Alterations of the erythrocyte membrane by in vitro action of Trichinella spiralis muscle larvae.

The complex life cycle of Trichinella spiralis includes the migration of newborn larvae through the bloodstream to their encystment in muscle. The parasite establishes an intimate contact with the erythrocytes of the host both during the migration of the newborn larvae and when encysting, as this parasite causes intense vascularization in the muscle cell. The goal of this work was to study the effects of various concentrations of T. spiralis muscle larvae (ML) on erythrocyte membranes. The treatment was performed by incubating human erythrocytes with equal volume of different concentrations of ML for 30 minutes, with controlled agitation (37°C). The control erythrocytes (with no contact with the larvae) were incubated in the same way with an equal volume of physiological solution. To evaluate the alterations to the erythrocytes by the action of the larvae and in the respective controls, an Erythrocyte Rheometer and a Digital Image Analysis technique were used. The results indicated that when the larval concentration was higher, the aggregation and erythrocyte membrane alterations were also higher. Also, the erythrocyte deformability index and the erythrocyte elasticity increased. The values of isolated cell coefficient varied from 0.51 in the treatment with 100 larvae/ml to 0.91 in the incubation with 1000 larvae/ml. This experiment shows that T. spiralis muscle larvae affect significantly the red blood cell aggregation and the erythrocyte viscoelastic properties.

Human presence impacts fungal diversity of inflated lunar/Mars analog habitat.

BACKGROUND: An inflatable lunar/Mars analog habitat (ILMAH), simulated closed system isolated by HEPA filtration, mimics International Space Station (ISS) conditions and future human habitation on other planets except for the exchange of air between outdoor and indoor environments. The ILMAH was primarily commissioned to measure physiological, psychological, and immunological characteristics of human inhabiting in isolation, but it was also available for other studies such as examining its microbiological aspects. Characterizing and understanding possible changes and succession of fungal species is of high importance since fungi are not only hazardous to inhabitants but also deteriorate the habitats. Observing the mycobiome changes in the presence of human will enable developing appropriate countermeasures with reference to crew health in a future closed habitat. RESULTS: Succession of fungi was characterized utilizing both traditional and state-of-the-art molecular techniques during the 30-day human occupation of the ILMAH. Surface samples were collected at various time points and locations to observe both the total and viable fungal populations of common environmental and opportunistic pathogenic species. To estimate the cultivable fungal population, potato dextrose agar plate counts method was utilized. The internal transcribed spacer region-based iTag Illumina sequencing was employed to measure the community structure and fluctuation of the mycobiome over time in various locations. Treatment of samples with propidium monoazide (PMA; a DNA intercalating dye for selective detection of viable microbial populations) had a significant effect on the microbial diversity compared to non-PMA-treated samples. Statistical analysis confirmed that viable fungal community structure changed (increase in diversity and decrease in fungal burden) over the occupation time. Samples collected at day 20 showed distinct fungal profiles from samples collected at any other time point (before or after). Viable fungal families like Davidiellaceae, Teratosphaeriaceae, Pleosporales, and Pleosporaceae were shown to increase during the occupation time. CONCLUSIONS: The results of this study revealed that the overall fungal diversity in the closed habitat changed during human presence; therefore, it is crucial to properly maintain a closed habitat to preserve it from deteriorating and keep it safe for its inhabitants. Differences in community profiles were observed when statistically treated, especially of the mycobiome of samples collected at day 20. On a genus level Epiccocum, Alternaria, Pleosporales, Davidiella, and Cryptococcus showed increased abundance over the occupation time.

Isolated, well-defined organovanadium(iii) on silica: single-site catalyst for hydrogenation of alkenes and alkynes.

A well-defined, isolated, single-site organovanadium(iii) catalyst on SiO2 [(SiO2)V(Mes)(THF)] was synthesized via surface organometallic chemistry, and fully characterized using a combination of analytical and spectroscopic techniques (EA, ICP, 1H NMR, TGA-MS, EPR, XPS, DR-UV/Vis, UV-Raman, DRIFTS, XAS). The catalyst exhibits unprecedented reactivity in liquid- and gas-phase alkene/alkyne hydrogenation. Kinetic poisoning experiments revealed that 100% of the V sites are active for hydrogenation.

Modulation of IL-12 and IFNγ by probiotic supplementation promotes protection against Toxocara canis infection in mice.

In this study, supplementation with the probiotic Saccharomyces boulardii promoted a reduction in intensity of infection by Toxocara canis and modulates cytokines mRNA expression in experimentally infected mice. IL-12 gene transcription had 40-fold increase in S. boulardii supplemented uninfected mice and sevenfold increase in supplemented infected mice comparing with not supplemented group. Regarding IFNγ, similar results were observed, since probiotic supplementation induced approximately 43-fold increase, but only in uninfected mice (P < 0·05). T. canis infection upregulated IL-10 expression while S. boulardii downregulated it and no change was observed for IL-4. Thus, based in these findings; we suggest that one possible mechanism responsible for S. boulardii protection effect against T. canis infection is by the modulation of cytokines expression, especially IL-12.

