A protocol for the preparation of fatty acid-functionalized nanohydroxyapatite was developed. These nanoparticles were characterized and used as building blocks for the preparation of Pickering water-in-oil emulsions, which in turn were used as a template for colloidosomes. It is possible to encapsulate proteins in these structures, and their low cellular toxicity was also demonstrated. These structures show structural fragility to pH and temperature changes.

Vanilla (Orchidaceae) is a highly valued spice used in a wide range of products. Although wild crop relatives of Vanilla planifolia, the primary cultivated species, may possess traits for crop improvement, they have received limited attention. This study presents an optimized and reproducible flow cytometry protocol adapted to Vanilla leaves, that effectively prevents nuclei clustering and raphide interference. Assessing nuclear C-DNA content and estimating DNA ploidy in succulent plant tissues pose significant challenges. The abundance of polysaccharides and raphides in Vanilla tissues often leads to inaccuracies in cytometric measurements and difficulties in instrument maintenance. To address this issue, we used a nuclei isolation buffer containing 2.0 % (v/v) detergent and utilized the clear upper phase of the filtrate after raphide sedimentation to obtain accurate nuclear DNA content values. We applied this protocol to estimate the C-DNA content of 12 Vanilla species native to Costa Rica and a widely cultivated commercial hybrid. The values ranged from 5.211 +/- 0.008 pg in V. planifolia to 9.0 +/- 1.3 pg in V. pompona, with no clear distinction between species of the subgenera Vanilla and Xanata. For the first time, we report the 2C-DNA content of V. costaricensis, V. dressleri, V. hartii, V. helleri, V. inodora, V. karenchristianae and the 'Vaitsy' hybrid. Our results confirm the occurrence of partial endoreplication in all samples analyzed. This study contributes to the understanding of less studied wild crop relatives of V. planifolia and provides an improved method for flow cytometric analysis that can be applied to tissues containing mucilaginous compounds and raphides of other plant species.

This study explores the development of biocompatible scaffolds for bone regeneration, utilizing polylactic acid (PLA) combined with calcium phosphate as a pH buffer and diatomaceous earth as a biocompatibilizer. These materials were extruded and 3D-printed to enhance cell adhesion and biodegradability after enough cell growth. The biocompatibility of the resulting composites, with different proportions of the components and sterilization methods, was tested according to the ISO 10993 protocol. The optimal performance, with nearly zero cytotoxicity, was observed with 20 PLA/1 CP/1 DE mass ratios and gamma sterilization. Tension analysis and scanning electron microscopy (SEM) were applied to the 3D-printed composites, which were also analyzed by differential scanning calorimetry (DSC) to understand the origin of the tension properties better, which were comparable to those of cancellous bone. Degradation tests under physiological conditions for 13 weeks showed no significant mass loss. Furthermore, it was observed that cell adhesion, viability, proliferation, and osteoconduction are possible in the scaffolds studied, opening opportunities for future studies to substantiate the use of 3D-printed silica-filled composites as an alternative to homologous implants for various bone regeneration applications.

Supplementary Table 2 from Elevated Systemic Levels of Inflammatory Cytokines in Older Women with Persistent Cervical Human Papillomavirus Infection

Supplementary Table 1 from Elevated Systemic Levels of Inflammatory Cytokines in Older Women with Persistent Cervical Human Papillomavirus Infection

A bio-assay guided fractionation of Piper garagaranum C. DC. led to the isolation of two prenylated hydroxy-benzoic acids (1-2) with anti-inflammatory and cytotoxic activities. The anti-inflammatory action was determined in an LPS stimulated RAW 264.7 murine macrophage assay with IC50 values for inhibition of NO production of (18 +/- 3) and (26 +/- 5) mu M, for 1 and 2, respectively. These compounds do not inhibit NO production by a competitive inhibition of the iNOS enzyme and show anti-inflammatory properties by lowering the expression of pro-inflammatory genes (TNF-alpha, IL-1 beta, CXCL2 and CCL2), as determined by qRT-PCR. Electrochemical measurements using cyclic voltammetry (CV) show that compound 1 exhibits anti-oxidant properties. This is the first phytochemical study of this plant, and we report a preliminary study of the biological activity of the isolated compounds.

