Adsorption behavior and activity of horseradish peroxidase onto polysaccharide-decorated particles

The adsorption behavior of horseradish peroxidase (HRP) onto hybrid particles of poly(methylmethacrylate) (PMMA) and carboxymethylcellulose (CMC) was investigated by means of spectrophotometry. Dispersions of PMMA/CMC particles were characterized by light scattering, zeta potential measurements and scanning electron microscopy before and after HRP adsorption. HRP adsorbed irreversibly onto PMMA/CMC particles; the adsorption isotherm showed an initial step and an adsorption plateau. The enzymatic activity of free HRP and immobilized HRP (plateau region) was monitored by means of spectrophotometry as a function of storing time. Upon adsorbing HRP there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. HRP-covered PMMA/CMC particles dispersions, which were dried and re-dispersed, retained 50% of their catalytic properties. These interesting findings were discussed in the light of a beneficial effect of a hydrated microenvironment for maintenance of enzyme conformation and activity.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae), Part 3: Analysis of the RAPD and ISSR molecular markers

The Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) molecular markers were used to complement the study of chromosomal polymorphism in Astyanax fasciatus (Teleostei, Characidae) from the Mogi-Guaçu River (Southeastern Brazil), analyzed in three collection sites along the river (Ouro Fino – MG, Cachoeira de Emas – SP and Barrinha – SP). Two cytotypes (or karyotypic types), denominated standard cytotypes, were previously characterized, one including 2n = 46 chromosomes and the other 2n = 48 chromosomes, where all the chromosomes of the complement form homologous pairs. Additionally, variant karyotypic forms with 2n = 45, 46 and 47 chromosomes were also detected, although with a lower frequency in relation to the standard cytotypes. RAPD turned out little informative in the analysis of the observed situation, indicating a high value of migrants per generation among the cytotypes. On the other hand, ISSR showed a small structure, especially among the standard cytotypes from the Barrinha region where the Nm was 0.4301 with a genetic identity of 0.6862 and genetic distance of 0.3765. However, the general results obtained do not discard the possibility of interbreeding between both standard cytotypes and/or their descendants as a source of chromosome variation. The association between the cytogenetic and molecular markers viabilized putative explanatory scenery for the origin and evolution of the forms seen in A. fasciatus.     

Host cell protein LAMP‐2 is the receptor for Trypanosoma cruzi surface molecule gp82 that mediates invasion

Host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is mediated by MT‐specific surface molecule gp82, which binds to a still unidentified receptor, inducing lysosome spreading and exocytosis required for the parasitophorous vacuole formation. We examined the involvement of the major lysosome membrane‐associated LAMP proteins in MT invasion. First, human epithelial HeLa cells were incubated with MT in the presence of antibody to LAMP‐1 or LAMP‐2. Antibody to LAMP‐2, but not to LAMP‐1, significantly reduced MT invasion. Next, HeLa cells depleted in LAMP‐1 or LAMP‐2 were generated. Cells deficient in LAMP‐2, but not in LAMP‐1, were significantly more resistant to MT invasion than wild‐type controls. The possibility that LAMP‐2 might be the receptor for gp82 was examined by co‐immunoprecipitation assays. Protein A/G magnetic beads cross‐linked with antibody directed to LAMP‐1 or LAMP‐2 were incubated with HeLa cell and MT detergent extracts. Gp82 bound to LAMP‐2 but not to LAMP‐1. Binding of the recombinant gp82 protein to wild‐type and LAMP‐1‐deficient cells, which was dose dependent and saturable, had a similar profile and was much higher as compared with LAMP‐2‐depleted cells. These data indicate that MT invasion is accomplished through recognition of gp82 by its receptor LAMP‐2.     

High Abundance of Escherichia During the Establishment of Fecal Microbiota in Brazilian Children

The sequence of bacterial events that occurs during the colonization of the gastrointestinal tract may affect the future health of the host. A clear understanding of the colonization process of the human neonatal gut in developing countries is lacking because the few available studies were mostly performed using culture techniques. Using molecular approaches, this study analyzed the fecal microbiota of children of low socioeconomic status in São Paulo, Brazil, during their first year of life. We collected fecal samples of healthy children at 3, 6, and 12 months of life. Total DNA was extracted directly from feces, and the bacteria-specific primers 27F-1492R were used to construct 16S rRNA libraries. Clones were randomly selected and partially sequenced. The main phylogenetic groups identified at 3 months were Streptococcus, unidentified bacteria, and Escherichia. At 6 months, Escherichia remained predominant, while the unidentified bacterial population increased significantly. At 12 months, a more complex composition of fecal microbiota was observed, represented by unidentified bacteria and microorganisms found at low rates at earlier ages. The genus Escherichia remained the most abundant microorganism (34 % relative abundance and 75 % prevalence). Principal component analysis (PCA) revealed changes in the composition of the microbiota at 6 months and an increase of diversity at 12 months of life. Bifidobacterium was identified by quantitative PCR (qPCR) and showed a high incidence in the microbiota at 3 months. The present results corroborate the global observation of inter-individual variability with an early establishment of microbial complexity at the end of the first year of life and highlight the presence of the Escherichia as abundant in microbiota composition of this group of children.     

