, LAP was combined with Tetronics, X-shaped amphiphilic PPO-PEO (poly (propylene oxide)-poly (ethylene oxide) block copolymers. ß-Lapachone (BLPC) was selected for its anticancer activity and its limited bioavailability due to very low aqueous solubility, with the aim to improve this by using LAP/Tetronic nano-hybrid systems. The nanocarriers were prepared over a range of Tetronic 1304 concentrations (1 to 20% w/w) and LAP (0 to 3% w/w). A combination of physicochemical methods was employed to characterize the hybrid systems, including rheology, particle size and shape (DLS, TEM), thermal analysis (TG and DSC), FTIR, solubility studies and drug release experiments. In vitro cytotoxicity assays were performed with BALB/3T3 and MCF-7 cell lines. In hybrid systems, a sol-gel transition can occur below physiological temperature. BLPC exhibits the most significant increase in solubility in formulations with a high concentration of T1304 (over 10% w/w) and 1.5% w/w LAP, or systems with only LAP (1.5%), with a 50 and 100-fold increase in solubilisation, respectively. TEM images showed spherical micelles of T1304, which elongated into wormlike micelles with concentration (20%) and in the presence of LAP, a finding that has not been reported before. A sustained release of BLPC over 140 hours was achieved in one of the formulations (10% T1304 with 1.5% laponite), which also showed the best selectivity index towards cancer cells (MCF-7) over BALB/3T3 cell lines. In conclusion, BLPC-loaded T1304/LAP nano-hybrid systems proved safe and highly effective and are thus a promising formulation for anticancer therapy.

Use of chemometrics to compare NIR and HPLC for the simultaneous determination of drug levels in fixed-dose combination tablets employed in tuberculosis treatment.

The World Health Organization recommends that TB treatment be administered using combination therapy. The methodologies for quantifying simultaneously associated drugs are highly complex, being costly, extremely time consuming and producing chemical residues harmful to the environment. The need to seek alternative techniques that minimize these drawbacks is widely discussed in the pharmaceutical industry. Therefore, the objective of this study was to develop and validate a multivariate calibration model in association with the near infrared spectroscopy technique (NIR) for the simultaneous determination of rifampicin, isoniazid, pyrazinamide and ethambutol. These models allow the quality control of these medicines to be optimized using simple, fast, low-cost techniques that produce no chemical waste. In the NIR - PLS method, spectra readings were acquired in the 10,000-4000cm-1 range using an infrared spectrophotometer (IRPrestige - 21 - Shimadzu) with a resolution of 4cm-1, 20 sweeps, under controlled temperature and humidity. For construction of the model, the central composite experimental design was employed on the program Statistica 13 (StatSoft Inc.). All spectra were treated by computational tools for multivariate analysis using partial least squares regression (PLS) on the software program Pirouette 3.11 (Infometrix, Inc.). Variable selections were performed by the QSAR modeling program. The models developed by NIR in association with multivariate analysis provided good prediction of the APIs for the external samples and were therefore validated. For the tablets, however, the slightly different quantitative compositions of excipients compared to the mixtures prepared for building the models led to results that were not statistically similar, despite having prediction errors considered acceptable in the literature.

Image-guided percutaneous intralesional administration of mesenchymal stromal cells in subjects with chronic complete spinal cord injury: a pilot study.

BACKGROUND AIMS: The potential of cell therapies to improve neurological function in subjects with spinal cord injury (SCI) is currently under investigation. In this context, the choice of cell type, dose, route and administration regimen are key factors. Mesenchymal stromal cells (MSCs) can be easily obtained, expanded and are suitable for autologous transplantation. Here we conducted a pilot study that evaluated safety, feasibility and potential efficacy of intralesional MSCs transplantation performed through image-guided percutaneous injection, in subjects with chronic complete SCI. METHODS: Five subjects with chronic traumatic SCI (>6 months), at thoracic level, classified as American Spinal Cord Injury Association impairment scale (AIS) grade A, complete injury, were included. Somatosensory evoked potentials (SSEP), spinal magnetic resonance imaging (MRI) and urodynamics were assessed before and after treatment. Autologous MSCs were injected directly into the lesion site through percutaneous injection guided by computerized tomography (CT). RESULTS: Tomography-guided percutaneous cell transplantation was a safe procedure without adverse effects. All subjects displayed improvements in spinal cord independence measure (SCIM) scores and functional independence measure (FIM), mainly due to improvements in bowel movements and regularity. Three subjects showed improved sensitivity to tactile stimulation. Two subjects improved AIS grade to B, incomplete injury, although this was sustained in only one of them during the study follow-up. CONCLUSION: Autologous bone marrow MSC transplantation, performed through CT-guided percutaneous injection, was shown to be safe and feasible. Further studies are required to demonstrate efficacy of this therapeutic scheme.

