Migration and ramification of microglia in quail embryo retina organotypic cultures

Organotypic cultures of retina explants preserve the complex cellular microenvironment of the retina and have been used as a tool to assess the biological functions of some cell types. However, studies to date have shown that microglial cells activate quickly in response to the retina explantation. In this study, microglial cells migrated and ramified in quail embryo retina organotypic cultures (QEROCs) according to chronological patterns bearing a resemblance to those in the retina in situ, despite some differences in cell density and ramification degree. Retinal explants from quail embryos at 9 days of incubation (E9) proved to be the best in vitro system for reproducing a physiological-like behavior of microglial cells when cultured in Eagle's basal medium supplemented with horse serum. During the first week in vitro, microglial cells migrated tangentially in the vitreal part of QEROCs, and some began to migrate radially from 3 days in vitro (div) onward, ramifying in the inner and outer plexiform layers, thus mimicking microglia development in the retina in situ, although reaching a lower degree of ramification after 7 div. From 8 div onward, microglial cells rounded throughout the explant thickness simultaneously with the nonphysiological appearance of dead photoreceptors and round microglia in the outernuclear layer. Therefore, E9 QEROCs can be used during the first week in vitro as a model system for experimental studies of molecules putatively involved in microglial migration and ramification.     

Genetic differentiation, structure, and a transition zone among populations of the pitcher plant moth Exyra semicrocea: implications for conservation

Pitcher plant bogs, or carnivorous plant wetlands, have experienced extensive habitat loss and fragmentation throughout the southeastern United States Coastal Plain, resulting in an estimated reduction to <3% of their former range. This situation has lead to increased management attention of these habitats and their carnivorous plant species. However, conservation priorities focus primarily on the plants since little information currently exists on other community members, such as their endemic arthropod biota. Here, we investigated the population structure of one of these, the obligate pitcher plant moth Exyra semicrocea (Lepidoptera: Noctuidae), using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. Examination of 221 individuals from 11 populations across eight southeastern US states identified 51 unique haplotypes. These haplotypes belonged to one of two divergent (～1.9-3.0%) lineages separated by the Mississippi alluvial plain. Populations of the West Gulf Coastal Plain exhibited significant genetic structure, contrasting with similarly distanced populations east of the Mississippi alluvial plain. In the eastern portion of the Coastal Plain, an apparent transition zone exists between two regionally distinct population groups, with a well-established genetic discontinuity for other organisms coinciding with this zone. The structure of E. semicrocea appears to have been influenced by patchy pitcher plant bog habitats in the West Gulf Coastal Plain as well as impacts of Pleistocene interglacials on the Apalachicola-Chattahoochee-Flint River Basin. These findings, along with potential extirpation of E. semicrocea at four visited, but isolated, sites highlight the need to consider other endemic or associated community members when managing and restoring pitcher plant bog habitats.     

Dynamics of molecular evolution and phylogeography of Barley yellow dwarf virus-PAV

Barley yellow dwarf virus (BYDV) species PAV occurs frequently in irrigated wheat fields worldwide and can be efficiently transmitted by aphids. Isolates of BYDV-PAV from different countries show great divergence both in genomic sequences and pathogenicity. Despite its economical importance, the genetic structure of natural BYDV-PAV populations, as well as of the mechanisms maintaining its high diversity, remain poorly explored. In this study, we investigate the dynamics of BYDV-PAV genome evolution utilizing time-structured data sets of complete genomic sequences from 58 isolates from different hosts obtained worldwide. First, we observed that BYDV-PAV exhibits a high frequency of homologous recombination. Second, our analysis revealed that BYDV-PAV genome evolves under purifying selection and at a substitution rate similar to other RNA viruses (3.158×10(-4) nucleotide substitutions/site/year). Phylogeography analyses show that the diversification of BYDV-PAV can be explained by local geographic adaptation as well as by host-driven adaptation. These results increase our understanding of the diversity, molecular evolutionary characteristics and epidemiological properties of an economically important plant RNA virus.     

A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues

Abstract/Background

Dengue is the most important arthropod borne viral disease worldwide in terms of morbidity and mortality and is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Brazil is responsible for approximately 80% of dengue cases in the Americas, and since the introduction of dengue in 1986, a total of 5,944,270 cases have been reported including 21,596 dengue hemorrhagic fever and 874 fatal cases. DENV can infect many cell types and cause diverse clinical and pathological effects. The goal of the study was to investigate the usefulness of NS1 capture tests as an alternative tool to detect DENV in tissue specimens from previously confirmed dengue fatal cases (n = 23) that occurred in 2002 in Brazil.

Methodology/Principal Findings

A total of 74 tissue specimens were available: liver (n = 23), lung (n = 14), kidney (n = 04), brain (n = 10), heart (n = 02), skin (n = 01), spleen (n = 15), thymus (n = 03) and lymph nodes (n = 02). We evaluated three tests for NS1 antigen capture: first generation Dengue Early ELISA (PanBio Diagnostics), Platelia NS1 (BioRad Laboratories) and the rapid test NS1 Ag Strip (BioRad Laboratories). The overall dengue fatal case diagnosis based on the tissues analyzed by Dengue Early ELISA, Platelia NS1 and the NS1 Ag Strip was 34.7% (08/23), 60.8% (14/23) and 91.3% (21/23), respectively. The Dengue Early ELISA detected NS1 in 22.9% (17/74) of the specimens analyzed and the Platelia NS1 in 45.9% (34/74). The highest sensitivity (78.3%; 58/74) was achieved by the NS1 Ag Strip, and the differences in the sensitivities were statistically significant (p<0.05). The NS1 Ag Strip was the most sensitive in liver (91.3%; 21/23), lung (71.4%; 10/14), kidney (100%; 4/4), brain (80%; 8/10), spleen (66.6%, 10/15) and thymus (100%, 3/3) when compared to the other two ELISA assays.