Cellular Senescence Induced by Prolonged Subculture Adversely Affects Glutamate Uptake in C6 Lineage

Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60–180 min of l-[³H]glu incubation, LPC6 presented an intracellular [³H] 55 % lower than EPC6. Both cultures showed a time-dependent increase of intracellular [³H] reaching maximal levels at 120 min. Cultures incubated with d-[³H]asp showed a time-dependent increase of [³H] until 180 min. Moreover, LPC6 have a d-[³H]asp-derived intracellular [³H] 30–45 % lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50 % of l-[³H]glu-derived intracellular [³H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high β-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.     

An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in Conifers

Background

Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.

Methodology/Principal Findings

We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.

Conclusion

Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.     

Carina? and Esteem?: A Systematic Review of Fully Implantable Hearing Devices

Objective

To review the outcomes of the fully implantable middle ear devices Carina and Esteem regarding the treatment of hearing loss.

Data Sources

PubMed, Embase, Scielo, and Cochrane Library databases were searched.

Study Selection

Abstracts of 77 citations were screened, and 43 articles were selected for full review. From those, 22 studies and two literature reviews in English directly demonstrating the results of Carina and Esteem were included.

Data Extraction

There were a total of 244 patients ranging from 18 to 88 years. One hundred and 10 patients were implanted with Carina and with 134 Esteem. There were registered 92 males and 67 females. Five studies provided no information about patients’ age or gender. From the data available, the follow-up ranged from 2 to 29.4 months.

Data Synthesis

The comparison of the results about word recognition is difficult as there was no standardization of measurement. The results were obtained from various sound intensities and different frequencies. The outcomes comparing to conventional HAs were conflicting. Nevertheless, all results comparing to unaided condition showed improvement and showed a subjective improvement of quality of life.

Conclusion

There are still some problems to be solved, mainly related to device functioning and price. Due to the relatively few publications available and small sample sizes, we must be careful in extrapolating these results to a broader population. Additionally, none of all these studies represented level high levels of evidence (i.e. randomized controlled trials).     

Choriodecidual Infection Downregulates Angiogenesis and Morphogenesis Pathways in Fetal Lungs from Macaca Nemestrina

Background

Intrauterine exposure to amniotic fluid (AF) cytokines is thought to predispose to bronchopulmonary dysplasia (BPD). We evaluated the effects of GBS exposure on RNA expression in fetal lung tissue to determine early molecular pathways associated with fetal lung injury that may progress to BPD.

Methods

Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received choriodecidual inoculation of either: 1) Group B Streptococcus (n = 5) or 2) saline (n = 5). Cesarean section and fetal necropsy was performed in the first week after GBS or saline inoculation regardless of labor. RNA was extracted from fetal lungs and profiled by microarray. Results were analyzed using single gene, Gene Set, and Ingenuity Pathway Analysis. Validation was by RT-PCR and immunohistochemistry.

Results

Despite uterine quiescence in most cases, fetal lung injury occurred in four GBS cases (intra-alveolar neutrophils, interstitial thickening) and one control (peri-mortem hemorrhage). Significant elevations of AF cytokines (TNF-α, IL-8, IL-1β, IL-6) were detected in GBS versus controls (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable by culture and PCR in the AF and fetal lungs. A total of 335 genes were differentially expressed greater than 1.5 fold (p<0.05) with GBS exposure associated with a striking upregulation of genes in innate and adaptive immunity and downregulation of pathways for angiogenesis, morphogenesis, and cellular growth and development.

Conclusions

A transient choriodecidual infection may induce fetal lung injury with profound alterations in the genetic program of the fetal lung before signs of preterm labor. Our results provide a window for the first time into early molecular pathways disrupting fetal lung angiogenesis and morphogenesis before preterm labor occurs, which may set the stage for BPD. A strategy to prevent BPD should target the fetus in utero to attenuate alterations in the fetal lung genetic program.     

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.