Introducción. La transformación del arroz (Oryza sativa L. ssp indica) mediada por Agrobacterium, representa una oportunidad para la investigación científica y el mejoramiento genético. Es necesaria la optimización del protocolo para obtener la mayor eficiencia de transformación. Objetivo. Evaluar diferentes factores que afectan la transformación genética en callos embriogénicos de arroz de la subespecie indica vía Agrobacterium tumefaciens. Materiales y métodos. Este estudio se realizó en San José, Costa Rica, entre 2012 y 2014. En seis tratamientos se evaluaron: el efecto de la edad del callo, la concentración de acetosiringona, condición luminosa, la presencia o ausencia de radícula y la cepa de Agrobacterium tumefaciens en la transformación genética de callos embriogénicos de arroz de la variedad CR5272 con el gen reportero gus. Se compararon la cepa de Agrobacterium LBA4404 con el plásmido pCAMBIA1305.2 y las cepas ATHV, GV3101 y LBA4404, con el plásmido pCAMBIA1303; mediante pruebas histoquímicas para la detección de la expresión transitoria del gen marcador que codifica para la β-glucuronidasa. Resultados. La evaluación de los seis tratamientos con la cepa LBA4404::pCAMBIA1305.2 resultó en expresión transitoria del gen GusPlus de 1,33-7,00 % para la variedad CR5272 y de 8,00 % para el control con la variedad Nipponbare (ssp. japonica). Las cepas con el plásmido pCAMBIA1303 presentaron una expresión transitoria del gen gusA entre el 100-65 % con un área promedio de 14,23 mm2 (ATHV), 8,81 mm2 (GV3101), y 8,83 mm2 (LBA4404), sin diferencias significativas entre ellas; sin embargo, sí hubo diferencias al compararlas con la cepa LBA4404::pCAMBIA1305.2 (85 %, 4,39 mm2). Conclusiones. La utilización de las condiciones: callos de seis días, concentración de acetosiringona de 76 µM, luz antes y después del cocultivo, presencia de radícula y la cepa ATHV::pCAMBIA 1303, mejoraron la eficiencia de transformación con Agrobacterium tumefaciens en la variedad de arroz CR5272.

Introduction. Agrobacterium-mediated transformation of rice (Oryza saliva L. ssp indica) represents an opportunity for scientific research and genetic improvement. Optimization of the protocol is necessary to obtain the highest transformation efficiency. Objective. To evaluate different factors that affect the genetic transformation in embryogenic rice callus of subspecies indica through Agrobacterium tumefaciens. Materials and methods. This study was performed in San Jose, Costa Rica between 2012 and 2014. The following were evaluated in six treatments: the effect of callus age, acetosyringone concentration, lighting condition, the presence or absence of radicle, and Agrobacterium tumefaciens strain on the genetic transformation of embryogenic calli of rice variety CR5272 with gus reporter gene. Agrobacterium strains LBA4404 with pCAMBIA1305.2 plasmid, and strains ATHV, GV3101, and LBA4404 with the pCAMBIA 1303 plasmid were compared; by histochemical tests for the detection of transient expression of the beta-glucuronidase reporter gene. Results. The evaluation of the six treatments with strain LBA4404::pCAMBIA 1305.2 resulted in transient expression of 1.33-7.00 % of the GusPlus gene for the CR5272 variety, and 8.00 % for the control with the Nipponbare (ssp. japonica) variety. The strains with the pCAMBIA1303 plasmid showed a transient expression of the gusA gene between 100-65 % with an average area of 14.23 mm(2) (ATHV), 8.81 mm(2) (GV3101), and 8.83 mm(2) (LBA4404) with no significant differences between them; however, there were differences when compared with strain LBA4404::pCAMBIA1305.2 (85 %, 4.39 mm(2)). Conclusions. The use of the conditions: six-day callus, acetosyringone concentration of 76 mu M, light before and after cocultivation, presence of radicle and the ATHV::pCAMBIA 1303 , improved the transformation efficiency with Agrobacterium tumefaciens in the rice variety C125272.

