All-trans-retinoid acid induces the differentiation of encapsulated mouse embryonic stem cells into GABAergic neurons

Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that (1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and (2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (～87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders.     

Systematic analysis of the regulation of type three secreted effectors in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

Background  

The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Higher-primate phylogeny--why can't we decide?

At present, no definitive agreement on either the correct branching order or differential rates of evolution among the higher primates exists, despite the accumulated integration of decades of morphological, immunological, protein and nucleic acid sequence data, and numerous reasonable theoretical models for the analysis, interpretation, and understanding of those data. Of the three distinct unrooted phylogenetic trees, that joining human with chimpanzee and the gorilla with the orangutan is currently favored, but the two alternatives that group humans with either gorillas or the orangutan rather than with chimpanzees also have support. This paper is a synthetic and critical review of the methodological literature and isolates some 20 specific reasons why uncertainty in the evolutionary understanding of our closest living relatives persists. Many of the difficulties are eliminated or ameliorated by Lake's new methods of phylogenetic invariants and operator metrics. In the companion paper these new methods are used to analyze both the nuclear and mitochondrial DNA of the higher primates.      

Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

The distribution and quantitation of 125I-alpha-bungarotoxin (alpha-BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetanic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, alpha-BTX was bound to approximately 90% of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of alpha-BTX relative to this membrane was determined to be 46,000 +/- 27% sites/mum2.     

Detection and estimation of aflatoxins using both chemical and biological techniques

Production of aflatoxins on both natural (rice and corn) and semisynthetic (YES) media was conducted using an identified toxin-producing strain ofAspergillus flavus. TheA flavus strain was able to produce 4 types of aflatoxins, namely B₁, B₂, G₁, and G₂ on rice, corn, and YES media. Quantitative data showed that the concentrations of aflatoxins B₁ and G₁ produced were 52, 40.3, and 39.6; and 64.7, 45.0, and 58.0jug for 50g of rice, corn, and YES media, respectively. In comparison, the yielded amounts of aflatoxins B₂ and G₂ were much lower: 11.5, 17.9, and 17.5; and 28.S, 40.3, and 39.5 μg for 50 g of rice, corn, and YES media, respectively. A bioassay was conducted using the following 5 standard bacterial strains:Bacillus megaterium. Bacillus subtilis, Streptococcus faecal is, Staphylococcus epidermidis, andParacoccus denitrificans as well as a field strain of Candida albicans. All strains exceptP denitrificans showed varied degrees of inhibition when applied with crude aflatoxins at 5 to 40μg/mL. The minimum concentration of crude aflatoxins needed to inhibitP denitrificans was 10μg/mL. Moreover,Candida albicans was not inhibited at any concentration of aflatoxins applied in this work. Both undiluted and diluted (1/10, 1/100, and 1/1000) bacterial broth cultures showed a direct relationship between the diameter of inhibition zones and the concentrations of crude aflatoxins. Mean diameters of (7.0–20.5), (5–14), (4.5–13.0), (3.0–12.0), and (1.5–11.0) mm were observed when various concentrations of aflatoxins were applied usingB megaterium, S epidermidis, S faecal is, B subtilis, andP denitrificans, respectively. Field trials were applied to testify the validity of our data. A 1/100 dilution was prepared from each strain of 4 different species to estimate aflatoxins in samples of contaminated corn. Both chemical and biological assays were carried out at the same time. Data revealed that the most sensitive organism inhibited by as low as 7.5μg aflatoxins/mL wasB megaterium giving an inhibition zone of 10.5 mm, followed byS epidermidis with an inhibition zone of 7.5mm. In relation, the other 2 organisms were less sensitive to crude aflatoxins. Similarly, the biological assay was applied to detect aflatoxins in some samples of wheat, corn, peanut, rice, and poultry rations. Of the 14 wheat and 10 corn samples, only 4 wheat and 2 corn samples were found to be positive. The same results were obtained using TLC analysis.     