Conclusions/Significance

This study shows the DENV NS1 capture assay as a rapid and valuable approach to postmortem dengue confirmation. With an increasing number of DHF and fatal cases, the availability of new approaches useful for cases confirmation plays an important tool for the disease surveillance.     

Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.     

Confirmation of cross-fertilization using molecular markers in ornamental passion flower hybrids

Several interspecific Passiflora hybrids are produced in the northern hemisphere for the ornamental plant market. In Brazil, production of passion flower hybrids is limited to the introgression of genes into the main cultivated species, yellow passion fruit, to be used as rootstocks. Confirmation of hybridization in the initial developmental stage is important for breeding perennial and sub-perennial plants, such as passion flowers, reducing time and costs in plant stock maintenance. In order to obtain F? hybrids with ornamental potential, four species of Passiflora (P. alata, P. gardneri, P. gibertii, and P. watsoniana) from the Active Germplasm Bank at UESC were hybridized. Flower buds, in pre-anthesis, of the genitors were previously protected, and the female buds were emasculated. To confirm hybridization, the genomic DNA of the genitor species and the supposed hybrids was extracted and RAPD primers were used to obtain molecular markers and select passion flower interspecific hybrids. Eight primers were used to confirm hybrids derived from P. gardneri with P. alata, P. watsoniana with P. alata, P. watsoniana with P. gardneri, and P. gardneri with P. gibertii; 75, 50, 45, and 46% of the informative bands, respectively, confirmed the hybrid nature of these plants. The RAPD technique was effective in the early identification of hybrids; this will be useful for development of hybrid Passiflora progeny.     

Differential mesenteric fat deposition in bovines fed on silage or concentrate is independent of glycerol membrane permeability

In the meat industry, the manipulation of fat deposition in cattle is of pivotal importance to improve production efficiency, carcass composition and ultimately meat quality. There is an increasing interest in the identification of key factors and molecular mechanisms responsible for the development of specific fat depots. This study aimed at elucidating the influence of breed and diet on adipose tissue membrane permeability and fluidity and their interplay on fat deposition in bovines. Two Portuguese autochthonous breeds, Alentejana and Barrosã, recognized as late- and early-maturing breeds, respectively, were chosen to examine the effects of breed and diet on fat deposition and on adipose membrane composition and permeability. Twenty-four male bovines from these breeds were fed on silage-based or concentrate-based diets for 11 months. Animals were slaughtered to determine their live slaughter and hot carcass weights, as well as weights of subcutaneous and visceral adipose depots. Mesenteric fat depots were excised and used to isolate adipocyte membrane vesicles where cholesterol content, fatty acid profile as well as permeability and fluidity were determined. Total accumulation of neither subcutaneous nor visceral fat was influenced by breed. In contrast, mesenteric and omental fat depots weights were higher in concentrate-fed bulls relative to silage-fed animals. Membrane fluidity and permeability to water and glycerol in mesenteric adipose tissue were found to be independent of breed and diet. Moreover, the deposition of cholesterol and unsaturated fatty acids, which may influence membrane properties, were unchanged among experimental groups. Adipose membrane lipids from the mesenteric fat depot of ruminants were rich in saturated fatty acids, and unaffected by polyunsaturated fatty acids dietary levels. Our results provide evidence against the involvement of cellular membrane permeability to glycerol on fat accumulation in mesenteric fat tissue of concentrate-fed bovines, which is consistent with the unchanged membrane lipid profile found among experimental groups.     

BMI and Metabolic Profile in Patients With Prolactinoma Before and After Treatment With Dopamine Agonists

Hyperprolactinemia might be related to weight gain, metabolic syndrome (MS), and insulin resistance (IR). Treatment with dopamine agonist (DA) has been shown to reduce body weight and improve metabolic parameters. The objectives of this study were to determine the prevalence of obesity, overweight, MS, and IR in patients with prolactinoma before and after therapy with DA and to evaluate the relation between prolactin (PRL), body weight, fat distribution, leptin levels, IR, and lipid profile before treatment. In addition, we investigated the correlation of the reduction in PRL levels with weight loss and metabolic profile improvement. Twenty‐two patients with prolactinoma completed 6 months of treatment with DA. These patients were submitted to clinical (BMI, waist circumference, blood pressure (BP)), laboratory evaluation (leptin, glucose, low‐density lipoprotein (LDL)‐cholesterol, and triglyceride (TG) levels) and abdominal computed tomography (CT) before and after treatment. The statistical analyses were done by nonparametric tests. At the beginning of the study, the prevalence of obesity, overweight, MS, and IR was 45, 27, 27, and 18%, respectively. After 6 months of treatment with DA, PRL levels normalized, but no significant difference in BMI was observed. However, there was a significant decrease on homeostasis model assessment of insulin resistance (HOMA_IR) index, glucose, LDL‐cholesterol, and TG levels. This study suggests a possible involvement of prolactinoma on the prevalence of obesity. We should consider that DA may be effective on improving metabolic parameters, and we speculate that a period longer than 6 months of treatment is necessary to conclude whether this drug can interfere in the body weight of patients with prolactinoma.