Polymeric Nanoparticles of Brazilian Red Propolis Extract: Preparation, Characterization, Antioxidant and Leishmanicidal Activity.

The ever-increasing demand for natural products and biotechnology derived from bees and ultra-modernization of various analytical devices has facilitated the rational and planned development of biotechnology products with a focus on human health to treat chronic and neglected diseases. The aim of the present study was to prepare and characterize polymeric nanoparticles loaded with Brazilian red propolis extract and evaluate the cytotoxic activity of "multiple-constituent extract in co-delivery system" for antileishmanial therapies. The polymeric nanoparticles loaded with red propolis extract were prepared with a combination of poly-ε-caprolactone and pluronic using nanoprecipitation method and characterized by different analytical techniques, antioxidant and leishmanicidal assay. The red propolis nanoparticles in aqueous medium presented particle size (200-280 nm) in nanometric scale and zeta analysis (-20 to -26 mV) revealed stability of the nanoparticles without aggregation phenomenon during 1 month. After freeze-drying method using cryoprotectant (sodium starch glycolate), it was possible to observe particles with smooth and spherical shape and apparent size of 200 to 400 nm. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and thermal analysis revealed the encapsulation of the flavonoids from the red propolis extract into the polymeric matrix. Ultra performance liquid chromatography coupled with diode array detector (UPLC-DAD) identified the flavonoids liquiritigenin, pinobanksin, isoliquiritigenin, formononetin and biochanin A in ethanolic extract of propolis (EEP) and nanoparticles of red propolis extract (NRPE). The efficiency of encapsulation was determinate, and median values (75.0 %) were calculated using UPLC-DAD. 2,2-Diphenyl-1-picryhydrazyl method showed antioxidant activity to EEP and red propolis nanoparticles. Compared to negative control, EEP and NRPE exhibited leishmanicidal activity with an IC50 value of ≅38.0 µg/mL and 31.3 µg/mL, 47.2 µg/mL, 154.2µg/mL and 193.2 µg/mL for NRPE A1, NRPE A2, NRPE A3 and NRPE A4, respectively. Nanoparticles loaded with red propolis extract in co-delivery system and EEP presented cytotoxic activity on Leishmania (V.) braziliensis. Red propolis extract loaded in nanoparticles has shown to be potential candidates as intermediate products for preparation of various pharmaceutical dosage forms containing red propolis extract in the therapy against negligible diseases such as leishmaniasis. Graphical Abstract Some biochemical mechanisms of cellular debridement of Leishmania (V.) braziliensis species by the flavonoids of red propolis extract (EEP) or NRPE loaded with red propolis extract.

Traditional uses, chemical constituents, and biological activities of Bixa orellana L.: a review.

Bixa orellana L., popularly known as "urucum," has been used by indigenous communities in Brazil and other tropical countries for several biological applications, which indicates its potential use as an active ingredient in pharmaceutical products. The aim of this work was to report the main evidence found in the literature, concerning the ethnopharmacology, the biological activity, and the phytochemistry studies related to Bixa orellana L. Therefore, this work comprises a systematic review about the use of Bixa orellana in the American continent and analysis of the data collected. This study shows the well-characterized pharmacological actions that may be considered relevant for the future development of an innovative therapeutic agent.

Gastric-resistant isoniazid pellets reduced degradation of rifampicin in acidic medium

Isoniazid and rifampicin are considered the first-line medication for preventing and treating tuberculosis. Rifampicin is degraded in the stomach acidic environment, especially when combined with isoniazid, factor contributing to treatment failure. In this study, gastric-resistant isoniazid pellets were obtained to physical contact of this drug with rifampicin and to bypass the stomach´s acidic environment. The pellets were fabricated using the extrusion-spheronization technique. The coating process was conducted in a fluid spray coater using Acrycoat L 100(r) solution as the coating agent. The pellets obtained were submitted to a dissolution test in HCl 0.1 N and phosphate buffer media. The results indicated that optimum gastric-resistance was only attained with the highest amount of coating material, with isoniazid almost fully released in phosphate buffer. The amount of rifampicin released from its mixture with non-coated isoniazid pellets in HCl 0.1 N was less than that released from its mixture with the enteric-coated pellets. Acrycoat L 100(r) was shown to be an effective enteric/gastric-resistant coating since the stability of rifampicin appeared to be enhanced when physical contact of this drug with isoniazid was prevented at low pH.