First record of a spawning aggregation for the tropical eastern Pacific endemic grouper Mycteroperca olfax in the Galapagos Marine Reserve.

This study provides direct and indirect evidence of temporally and spatially consistent spawning aggregations for the grouper Mycteroperca olfax. Recently reported declines in population numbers, probably related to the direct targeting of aggregations by artisanal fishermen, highlight the urgent need for species-specific management actions in the Galapagos Marine Reserve, such as minimum and maximum landing sizes, and the importance of protecting key aggregation sites with the declaration of no-take areas and the establishment of total fishing bans during the reproductive season.

Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker.

The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P<0.01) and MIn (45.8%; P<0.05) of the oocytes (N=59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P<0.05), whereas MIn remained compromised in this group (N=67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.

NanoSMGT: transfection of exogenous DNA on sex-sorted bovine sperm using nanopolymer.

The objective was to introduce exogenous DNA into commercially sex-sorted bovine sperm using nanopolymer for transfection. In the first experiment, the optimal concentration and ratio of linear-to-circular plasmid was determined for NanoSMGT in unsorted sperm. A second experiment was conducted to transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake occurred in a dose-dependent manner (P < 0.05). The optimal amount of DNA was 10 µg/10(6) cells. The ratios of linear-to-circular plasmid do not influence the uptake by unsorted sperm cells and none of the tested treatments affected sperm motility and viability. Commercially sex-sorted bovine sperm were able to uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted sperm had decreased DNA uptake in comparison to unsorted sperm (P < 0.05). Neither sperm motility nor viability were affected by nanotransfection. In conclusion, nanopolymer efficiently introduced exogenous DNA into commercially sex-sorted bovine sperm; we inferred that these sperm could be used for production of embryos of the desired sex, a technique named NanoSMGT.

Frizzled-related protein variants are risk factors for hip osteoarthritis.

OBJECTIVE: To examine the association of the Arg200Trp and Arg324Gly variants of FRZB with the risk and phenotype of radiographic osteoarthritis (OA) of the hip and serum levels of Frizzled-related protein (FRP) in a prospective cohort of elderly Caucasian women. METHODS: Radiographic hip OA status of patients was defined by the presence of severe joint space narrowing (JSN) (feature grade>or=3), a summary grade>or=3, or definite osteophytes (grade>or=2) and JSN (grade>or=2) in the same hip. Genotypes were obtained in 569 patients with radiographic OA of the hip and in 1,317 and 4,136 controls for the Arg200Trp and Arg324Gly variants, respectively. Serum FRP levels were measured by enzyme-linked immunosorbent assay. Multivariate logistic regression was performed. RESULTS: The minor allele frequency for the Arg200Trp polymorphism was 0.12 in the control group compared with 0.14 in the group with radiographic OA of the hip (P=0.12), and the minor allele frequency for the Arg324Gly variant was 0.083 in the control group compared with 0.088 in the group with radiographic OA of the hip (P=0.63). The multilocus genotypes available in 1,886 subjects suggested that inheritance of both minor alleles was a risk factor for developing OA characterized by JSN (P<0.01). Patients with radiographic OA of the hip who were homozygous for the Arg200Trp minor allele had higher serum FRP levels than controls who were homozygous for the major allele. CONCLUSION: Our data confirm findings of another study, that a rare haplotype with both Arg200Trp and Arg324Gly FRZB variants contributes to the genetic susceptibility to hip OA among Caucasian women, and that these polymorphisms may contribute to increased serum levels of proteins as biomarkers of OA.

Leaves of Cercocarpus mixteca n. sp. (Rosaceae) from Oligocene sediments, near Tepexi de Rodríguez, Puebla.

A new species of Cercocarpus, Cercocarpus mixteca Velasco de León & Cevallos-Ferriz, is described based on leaf impressions from the Los Ahuehuetes locality, near Tepexi de Rodríguez, Puebla, Mexico. The lamina is obovate, 1.3cm in length by 0.5cm in width, has a serrate margin in its distal fourth, craspedodromous venation with a single straight mid-vein and two to four pairs of secondary ones, and areols that tend to be quadrangular in shape. A phenetic analysis of the agglomerative, non-hierarchical type, with mean linkage, is applied using 22 OTUs and 34 character states. The morphological characters observed on the leaves of the new fossil plants support the recognition of a new taxon closely related to the extant Cercocarpus paucidentatus growing naturally in northern Mexico. Its microphyll size corresponds with the temperate to xeric climate postulated for the Los Ahuehuetes locality; this further suggests that some taxa, like Cercocarpus, have a long history in low latitude North America. In this particular case, the extant Cercocarpus fothergilloides and Cercocarpus macrophyllus could, as they were able to colonise new humid and xeric areas, represent descendants of C. mixteca.

Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography.

