Studies onMimosa tenuiflora callus culture. Interaction of kinetin and 2,4 dichlorophenoxyacetic acid in initiation and growth

Summary Influence of growth regulators during initiation ofMimosa tenuiflora calli tissue varied according to explant source. KN (Kinetin) affected hypocotyls, while an interaction between different combinations of 2,41) (2,4 dichlorophenoxyacetic acid) and KN induced cotyledons,stems and leaves. Calli growth after 60 days was influenced by both hormones in cotyledons, by 2,4D in hypocotils and by an interaction of the two regulators in stems and leaves. The indole alkaloid Nb-Nb dimethyltryptamine was found to be present in some cultures at a level comparable to that of the source plant.     

22-Oxocholestanes SPGP4 and SPGP8: in Silico and in Vitro Study as Activators of Plant Growth Promotion

The 22-oxocholestanes compounds have shown an outstanding plant growth promoting activity; they have similar bioactivity as brassinosteroids, so they are normally named as brassinosteroid analogs thinking that they also impact on the known receptor BRI1. However, in silico studies allow us to predict interactions with other receptors and thus it's possible to evaluate them, through receptors of gibberellins, auxins, jasmonates, strigolactones and the protein associated with the BRI1 gene. This article describes the bioactivity of structures SPGP4 and SPGP8 as plant growth-promoting compounds. Both structures present coupling energies and interactions at the same level as epibrassinolide in the protein associated with BRI1 gene. Additionally, interactions through the auxin pathway and to strigolactone receptor were found using selected tests. In the rice lamina tilt, a higher effect was obtained when SPGP4 and SPGP8 were compared to epibrassinolide, although in a lesser level vis à vis to homobrassinolide. In the same way, when SPGP4 and SPGP8 were tested in the Growth Root Model an activity as strigonolactones was observed, enhancing the relationship between the main and secondary roots. However, the growth of coleptiles, when applying auxins, compounds SPGP4 and SPGP8 did not reach the same level as controls. In the tests associated to gibberellins and jasmonic acid, an increased bioactivity was observed, although this behavior was not reflected from the in silico study, possibly due to secondary signaling cascades. This work demonstrates that the 22-oxocolestane compounds SPGP4 and SPGP8 could be used as plant growth hormones, promoting several pathways.     

The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

Design of a microfluidic mixer channel: First steps into creating a fluorescent dye-based biosensor for mAb aggregate detection

A major challenge in the transition to continuous biomanufacturing is the lack of process analytical technology (PAT) tools which are able to collect real-time information on the process and elicit a response to facilitate control. One of the critical quality attributes (CQAs) of interest during monoclonal antibodies production is aggregate formation. The development of a real-time PAT tool to monitor aggregate formation is then crucial to have immediate feedback and process control. Miniaturized sensors placed after each unit operation can be a powerful solution to speed up an analytical measurement due to their characteristic short reaction time. In this work, a micromixer structure capable of mixing two streams is presented, to be employed in the detection of mAb aggregates using fluorescent dyes. Computational fluid dynamics (CFD) simulations were used to compare the mixing performance of a series of the proposed designs. A final design of a zigzag microchannel with 45° angle was reached and this structure was subsequently fabricated and experimentally validated with colour dyes and, later, with a FITC-IgG molecule. The designed zigzag micromixer presents a mixing index of around 90%, obtained in less than 30 seconds. Therefore, a micromixer channel capable of a fast and efficient mixing is hereby demonstrated, to be used as a real-time PAT tool for a fluorescence based detection of protein aggregation.     

Evidence for the Implication of Phosphoinositol Signal Transduction in μ-Opioid Inhibition of DNA Synthesis

An opioid receptor agonist, [D-Ala2,Me-Phe4,Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the mu-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The mu-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly mu-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.     

Aspirin-triggered resolvin D1 reduces parasitic cardiac load by decreasing inflammation in a murine model of early chronic Chagas disease

BackgroundChagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and is widely distributed worldwide because of migration. In 30% of cases, after years of infection and in the absence of treatment, the disease progresses from an acute asymptomatic phase to a chronic inflammatory cardiomyopathy, leading to heart failure and death. An inadequate balance in the inflammatory response is involved in the progression of chronic Chagas cardiomyopathy. Current therapeutic strategies cannot prevent or reverse the heart damage caused by the parasite. Aspirin-triggered resolvin D1 (AT-RvD1) is a pro-resolving mediator of inflammation that acts through N-formyl peptide receptor 2 (FPR2). AT-RvD1 participates in the modification of cytokine production, inhibition of leukocyte recruitment and efferocytosis, macrophage switching to a nonphlogistic phenotype, and the promotion of healing, thus restoring organ function. In the present study, AT-RvD1 is proposed as a potential therapeutic agent to regulate the pro-inflammatory state during the early chronic phase of Chagas disease.Methodology/Principal findingsC57BL/6 wild-type and FPR2 knock-out mice chronically infected with T. cruzi were treated for 20 days with 5 μg/kg/day AT-RvD1, 30 mg/kg/day benznidazole, or the combination of 5 μg/kg/day AT-RvD1 and 5 mg/kg/day benznidazole. At the end of treatment, changes in immune response, cardiac tissue damage, and parasite load were evaluated. The administration of AT-RvD1 in the early chronic phase of T. cruzi infection regulated the inflammatory response both at the systemic level and in the cardiac tissue, and it reduced cellular infiltrates, cardiomyocyte hypertrophy, fibrosis, and the parasite load in the heart tissue.Conclusions/SignificanceAT-RvD1 was shown to be an attractive therapeutic due to its regulatory effect on the inflammatory response at the cardiac level and its ability to reduce the parasite load during early chronic T. cruzi infection, thereby preventing the chronic cardiac damage induced by the parasite.     