Isoniazida e rifampicina são fármacos de primeira escolha para a prevenção e tratamento da tuberculose. A rifampicina degrada-se em condições ácidas do estômago, principalmente na presença da isoniazida, o que contribui para a falha do tratamento. O presente trabalho teve como objetivo a obtenção de péletes de isoniazida gastrorresistentes, visando a evitar contato da rifampicina com isoniazida e consequente degradação no meio ácido estomacal. Os péletes foram produzidos pela técnica de extrusão-esferonização. O processo de revestimento foi conduzido em leito fluidizado com solução orgânica de Acrycoat L 100(r). Os péletes obtidos foram submetidos ao teste de dissolução em HCl 0,1 N e tampão fosfato. Os resultados indicam que a gastrorresistência foi obtida somente com a maior quantidade de revestimento, sendo a isoniazida liberada completamente no meio tampão fosfato. A quantidade de rifampicina dissolvida em meio ácido, quando associada a péletes de isoniazida não revestidos, foi menor do que a observada na presença de péletes de liberação entérica. O polímero Acrycoat L 100(r) mostrou-se eficiente para o recobrimento com a função de gastrorresistência, indicando que a instabilidade da rifampicina pode ser reduzida nas associações com a isoniazida através do revestimento entérico da isoniazida.

Strategies used for to improve aqueous solubility of simvastatin: a systematic review / Estratégias utilizadas para melhoria da solubilidade da sinvastatina – revisão sistemática

A great variety of molecules with potential pharmacological activity have been discovered. However, the great majority of these molecules have poor bioavailability, mainly associated with their low solubility in water. Lately, a great deal of attention has been paid to existing drugs that are used chronically in the treatment of high levels of cholesterol and triglycerides. Among these drugs, simvastatin is the most frequently used, despite its poor solubility in water. This review discusses some of the strategies that have been used to enhance the aqueous solubility of simvastatin over the last 12 years (January 2000 - April 2012). Techniques employing solid dispersions, microencapsulation, supercritical fluid and the cyclodextrin inclusion system are described and systematically compared.

Uma grande variedade de moléculas com potencial atividade farmacológica tem sido descoberta, mas em sua grande maioria com problemas de baixa biodisponibilidade, algumas vezes por apresentarem baixa solubilidade em água. Desse modo os olhares têm se voltado às moléculas já existentes e, nesse contexto, inclui-se a sinvastatina, com baixa biodisponibilidade e solubilidade aquosa, de uso crônico em pacientes com taxas aumentadas de colesterol e triglicerídeos. Esse trabalho tem por objetivo relacionar a literatura científica em que foi feita alguma modificação de solubilidade da sinvastatina num período de 12 anos (janeiro de 2000 - abril 2012), utilizando para isso técnicas como formação de dispersões sólidas, microemulsões, nanopartículas, fluido supercrítico e complexos de inclusão com ciclodextrinas. Além de relacionar, este artigo compara e avalia os resultados como gráficos, destacando ainda aspectos como técnicas de caracterização e estudos de estabilidade. Portanto, este trabalho tem características de uma revisão sistemática com coleta e análise de dados, de modo simples, ajudando na busca das informações.

Aborto provocado: redução da frequência e gravidade das complicações. Consequência do uso de misoprostol / Artificial abortion: is reduction in the frequency and severity of complications a result of the use of misoprostol

OBJECTIVES: to ascertain the frequency and severity of complications resulting from artificial abortions and their possible association with the use of misoprostol. METHODS: a cross-sectional study was carried out. For ten months, a checklist (of World Health Organization criteria) was applied to all 543 women admitted to hospital for abortion at two hospitals in the city of Campinas, in the State of São Paulo, Brazil. Those classified as having a possibly, pro-bably or certainly artificial abortion were asked to fill in the questionnaire. RESULTS: of all the women admitted to hospital, 259 (48 percent) were classified as possibly, probably or certainly having an induced abortion and these filled in the questionnaire; 25 women stated that they had induced the abortion and, of these, nine mentioned the use of misoprostol. Infections and hemorrhaging were complications in 10 percent and 13 percent of the 259 women. Those who used misoprostol had fewer complications than those who used other methods, although this difference was not statistically significant, perhaps for reason of the low frequency for complications. CONCLUSIONS: the data show a reduction in the frequency and severity of complications arising from abortion, although it is not possible to point to the use of misoprosol as being responsible for this.