Psidium friedrichsthalianum (O. Berg) Nied is a species found from southern Mexico, Central America; and there are reports that it is also found in Venezuela and Ecuador. It is a common fruit component of the Costa Rican diet, and it is valued industrially for its high content of polyphenols, mainly proanthocyanidins (PACs). This crop is not completely domesticated and there are no improved varieties produced through plant breeding. Genome size or ploidy levels have not been investigated in Costa Rican populations of Psidium friedrichsthalianum. Information about chromosome number and genome size is paramount for plant breeding strategies. Therefore, the main objective of our study was to determine chromosome number using pollen meiocytes and genome size by flow cytometry in six populations of P. friedrichsthalianum in Costa Rica. We found x = 11 bivalent chromosomes in all meiocytes analysed, classifying these populations as diploid. All populations had an average nuclear DNA content of 2C = 1.960 +/- 0.005 pg. No statistically significant differences in nuclear DNA content were found among populations. We conclude that the consistency in chromosome number and genome size among populations suggests a common origin among them. Our estimates of the number of chromosomes and genome size of P. friedrichsthalianum determined in this study will be essential for future breeding programs, hybridization practices and development of QTL (Quantitative Trait Loci).

Nearly half of the world’s population relies on combustion of solid biofuels to cover fundamental energy demands. Epidemiologic data demonstrate that particularly long-term emissions adversely affect human health. However, pathological molecular mechanisms are insufficiently characterized. Here we demonstrate that long-term exposure to fine particulate matter (PM2.5) from biomass combustion had no impact on cellular viability and proliferation but increased intracellular reactive oxygen species (ROS) levels in bronchial epithelial BEAS-2B cells. Exposure to PM2.5 induced the nuclear factor erythroid 2-related factor 2 (Nrf2) and mediated an anti-oxidative response, including enhanced levels of intracellular glutathione (GSH) and nuclear accumulation of heme oxygenase-1 (HO-1). Activation of Nrf2 was promoted by the c-Jun N-terminal kinase JNK1/2, but not p38 or Akt, which were also induced by PM2.5. Furthermore, cells exposed to PM2.5 acquired chemoresistance to doxorubicin, which was associated with inhibition of apoptosis and elevated levels of GSH in these cells. Our findings propose that exposure to PM2.5 induces molecular defense mechanisms, which prevent cellular damage and may thus explain the initially relative rare complications associated with PM2.5. However, consistent induction of pro-survival pathways may also promote the progression of diseases. Environmental conditions inducing anti-oxidative responses may have the potential to promote a chemoresistant cellular phenotype.

We studied the adsorption ability and tolerance of the thermophilic filamentous cyanobacteria Letolyngbya 7M towards Paraquat and Bromacil. Adsorption isotherms at pH = 7.0 showed an adsorption capacity of 24.4 mg/g and 66.8 mg/g, respectively, and a good fit to the Langmuir model (R2 = 0.97 and 0.99, respectively). To evaluate the effect of both herbicides on photosynthetic pigments and viability of cyanobacteria, cell autoflorescence and esterase activity was determined using flow cytometry. Autofluorescence was less sensitive to changes in cell viability, as it was only slightly reduced at high Paraquat and Bromacil concentrations. Herbicide effect on esterase activity is dose-dependent. Bromacil did not cause a significant effect on either chlorophyll a content or cell viability. This study demonstrates the potential of Leptolyngbya 7M to remove Paraquat and Bromacil herbicides from aqueous solution under laboratory conditions.

Violacein, an indole-derived, purple-colored natural pigment isolated from Chromobacterium violaceum has shown multiple biological activities. In this work, we studied the effect of violacein in different immune cell lines, namely THP-1, MonoMac 6, ANA-1, Raw 264.7 cells, as well as in human peripheral blood mononuclear cells (PBMCs). A stimulation of TNF-α production was observed in murine macrophages (ANA-1 and Raw 264.7), and in PBMCs, IL-6 and IL-1β secretion was detected. We obtained evidence of the molecular mechanism of activation by determining the mRNA expression pattern upon treatment with violacein in Raw 264.7 cells. Incubation with violacein caused activation of pathways related with an immune and inflammatory response. Our data utilizing TLR-transfected HEK-293 cells indicate that violacein activates the human TLR8 (hTLR8) receptor signaling pathway and not human TLR7 (hTLR7). Furthermore, we found that the immunostimulatory effect of violacein in PBMCs could be suppressed by the specific hTLR8 antagonist, CU-CPT9a. Finally, we studied the interaction of hTLR8 with violacein in silico and obtained evidence that violacein could bind to hTLR8 in a similar fashion to imidazoquinoline compounds. Therefore, our results indicate that violacein may have some potential in contributing to future immune therapy strategies.