Telomere length in healthy newborns is not affected by adverse intrauterine environments

Different intrauterine exposures are associated with different metabolic profiles leading to growth and development characteristics in children and also relate to health and disease patterns in adult life. The objective of this work was to evaluate the impact of four different intrauterine environments on the telomere length of newborns. This is a longitudinal observational study using a convenience sample of 222 mothers and their term newborns (>37 weeks of gestational age) from hospitals in Porto Alegre, Rio Grande do Sul (Brazil), from September 2011 to January 2016. Sample was divided into four groups: pregnant women with Gestational Diabetes Mellitus (DM) (n=38), smoking pregnant women (TOBACCO) (n=52), mothers with small-for-gestational age (SGA) children due to idiopathic intrauterine growth restriction (n=33), and a control group (n=99). Maternal and newborn genomic DNA were obtained from epithelial mucosal cells. Telomere length was assessed by qPCR, with the calculation of the telomere and single copy gene (T/S ratio). In this sample, there was no significant difference in telomere length between groups (p>0.05). There was also no association between childbirth weight and telomere length in children (p>0.05). For term newborns different intrauterine environments seems not to influence telomere length at birth.     

Carbon Partitioning in Soybean Infected with Meloidogyne incognita and M. javanica

Seven-day-old seedlings of two cultivars (Cristalina and UFV ITM1) of Glycine max were inoculated with 0, 3,000, 9,000, or 27,000 eggs of Meloidogyne incognita race 3 or M. javanica and maintained in a greenhouse. Thirty days later, plants were exposed to ¹⁴CO₂ for 4 hours. Twenty hours after ¹⁴CO₂ exposure, the root fresh weight, leaf dry weight, nematode eggs per gram of root, total and specific radioactivity of carbohydrates in roots, and root carbohydrate content were evaluated. Meloidogyne javanica produced more eggs than M. incognita on both varieties. A general increase in root weight and a decrease in leaf weight with increased inoculum levels were observed. Gall tissue appeared to account for most of the root mass increase in seedlings infected with M. javanica. For both nematodes there was an increase of total radioactivity in the root system with increased levels of nematodes, and this was positively related to the number of eggs per gram fresh weight and to the root fresh weight, but negatively related to leaf dry weight. In most cases, specific radioactivities of sucrose and reducing sugars were also increased with increased inoculum levels. Highest specific radioactivities were observed with reducing sugars. Although significant changes were not observed in endogenous levels of carbohydrates, sucrose content was higher than reducing sugars. The data show that nematodes are strong metabolic sinks and significantly change the carbon distribution pattern in infected soybean plants. Carbon partitioning in plants infected with nematodes may vary with the nematode genotype.     

B1‐type cyclins control microtubule organization during cell division in Arabidopsis

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development‐specific requirements of B1‐type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1‐type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1‐type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA‐TUBULIN COMPLEX PROTEIN 3‐INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1‐type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.     

Active immunization combined with cisplatin confers enhanced therapeutic protection and prevents relapses of HPV-induced tumors at different anatomical sites

The active immunotherapy concept relies on the use of vaccines that are capable of inducing antitumor immunity, reversion of the suppressive immunological environment, and long-term memory responses. Previously, antitumor vaccines based on a recombinant plasmid (pgDE7h) or a purified protein (gDE7) led to regression of early-established human papillomavirus (HPV)-associated tumors in a preclinical model. In this work, the anticancer vaccines were combined with cisplatin to treat HPV-induced tumors at advanced growth stages. The antitumor effects were evaluated in terms of tumor regression, induction of specific CD8⁺ T cells, and immune modulation of the tumor microenvironment. Acute toxicity induced by the treatment was measured by weight loss and histological alterations in the liver and kidneys. Our results revealed that the combination of cisplatin with either one of the tested immunotherapies (pgDE7h or gDE7) led to complete tumor regression in mice. Also, the combined treatment resulted in synergistic effects, particularly among mice immunized with gDE7, including activation of systemic and tumor-infiltrating E7-specific CD8⁺ T cells, tumor infiltration of macrophages and dendritic cells, and prevention of tumor relapses at different anatomical sites. Furthermore, the protocol allowed the reduction of cisplatin dosage and its intrinsic toxic effects, without reducing antitumor outcomes. These results expand our knowledge of active immunotherapy protocols and open perspectives for alternative treatments of HPV-associated tumors.