OBJETIVOS: verificar a frequência e a gravidade das complicações por abortos provocados e suas possíveis associações com o uso de misoprostol. MÉTODOS: estudo de corte transversal. Durante dez meses aplicou-se uma lista de verificação (critérios da World Health Organization) a todas as 543 mulheres internadas por aborto em dois hospitais na cidade de Campinas, São Paulo. Àquelas classificadas como aborto possível, provável ou certamente provocado foi aplicado também um questionário. RESULTADOS: dentre todas as mulheres internadas, 259 (48 por cento) foram classificadas como aborto possível, provável ou certamente induzido e responderam ao questionário; 25 mulheres declararam a indução do aborto e, destas, nove referiram uso de misoprostol. Complicações infecciosas e hemorrágicas ocorreram respectivamente em 10 por cento e 13 por cento das 259 mulheres. As que usaram misoprostol se complicaram menos que as que usaram outros métodos, porém essa diferença não foi estatisticamente significativa, talvez pela baixa freqüência de complicações. CONCLUSÕES: os dados mostram redução da freqüência e da gravidade das complicações do aborto, mas não permitem avaliar o papel do misoprostol.

Validade e fidedignidade da Escala de Depressão Gerißtrica na identificação de idosos deprimidos em um hospital geral / Validity and reliability of the Geriatric Depression Scale for the identification of depressed patients in a general hospital

OBJETIVO: O objetivo deste estudo foi avaliar validade e fidedignidade da Escala de Depressão Gerißtrica (EDG) nas versões de 30 e 15 itens na identificação de suspeitas de depressão em idosos nas enfermarias de clínica médica do Hospital Universitßrio Lauro Wanderley, em João Pessoa, Paraíba. MÉTODOS: Realizou-se um estudo observacional e transversal, com aplicação da EDG-30 e da EDG-15 por uma entrevistadora treinada, efetuando-se no mesmo dia exame mental dos pacientes por um psiquiatra de acordo com critérios da CID-10. A fidedignidade das duas versões da EDG foi avaliada por meio da técnica do teste-reteste e da equivalência interobservadores. RESULTADOS: A concordância entre a aplicação da EDG30 e o exame psiquißtrico foi significativa e moderada (kappa = 0,48; p = 0,04), enquanto a EDG-15 não apresentou concordância estatística (p = 0,62). A EDG-30 apresentou sensibilidade de 83 por cento e especificidade de 57 por cento, mas a EDG-15 apresentou sensibilidade de 50 por cento e especificidade de 62 por cento. Os escores da EDG-30 apresentaram correlação estatisticamente significativa entre si no teste-reteste e na equivalência entre observadores, porém a versão de 15 itens não foi fidedigna. CONCLUSÕES: Conclui-se que a EDG-30 é mais sensível e fidedigna que a EDG-15. Assim, a EDG-30 é útil para detectar como negativos os pacientes que realmente não apresentem depressão, porém são necessßrios estudos posteriores para adequação dos itens dessa escala à nossa clientela, a fim de aumentar sua especificidade e valor preditivo positivo.

OBJECTIVE: The aim of this study was to evaluate the validity and reliability of the Geriatric Depression Scale (GDS) in the versions of 30 and 15 items for the identification of suspicion of depression in the elderly in the infirmary of general practice of the Hospital Universitßrio Lauro Wanderley, in João Pessoa, Paraíba. METHODS: An observational and transversal study, with the application of GDS-30 and GDS-15 by a trained interviewer, was held at the same day the mental exam of the patients by a psychiatrist, according to the criteria of ICD-10. The concordance of the two versions of the GDS was evaluated by the test-retest method and inter-observers equivalence. RESULTS: The concordance between the application of the GDS-30 and the psychiatric exam was significant and moderated (kappa = 0.48, p = 0.04), while the GDS-15 didn't present statistics concordance (p = 0.62). The GDS-30 presented sensitivity of 83 percent and specificity of 57 percent, but the GDS-15 presented sensitivity of 50 percent and specificity of 62 percent. Scores of GDS-30 presented significant statistically correlation in the test-retest method and inter-observers equivalence, but the version of 15 items weren't reliable. CONCLUSION: The conclusion was that the GDS-30 is more sensitive and reliable than the GDS-15, however its sensitivity was inferior to the numbers related in other studies, while his specificity was not high. Therefore, the GDS-30 is useful to detect as negatives the patients who really do not have depression, however further studies are necessary to adjust the items of this scale to our clientele, in order to enhance its specificity and positive predictive value.