Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.

Relationships between gender, age, family conditions, physical and mental health, and social isolation of elderly caregivers

Background: In an aging population an increasing number of elderly caregivers will be called upon to provide care over a long period, during which time they will be burdened both by caregiving and by the physiological effects of their own aging. Among them there will be more aged male caregivers, who will probably be less prepared than women to become caregivers. The aim of this study was to investigate the relationship between caregivers' gender, age, family income, living arrangements and social support as independent variables, and depressive symptoms, comorbidities, level of frailty, grip strength, walking speed and social isolation, as dependent variables. Methods: 176 elderly people (123 women) were selected from a sample of a population-based study on frailty (n = 900), who had cared for a spouse (79.3%) and/or parents (31.4%) in the past five years (mean age = 71.8 +/- 4.86 years; mean monthly family income in minimum wages = 4.64 +/- 5.14). The study used questionnaires and self-report scales, grip strength and walking speed tests. Results: 65% of participants evaluated caregiving as being very stressful. Univariate analyses of regression showed low family income as a risk factor for depression; being female and low perceived social support as a risk for comorbidities; being 80 years of age and above for low grip strength; and being male for social isolation indicated by discontinuity of activities and social roles. In multivariate analyses of regression, poverty arose as a risk factor for depression and being female for comorbidities. Conclusions: Gender roles, age, income and social support interacted with physical and emotional health, and with the continuity of social participation of elderly caregivers. Special attention must be given to male caregivers.

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODS; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 degrees C for 1 h at pH from 8 to 11.5, while maintaining its biological activity. (C) 2011 Elsevier B.V. All rights reserved.

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espirito Santo state, southeastern Brazil

Prevalence of Toxoplasma gondii infection in 510 free-range (FR) chickens (380 from 33 small farms, and 130 from a slaughter house for FR chickens) from Espirito Santo state, southeastern Brazil, was investigated. Antibodies to T. gondii were sought using commercial indirect haemagglutination (IHAT, Imuno-HAI Toxo (R), Wama Diagnostica, Sao Paulo, Brazil, cut-off 1:16) and the modified agglutination test (MAT, cut-off 1:25) tests. Attempts were made to isolate viable T. gondii from seropositive chickens by bioassay in mice. Pooled samples of brain, heart and quadriceps muscle of one thigh (total 40 g) from 64 chickens with IHAT titers of >= 1:16 were minced, digested in pepsin and bioassayed in mice. Antibodies to T. gondii were found in 40.4% (206/510) FR chickens by IHAT (titer >= 1:16) and 38.8% (198/510) by MAT (titer >= 1:25); concordance between IHAT and MAT was 81.6% (kappa index = 0.614). Viable T. gondii was isolated (designated TgCkBr234-281) from 48 of 64(75%) seropositive (IHAT titers >= 1:32) FR chickens. Most isolates of T. gondii were virulent for mice; 100% of mice inoculated with 44 of 48 isolates died of toxoplasmosis within 30 days post inoculation (p.i). An epidemiological investigation revealed that people living in rural areas have little knowledge about the parasite and about the risk of acquiring it from raw meat. Results indicated that the locally available IHAT was useful for screening of chicken sera for T. gondii antibodies. (C) 2012 Elsevier B.V. All rights reserved.

Candida albicans Isolation from Buccal Mucosa of Patients with HIV Wearing Removable Dental Prostheses

Purpose: The aim of this study was to evaluate the influence of complete or partial removable dental prostheses (RDPs) on the frequency of Candida albicans isolated from the mouth and the presence of oral candidiasis in human immunodeficiency virus positive (HIV+) patients correlated with CD4 levels. Materials and Methods: One hundred ninety-three HIV+ patients were evaluated; 68 had RDPs and 125 did not. CD4 cell count was obtained after blood sampling and performed on the day of clinical examination. The material was collected from the buccal mucosa for isolation of yeasts with a sterile swab and seeded onto Sabouraud dextrose agar with chloramphenicol. C albicans strains were identified by testing germ tubes and chlamydospore formation and biochemical (zymogram, auxanogram) characteristics. The results were subjected to the Fischer exact test and chi-square tests. Results: C albicans were isolated from 45(66.17%) patients who had RDPs and 48 (38.4%) who did not (P = .0003). The presence of oral candidiasis was observed in 14 patients (7.25%), and 10 of the 14 (71.43%) were RDP users. The absence of candidiasis occurred in 121 (67.59%) nonusers and 58 (32.40%) users of RDPs (P = .0065). The mean CD4 cell count was lower in patients with oral candidiasis regardless of the use of RDPs. Conclusion: The use of RDPs was an important factor in the isolation of C albicans among HIV+ patients, and CD4 level seems to play a role in the presence of oral candidiasis. Int J Prosthodont 2012;25:127-131.

