Isolation of radial glia-like neural stem cells from fetal and adult mouse forebrain via selective adhesion to a novel adhesive peptide-conjugate

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.     

Defining natural history: assessment of the ability of college students to aid in characterizing clinical progression of Niemann-Pick disease, type C

Niemann-Pick Disease, type C (NPC) is a fatal, neurodegenerative, lysosomal storage disorder. It is a rare disease with broad phenotypic spectrum and variable age of onset. These issues make it difficult to develop a universally accepted clinical outcome measure to assess urgently needed therapies. To this end, clinical investigators have defined emerging, disease severity scales. The average time from initial symptom to diagnosis is approximately 4 years. Further, some patients may not travel to specialized clinical centers even after diagnosis. We were therefore interested in investigating whether appropriately trained, community-based assessment of patient records could assist in defining disease progression using clinical severity scores. In this study we evolved a secure, step wise process to show that pre-existing medical records may be correctly assessed by non-clinical practitioners trained to quantify disease progression. Sixty-four undergraduate students at the University of Notre Dame were expertly trained in clinical disease assessment and recognition of major and minor symptoms of NPC. Seven clinical records, randomly selected from a total of thirty seven used to establish a leading clinical severity scale, were correctly assessed to show expected characteristics of linear disease progression. Student assessment of two new records donated by NPC families to our study also revealed linear progression of disease, but both showed accelerated disease progression, relative to the current severity scale, especially at the later stages. Together, these data suggest that college students may be trained in assessment of patient records, and thus provide insight into the natural history of a disease.     

An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50 kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83–122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87–99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.     

Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.     

Reproductive strategy of Psectrogaster rhomboides Eigenmann & Eigenmann, 1889, a freshwater fish from Northeastern Brazil

Several environmental parameters related to the spawning season of the freshwater fish, Psectrogaster rhomboides were identified. The hypothesis predicts synchrony of the spawning season with the rainy period, presenting a seasonal reproductive strategy. This study investigated the total body length and body mass, sex ratio, body size at first sexual maturity, gonadosomatic index, condition factor, fecundity and reproductive period of P. rhomboides in the natural environment. Twelve monthly samplings (mean n = 20) were carried out from March 2009 to February 2010. Fish were captured using 4 cm diameter cast nets and environmental parameters such as rainfall, temperature, dissolved oxygen concentration, pH and electrical conductivity of the water were registered. The sampled population of P. rhomboides (n = 444) showed a sex ratio of 1 : 1. Females attained first sexual maturity at 15 cm total length and males at 15.3 cm total length. Spawning occurred from March to May, with high gonadosomatic index values during the rainy season. This species is a total spawner, with a mean fecundity of 6188 (±367) mature oocytes. P. rhomboides presents a seasonal reproductive strategy, providing a population increase and exploitation of the environmental resources during the rainy period. Rainfall (196.5 ± 4.9 mm) and the concentration of dissolved oxygen (7.82 ± 0.7018 mg L^?1) were influential factors during the spawning season. Gonadosomatic index and the condition factor were negatively correlated during the gonadal development cycle of this species. The monthly gonadosomatic index values show that spawning begins when the rainy season is at its maximum and ends synchronously (females r = 0.9171; males r = 0.889) with the end of the rainy season.     

Information‐gathering as a response to manipulated signals in the eastern fence lizard,Sceloporus undulatus

Colorful visual signals are used across taxa to convey information during agonistic male‐male encounters, which are important for sexual selection. Although much is known about the information content of color signals, less is known about how receivers interpret this information. Here, using territorial Sceloporus undulatus lizards in a natural setting, we examined receiver response to intruders with different color patch sizes to determine (a) if patch size conveys information assessed during male‐male interactions and (b) if/how receivers modulate their behavioral responses to different types of behavioral signals. We found that larger lizards had longer and wider patches, indicating that the size of the patches may be one of the many characteristics of these patches that is used by males to convey information. Free‐ranging subject males also produced more headbob displays in response to intruders with small patches and took marginally longer to react to intruders with large patches. However, we found no differences in the aggressiveness of the response (i.e., fullshows). This indicates that patch size conveys information that is employed during territorial disputes, but that the response is primarily in terms of timing, allowing lizards to gather more information about intruders, instead of aggressive behavior (i.e., fullshows).     

The Effect of Zn(II) Ions and Reactive Oxygen on the Uptake of Zinc and Production of Carotenoids by Selected Red Yeasts

Three strains of red yeast Rhodosporidium kratochvilovae, Rhodotorula glutinis and Sporidiobolus salmonicolor were studied for their responses to the presence metal stress, oxidative stress and a combination of these stress factors. For all yeast strains, the production of β‐carotene increased in stress conditions. The combination of H₂O₂ and Zn²⁺ significantly activated the pathways for the production of torularhodin in the strain R. glutinis (from 250 to 470 μg g^?1 DCW) as well as β‐carotene (from 360 to 1100 μg g^?1 DCW) and torulene (from 100 to 360 μg g^?1 DCW) in Sp. salmonicolor. Strains of R. glutinis and Rh. kratochvilovae bound the majority of Zn(II) ions to the fibrillar part of the cell walls, whereas the strain Sp. salmonicolor bound them to both extracellular polymers and the fibrillar part of the cell walls. A decrease in the ability of yeasts to tolerate higher concentrations of Zn(II) in the presence of free radicals (hydrogen peroxide) was also found.     

High-dose methylprednisolone influences the physiology and virulence of Candida albicans ambiguously and enhances the candidacidal activity of the polyene antibiotic amphotericin B and the superoxide-generating agent menadione

Although exposure of Candida albicans cells to high-dose (4 mM) methylprednisolone stimulated microbial growth, germination rate in serum and phospholipase release, it also promoted the recognition of C. albicans cells by polymorphonuclear leukocytes. Pretreatment of C. albicans cells with methylprednisolone did not result in any increase in the pathogenicity of the fungus in intraperitoneal and intravenous mouse assays. Therefore, the virulence of C. albicans is unlikely to increase in patients treated with comparably high-dose methylprednisolone on skin and mucosal membranes. Methylprednisolone treatments also increased the production of conjugated dienes and thiobarbituric acid-reactive substances, and the menadione sensitivity of C. albicans cells, which can be explained by a significant decrease in the specific activities of several antioxidant enzymes. The combination of methylprednisolone with oxidants, e.g. in topical applications, may be of clinical importance when the predisposition to candidiasis is high. Methylprednisolone treatments negatively affected membrane fluidity and decreased the antifungal effects of both the polyene antibiotic nystatin and the ergosterol biosynthesis inhibitor lovastatin, and also enhanced the deleterious effects of the polyene antimycotic amphotericin B on C. albicans cells. These corticosteroid-polyene drug interactions should be considered in the treatment of C. albicans infections in patients with prolonged topical application of corticosteroids.