T-cell activation without proliferation in juvenile idiopathic arthritis

A study was done to determine if the differentiation and activation phenotype of T cells in synovial fluid (SF) from patients with juvenile idiopathic arthritis (JIA) is associated with T-cell proliferation in situ. Mononuclear cells were isolated from 44 paired samples of peripheral blood and SF. Differentiation and activation markers were determined on CD4 and CD8 T cells by flow cytometry. Cell-cycle analysis was performed by propidium iodide staining, and surface-marker expression was also assessed after culture of the T cells under conditions similar to those found in the synovial compartment. The majority of the T cells in the SF were CD45RO⁺CD45RB^dull. There was greater expression of the activation markers CD69, HLA-DR, CD25 and CD71 on T cells from SF than on those from peripheral blood. Actively dividing cells accounted for less than 1% of the total T-cell population in SF. The presence or absence of IL-16 in T-cell cultures with SF or in a hypoxic environment did not affect the expression of markers of T-cell activation. T cells from the SF of patients with JIA were highly differentiated and expressed early and late markers of activation with little evidence of in situ proliferation. This observation refines and extends previous reports of the SF T-cell phenotype in JIA and may have important implications for our understanding of chronic inflammation.     

Anatomical and chemical characteristics of the seed coat of <i>Medicago sativa</i> L. (alfalfa) cv. Baralfa 85 seeds and their association with seed dormancy

Seeds of alfalfa (Medicago sativa L.) can exhibit seedcoat imposed dormancy, which produces hard seeds within a seed lot. These seeds do not germinate because they do not imbibe water due to a barrier to water entry in the seed coat. The aim of this work was to analyze the anatomical and chemical characteristics of the testa of alfalfa seeds with respect to water permeability levels. The anatomy of seeds of the cv. Baralfa 85 was studied and structural substances, polyphenols, tannins and cutin present in the testa of seeds of different water permeability levels were determined. The anatomical characteristics of the seed coat and the proportions of components were found to determine the permeability level of the seed coat, an aspect that is associated with the physical seed dormancy level. Anatomically, increased thickness of the testa was associated with a lower permeability level. The difference may be attributed to the variation in cuticle thickness, length of macrosclereids and thickness of the cell wall, and presence and development of osteosclereids. From the physiological and chemical points of view, the mechanism of physical dormancy of the testa is explained by a greater amount of components that repel water and cement the cell wall, such as polyphenols, lignins, condensed tannins, pectic substances, and a lower proportion of cellulose and hemicellulose.     

Current medical treatment of estrogen receptor-positive breast cancer

Approximately 80% of breast cancers(BC) are estrogen receptor(ER)-positive and thus endocrine therapy(ET) should be considered complementary to surgery in the majority of patients. The advantages of oophorectomy, adrenalectomy and hypophysectomy in women with advanced BC have been demonstrated many years ago, and currently ET consist of(1) ovarian function suppression(OFS), usually obtained using gonadotropinreleasing hormone agonists(Gn RHa);(2) selective estrogen receptor modulators or down-regulators(SERMs or SERDs); and(3) aromatase inhibitors(AIs), or a combination of two or more drugs. For patients aged less than 50 years and ER+ BC, there is no conclusive evidence that the combination of OFS and SERMs(i.e., tamoxifen) or chemotherapy is superior to OFS alone. Tamoxifen users exhibit a reduced risk of BC, both invasive and in situ, especially during the first 5 years of therapy, and extending the treatment to 10 years further reduced the risk of recurrences. SERDs(i.e., fulvestrant) are especially useful in the neoadjuvant treatment of advanced BC, alone or in combination with either cytotoxic agents or AIs. There are two types of AIs: type Ⅰ are permanent steroidal inhibitors of aromatase, while type Ⅱ are reversible nonsteroidal inhibitors. Several studies demonstrated the superiority of the third-generation AIs(i.e., anastrozole and letrozole) compared with tamoxifen, and adjuvant therapy with AIs reduces the recurrence risk especially in patients with advanced BC. Unfortunately, some cancers are or became ET-resistant, and thus other drugs have been suggested in combination with SERMs or AIs, including cyclin-dependent kinase 4/6 inhibitors(palbociclib) and mammalian target of rapamycin(m TOR) inhibitors, such as everolimus. Further studies are required to confirm their real usefulness.