Influence of caffeine on the survival of leaf-cutting ants Atta sexdens rubropilosa and in vitro growth of their mutualistic fungus

BACKGROUND: Leaf-cutting ants collect plant fresh material for the cultivation of their mutualistic fungus. Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) cause great economic losses through their foraging activity, mainly in agriculture. The main control method is the application of granulated toxic baits incorporated with an active ingredient (AI). The present goal is to evaluate the effect of caffeine on in vitro growth of the mutualistic fungus and on the survival of the leaf-cutting ants, aiming to verify the potential toxicity of this secondary metabolite over these organisms. RESULTS: Three distinct patterns of fungal growth correlated with caffeine concentration were observed: (1) no effect (0.01% caffeine); (2) intermediate growth reduction (0.05% caffeine); (3) drastic growth reduction (0.10 and 0.50% caffeine). The highest caffeine concentration causes fungus death in the first week. As for insect survival, caffeine does not seem to exert any effect. The treatments with diet containing caffeine showed similar values of M50, irrespective of caffeine concentration. CONCLUSION: As caffeine was shown to reduce growth of the mutualistic fungus of Atta sexdens rubropilosa, but with no conclusive effect on insect survival, a hypothetical explanation for the selection of different Coffea species by this leaf-cutting ant species might be associated with caffeine toxicity to the fungus. Copyright (C) 2011 Society of Chemical Industry

Microbial transformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus

The biotransformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus MT 5.3 yielded a rare derivative that was elucidated by spectrometric methods. The fungus led to the formation of a different product through an unusual epoxidation reaction between C4 and C5, formation of a C3,C10 ether bridge, and a methoxylation of the C1 of tagitinin C. The chemical structure of the product, namely 1 beta-methoxy-3 alpha-hydroxy-3,10 beta-4,5 alpha-diepoxy-8 beta-isobutyroyloxygermacr-11(13)-en-6 alpha,12-olide, is the same as that of a derivative that was recently isolated from the flowers of a Brazilian population of Mexican sunflower (Tithonia diversifolia), which is the source of the substrate tagitinin C. The in vitro cytotoxic activity of the substrate and the biotransformed product were evaluated in HL-60 cells using an MTT assay, and both compounds were found to be cytotoxic. We show that soil fungi may be useful in the biotransformation of sesquiterpene lactones, thereby leading to unusual changes in their chemical structures that may preserve or alter their biological activities, and may also mimic plant biosynthetic pathways for production of secondary metabolites.

Effects of exogenous calcium or oxalic acid on Pinus taeda treatment with the white-rot fungus Ceriporiopsis subvermispora

Pinus taeda wood chips were treated with the biopulping fungus Ceriporiopsis subvermispora in calcium-or oxalic acid-amended cultures. The secretion of hydrolytic and oxidative enzymes was inhibited only in the cultures having the highest concentration of calcium (1400 mg kg(-1) wood). Calcium decreased the availability of free oxalic acid, inhibited fungal growth, and reduced lignin mineralization and transformations. Oxalic acid amendment in the cultures was found not to affect the lignin mineralization and transformations; however, it did inhibit the depolymerization reactions detectable in the residual lignin that was retained in the biotreated wood. C. subvermispora presented catabolic activity for oxalic acid in the cultures amended with 1660 mg acid kg(-1) wood, whereas oxalic acid was synthesized when it was amended at low amounts or initially absent in the cultures. These data suggest one ideal ratio of oxalic acid in C. subvermispora cultures and indicate that its exogenous addition does not necessarily accompany the further degradation of lignin. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Enterobacteria Isolation in Ostrich Eggs (Struthio camelus)

This study was conducted to determine the presence of enterobacteria in the eggs of ostriches reared on a farm with a history of reproductive failure. Ninety samples from twenty eggs were submitted to bacteriological tests. The results showed Enterobacteria growth in 100% of the eggs. The microorganisms isolated were Hafnia alvei in 50% (10/20), Serratia spp. in 20% (4/20), Escherichia coli in 15% (3/20), and Citrobacter freundii in 15% (3/20). All eggs presented poor eggshell quality, which favored enterobacteria contamination. Hafnia alvei was present only in the internal egg structures (albumen and yolk sac), suggesting the possibility of vertical infection.

Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

Isolation and characterization of the flour and starch of plantain bananas (Musa paradisiaca)

Plantain bananas of the variety Terra (Musa paradisiaca) may have industrial value due to their high starch content. In this research, the flour and starch of such unripe fruit were isolated and their chemical, physicochemical, and structural characteristics were determined. Banana flour and starch had a dry basis yield of 50.6 and 28.5%, and an average granule size of 31.7 and 47.3?mu m, respectively. Both raw materials revealed a C-type pattern and high gelatinization temperatures. The peak viscosity was greater for flour (378.0 RVU) than for starch (252.6 RVU), although the final viscosity was lower. At temperatures above 65 degrees C, the swelling power of banana flour was lower than that of starch, while the solubility of flour was greater than that of starch at all temperatures. Furthermore, the presence of other components in banana flour influenced its physicochemical properties. In general, the flour and starch processed from unripe bananas have numerous possible uses as ingredients in food systems and for other industrial purposes.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Source of slow lithium atoms from Ne or H-2 matrix isolation sublimation

We have studied, via laser absorption spectroscopy, the velocity distribution of Li-7 atoms released from cryogenic matrices of solid neon or molecular hydrogen. The Li atoms are implanted into the Ne or H-2 matrices - grown onto a sapphire substrate - by laser ablation of a solid Li or LiH precursor. A heat pulse is then applied to the sapphire substrate sublimating the matrix together with the isolated atoms. With a NiCr film resistor deposited directly onto the sapphire substrate we are able to transfer high instantaneous power to the matrix, thus reaching a fast sublimation regime. In this regime the Li atoms can get entrained in the released matrix gas, and we were also able to achieve matrix sublimation times down to 10 mu s for both H-2 or Ne matrix, enabling us to proceed with the trapping of the species of our interest such as atomic hydrogen, lithium, and molecules. The sublimation of the H-2 matrix, with its large center-of-mass velocity, provides evidence for a new regime of one-dimensional thermalization. The laser ablated Li seems to penetrate the H-2 matrix deeper than it does in Ne. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4704125]

Comparative EST analysis provides insights into the basal aquatic fungus Blastocladiella emersonii

Abstract Background Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. In this sense, some ancestral characteristics of fungi and animals or fungi and plants could have been retained in this aquatic fungus and lost in members of late-diverging fungal species. To identify in B. emersonii sequences associated with these ancestral characteristics two approaches were followed: (1) a large-scale comparative analysis between putative unigene sequences (uniseqs) from B. emersonii and three databases constructed ad hoc with fungal proteins, animal proteins and plant unigenes deposited in Genbank, and (2) a pairwise comparison between B. emersonii full-length cDNA sequences and their putative orthologues in the ascomycete Neurospora crassa and the basidiomycete Ustilago maydis. Results Comparative analyses of B. emersonii uniseqs with fungi, animal and plant databases through the two approaches mentioned above produced 166 B. emersonii sequences, which were identified as putatively absent from other fungi or not previously described. Through these approaches we found: (1) possible orthologues of genes previously identified as specific to animals and/or plants, and (2) genes conserved in fungi, but with a large difference in divergence rate in B. emersonii. Among these sequences, we observed cDNAs encoding enzymes from coenzyme B12-dependent propionyl-CoA pathway, a metabolic route not previously described in fungi, and validated their expression in Northern blots. Conclusion Using two different approaches involving comparative sequence analyses, we could identify sequences from the early-diverging fungus B. emersonii previously considered specific to animals or plants, and highly divergent sequences from the same fungus relative to other fungi.

Evaluating methods for the isolation of marine-derived fungal strains and production of bioactive secondary metabolites

In the present investigation we evaluate methods for the isolation and growth of marine-derived fungal strains in artificial media for the production of secondary metabolites. Inoculation of marine macroorganisms fragments in Petri dishes proved to be the most convenient procedure for the isolation of the largest number of strains. Among the growth media used, 3% malt extract showed the best result for strains isolation and growth, and yielded the largest number of strains from marine macroorganisms. The percentage of strains isolated using each of the growth media which yielded cytotoxic and/or antibiotic extracts was in the range of 23-35%, regardless of the growth media used. Further investigation of extracts obtained from different marine-derived fungal strains yielded several bioactive secondary metabolites, among which (E)-4-methoxy-5-(3-methoxybut-1-enyl)-6-methyl-2H-pyran-2-one is a new metabolite isolated from the Penicillium paxilli strain Ma(G)K.