Chromosome Segregation Is Biased by Kinetochore Size

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Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.     

Examining the reaction between antioxidant compounds and 2,2-diphenyl-1-picrylhydrazyl (DPPH) through a computational investigation

In this work, we present a computational investigation on the reactions between two well-known antioxidants (quercetin and morin) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). A density functional theory (DFT) approach with the B3LYP functional and the 6-31G(d,p) basis set was used for the simulations. The structural and energetic parameters (Gibbs free-energy, ΔG, and Gibbs free-energy of activation, ΔG⁺⁺) were determined to provide information on the antioxidant activity as well as to evaluate the contributions of each hydroxyl group to the referred property. According to the results obtained, quercetin presented three hydroxyls as being thermodynamically spontaneous in the reaction with DPPH (4\(^{\prime }\)-ArOH, 3\(^{\prime }\)-ArOH, and 3-ArOH, with ΔG = -4.93 kcal/mol, -2.89 kcal/mol, and -1.87 kcal/mol, respectively) against only two in the case of morin (2\(^{\prime }\)-ArOH and 3-ArOH, with ΔG = -7.56 kcal/mol and -4.57 kcal/mol, respectively). Hence, quercetin was found to be a more efficient antioxidant, which is in agreement with different experimental and computational investigations of bond dissociation enthalpies (BDEs). However, the order of contribution of the OH groups of each compound to the antioxidant potential present some differences when compared to what was seen in the previous investigations, especially for morin. These findings are in contrast to what was observed in studies based on the determinations of BDEs. Therefore, experimental investigations on the hydrogen-atom transfer mechanism (HAT) for both compounds are encouraged in order to clarify these observations.     

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67

Cryptococcus neoformans is an opportunistic human pathogen that causes life‐threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal–host relationship is discussed.     

Larval development of mandi-pintado Pimelodus britskii (Siluriformes: Pimelodidae)

This study investigated the morphology, morphometric and meristic characters of 117 larval Pimelodus britskii showing early development of head, eye, barbel and snout. Body and mouth pigmentation increased throughout development; the mouth was ventrally situated in the yolk-sac stage, becoming subterminal afterwards, and an embryonic fin was visible in all four stages observed. Post-flexion larval P. bristskii are distinguished from larval P. ortmanni by having 47–50 myomeres (v. 36).     

Antiproliferative terpenoids and alkaloids from the roots of Maytenus vitis-idaea and Maytenus spinosa

Investigation of the organic extracts of the roots of Maytenus vitis-idaea and Maytenus spinosa, collected in the province of Salta, Argentina, led to isolation of eighteen compounds belonging to several classes. From M. vitis-idaea, eight methylenequinone celastroids (1–8) were isolated, four of which (4–7) were hitherto unknown. Additionally, from M. spinosa, two known celastroids, a known celastroid dimer (9), three pentacyclic triterpenoids (10–12) and six β-dihydroagarofuran sesquiterpenoid alkaloids (13–18) were identified. Compounds 4–7 were active against six solid tumor cell lines at micromolar concentrations.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Application of flow cytometry for the identification of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples

Staphylococcus epidermidis is considered to be one of the most common causes of nosocomial bloodstream infections, particularly in immune-compromised individuals. Here, we report the development and application of a novel peptide nucleic acid probe for the specific detection of S. epidermidis by fluorescence in situ hybridization. The theoretical estimates of probe matching specificity and sensitivity were 89 and 87%, respectively. More importantly, the probe was shown not to hybridize with closely related species such as Staphylococcus aureus. The method was subsequently successfully adapted for the detection of S. epidermidis in mixed-species blood cultures both by microscopy and flow cytometry.     

DNA barcoding of freshwater ichthyoplankton in the Neotropics as a tool for ecological monitoring

Quantifying and classifying ichthyoplankton is one of the most effective ways of monitoring the recruitment process in fishes. However, correctly identifying the fish based on morphological characters is extremely difficult, especially in the early stages of development. We examined ichthyoplankton from tributaries and reservoirs along the middle stretch of the Paranapanema River, one of the areas most impacted by hydroelectric projects in the Neotropics. Matching DNA sequences of the COI gene (628–648 bp) allowed us to identify 99.25% of 536 samples of eggs (293) and larvae (243) subjected to BOLD‐IDS similarity analysis with a species‐level threshold of 1.3%. The results revealed 37 species in 27 genera, 15 families and four orders, some 23.8% of documented fish species in the Paranapanema River. Molecular identification meant that we could include data from egg samples that accounted for about 30% of the species richness observed. The results in this study confirm the efficacy of DNA barcoding in identifying Neotropical ichthyoplankton and show how the data produced provide valuable information for preparing plans for conserving and managing inland waters.