Bioassay-guided fractionation of Acnistus arborescens (Solanaceae) organic extract afforded two anti-inflammatory withanolides: 2,3-Dihydro-3 beta-O-sulfate withacnistin (1) and 2,3-Dihydro-3 beta-O-sulfate withaferin A (2) showing IC50 values of 1.0 +/- 0.1 and 14 +/- 1 mu M respectively in a LPS-stimulated RAW 264.7 murine macrophage assay. These compounds were also evaluated against two selected cancer cell lines showing significant cytotoxicity. Although there are several reports dealing with the chemistry of this species, we report for the first time the isolation of O-sulfated withanolides.

Simple surface modification of nano-hydroxyapatite, through acid-basic reactions, allows expanding the properties of this material. Introduction of organic groups such as hydrophobic alkyl chains, carboxylic acid, and amide or amine basic groups on the hydroxyapatite surface systematically change the polarity, surface area, and reactivity of hydroxyapatite without modifying its phase. Physical and chemical properties of the new derivative particles were analyzed. The biocompatibility of modified Nano-Hap on Raw 264.7 cells was also assessed.

We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case-control study within a 10,049 woman population-based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46-74 and with HPV results at their 5th year anniversary visit were considered, and all women (n = 87) with persistent HPV infections, all women (n = 196) with transient HPV infections and a random sample of HPV DNA-negative women (n = 261) frequency-matched to cases on age were selected for this study. A median of 3 years after the case-control matching visit, cervical cells were collected for liquid-based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for 3 marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95% CI = 2.2-13.3) for CD69(+)CD4(+), 2.6 (95% CI = 1.2-5.9) for HLADR(+)CD3(+)CD4(+) and 2.3 (95% CI = 1.1-4.7) for CD45RO(+)CD27(-)CD8(+). A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO(+)CD27(+)CD4(+) (OR = 0.36; 95% CI = 0.17-0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies.

Background: mRNA expression signatures are frequently used as surrogate measures of cellular function and pathway changes. Few studies have directly compared results obtained using gene expression and multiplex protein assays for corresponding gene products.Methods: We used data available from a clinical trial of a human papillomavirus-16 vaccine that tracked gene expression and cytokine/chemokine production by peripheral blood mononuclear cells stimulated in culture with various antigens to evaluate the degree to which gene expression levels reflect observed levels of cytokines/chemokines. Twenty-six women enrolled in a phase II clinical trial of a human papillomavirus-16 vaccine were evaluated for gene expression (using the Affymetrix Human Genome Focus Array) and cytokine/chemokine levels (using a bead-based 22-plex cytokine assay developed by Linco Research, Inc.) before and after vaccination.Results: Our results suggest the presence of a wide range of correlations between mRNA expression and secreted protein levels. The strongest correlation was observed for IFN-gamma (R = 0.90 overall levels; R = 0.69 when vaccine induced changes were evaluated). More modest overall correlations ranging from 0.40 to 0.80 were observed for MIP1A, IP10, TNF-alpha, MCP1, IL-2, GM-CSF, IL-5, RANTES, and IL-8. Weaker or no correlation was observed between gene expression and protein levels for the remaining cytokines/chemokines evaluated.Conclusion: The degree of correlation between gene expression and protein levels varied among different cytokines/chemokines. Impact: Researchers should be cautious when using mRNA expression array results as a proxy for protein levels using existing technologies. Cancer Epidemiol Biomarkers Prev;19 (4); 978-81. (C)2010 AACR.

Abstract Background: Defects in lymphoproliferative responses to mitogens/antigens in women >45 years old with a persistent type-specific human papillomavirus (HPV) infection have been reported. Methods: To determine whether these defects were associated with altered cytokine profiles, plasma and peripheral blood mononuclear cell (PBMC) culture supernatants from 50 cases (oversampled for their reduced lymphoproliferative ability) and 50 uninfected controls (oversampled for their robust lymphoproliferative ability) were examined for 24 cytokines using multiplexed bead–based immunoassays and ELISA. Results: The following plasma cytokines were significantly increased in cases relative to controls (cases versus controls; median pg/mL): interleukin (IL)-6, 393.1 versus 14.5; IL-8, 1,128.5 versus 43.9; tumor necrosis factor-α (TNF-α), 164.1 versus 9.2; macrophage inflammatory protein-1α (MIP-1α), 1,368.9 versus 25.5; granulocyte macrophage colony-stimulating factor (GM-CSF), 13.8 versus 7.3; IL-1β, 8.3 versus 1.6 (all P < 0.0001); and IL-1α, 218.2 versus 169.5 (P = 0.02). We focused our analysis on the cytokines IL-6, IL-8, TNF-α, and MIP-1α due to their high fold change (>10) and highly statistically significant difference between cases and controls. Length of persistence or type of infection (high risk and low risk) did not affect these differences. IL-6, TNF-α, and MIP-1α levels were also increased in unstimulated PBMC culture supernatants from cases compared with controls (P < 0.05), however, the cytokine levels from phytohemagglutinin-stimulated PBMC culture supernatants were significantly lower in the cases (P < 0.0001). Conclusions: Persistent HPV infection in older women with evidence of immune deficit is associated with an increase in systemic inflammatory cytokines. Impact: Future studies are needed to determine whether the inflammatory profile is age dependent and to examine the role that inflammatory cytokines play in HPV-induced progression from infection to cervical cancer. Cancer Epidemiol Biomarkers Prev; 19(8); 1954–9. ©2010 AACR.

The properties of porous silicon make it an interesting material for biological applications. However, porous silicon is not an appropriate surface for cell growth. Surface modification is an alternative that could afford a bioactive material. In this work, we report a method to yield materials by modification of the porous silicon surface with hydroxyapatite of nanometric dimensions, produced using an electrochemical process and coated on macroporous silicon substrates by cathodic bias.The chemical nature of the calcium phosphate deposited on the substrates after the experimental process and the amount of cell growth on these surfaces were characterized. (C) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ownload kground: Defects in lymphoproliferative responses to mitogens/antigens in women >45 years old with istent type-specific human papillomavirus (HPV) infection have been reported. thods: To determine whether these defects were associated with altered cytokine profiles, plasma and eral blood mononuclear cell (PBMC) culture supernatants from 50 cases (oversampled for their reduced oproliferative ability) and 50 uninfected controls (oversampled for their robust lymphoproliferative ) were examined for 24 cytokines using multiplexed bead–based immunoassays and ELISA. ults: The following plasma cytokines were significantly increased in cases relative to controls (cases controls; median pg/mL): interleukin (IL)-6, 393.1 versus 14.5; IL-8, 1,128.5 versus 43.9; tumor necrosis α (TNF-α), 164.1 versus 9.2; macrophage inflammatory protein-1α (MIP-1α), 1,368.9 versus 25.5; grane macrophage colony-stimulating factor (GM-CSF), 13.8 versus 7.3; IL-1β, 8.3 versus 1.6 (all P < 0.0001); -1α, 218.2 versus 169.5 (P = 0.02). We focused our analysis on the cytokines IL-6, IL-8, TNF-α, and MIP-1α their high fold change (>10) and highly statistically significant difference between cases and controls. of persistence or type of infection (high risk and low risk) did not affect these differences. IL-6, TNF-α, IP-1α levels were also increased in unstimulated PBMC culture supernatants from cases compared ontrols (P < 0.05), however, the cytokine levels from phytohemagglutinin-stimulated PBMC culture atants were significantly lower in the cases (P < 0.0001). clusions: Persistent HPV infection in older women with evidence of immune deficit is associated with rease in systemic inflammatory cytokines. act: Future studies are needed to determine whether the inflammatory profile is age dependent and to Imp examine the role that inflammatory cytokines play in HPV-induced progression from infection to cervical cancer. Cancer Epidemiol Biomarkers Prev; 19(8); 1954–9. ©2010 AACR.