Very high flux ion-exchange membranes were utilized for a novel purification of antisense oligonucleotides (20-mer). Strong anion-exchange membranes were produced by attaching polymeric ligands onto a microporous cellulosic matrix. The oligonucleotides purified were therapeutic single-stranded phosphorothioates deoxyribonucleotides. Although small-scale membrane devices (15 cm2) had similar resolution to traditional chromatographic columns; their throughputs were superior. Greater than a 1300-fold scale-up produced very similar purity and yields of the phosphorothionate product. Scale-up experiments were conducted with a 2 m2 surface area membrane module. These modules were easily capable of very high throughputs of 0.5 to 2 l/min. High purity and yields were achieved by both step and linear gradient elution.

Genomic variability of Haemophilus influenzae isolated from Mexican children determined by using enterobacterial repetitive intergenic consensus sequences and PCR.

Genomic fingerprints from 92 capsulated and noncapsulated strains of Haemophilus influenzae from Mexican children with different diseases and healthy carriers were generated by PCR using the enterobacterial repetitive intergenic consensus (ERIC) sequences. A cluster analysis by the unweighted pair-group method with arithmetic averages based on the overall similarity as estimated from the characteristics of the genomic fingerprints, was conducted to group the strains. A total of 69 fingerprint patterns were detected in the H. influenzae strains. Isolates from patients with different diseases were represented by a variety of patterns, which clustered into two major groups. Of the 37 strains isolated from cases of meningitis, 24 shared patterns and were clustered into five groups within a similarity level of 1.0. One fragment of 1.25 kb was common to all meningitis strains. H. influenzae strains from healthy carriers presented fingerprint patterns different from those found in strains from sick children. Isolates from healthy individuals were more variable and were distributed differently from those from patients. The results show that ERIC-PCR provides a powerful tool for the determination of the distinctive pathogenicity potentials of H. influenzae strains and encourage its use for molecular epidemiology investigations.

[A new trichromic safranin stain for the detection of Cryptosporidium parvum, Cyclospora cayetanensis, species of Microsporidia and Isospora belli in fecal material]. / Una nueva coloración safranina tricrómica para la detección de Cryptosporidium parvum, Cyclospora cayetanensis, especies de Microsporidia e Isospora belli en materia fecal.

Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis and Microsporidia are frequent pathogens in the immunodeficient host, which may cause multiple infections. The above mentioned parasites are found in feces by the application of different specific tintorial techniques. The objective of this work was the development of a stain for the simultaneous detection of these parasites, reducing costs as well as the time taken to make the diagnosis. The safranin-trichrome stain is simple, chip and its results are similar to those of specific tints. All microorganisms are easy to detect and besides being perfectly distinguishable from fungi and faecal elements.

Ribosomal RNA synthesis in Streptomyces lividans under heat shock conditions.

Clones containing rRNA genes were isolated from a gene library of Streptomyces lividans when RNA produced under heat shock conditions was used as a probe. Two of the clones carried entire rRNA operons rrnA and rrnF, respectively, the expression of both operons being under the control of four different promoters. At least two of the promoters were fully functional when the temperature increased from 30 to 45 degrees C, ensuring transcription of the rRNA genes under the heat shock. A third clone carried a partial rRNA operon in which expression was controlled by a main promoter that was functional at both 30 and 45 degrees C.

[Parasitosis in an adult population with chronic gastrointestinal disorders]. / Parasitosis en una población adulta con trastornos gastrointestinales crónicos.

We worked with 185 middle-class patients above 18 years of age, both sexes, who presented diarrhea and/or chronic gastrointestinal disorders. The faeces were collected serially in formol 10% and processed in the following way: direct microscopy, with and without wet staining, concentration by Ritchie's method, 1% safranine technique for a specific investigation of Cryptosporidium sp., and faecal sieving macroparasites. Twenty eight point six of the studied patients showed at least one enteroparasite in their faeces, 48 harboured one parasite and 5 harboured two parasites. The following parasites were found and their corresponding percentages in the entire studied population are given below: Blastocystis hominis 15.7%, Giardia lamblia 7.5%, Cryptosporidium sp. 1.6%, Entamoeba coli 3.3%, Chilomastix mesnilii 1.1%, Ancylostoma duodenale-Necator americanus 0.5%, Ascaris lumbricoides 0.5%, Enterobious vermicularis 0.5% y Endolimax nana 0.5%. The most frequently found enteroparasites in the positive patients were B. hominis and G. lamblia. Cryptosporidium sp. was diagnosed in only three patients. The source of infection could be presumed in all of them. The symptomatology coincided with that described for this coccid in the bibliography. In spite of the fact that they were HIV seronegative patients the diarrhea was not self-limiting, but the immunologic profile of their relatives remained unknown and no other cause of immunosuppression could be detected with justified chronicity. The treatment with spiramycin was effective. Giardiasis was found in 17 patients, and the source of infection could not be inferred in any of them. They all had chronic diarrhea and their most frequent symptoms were abdominal pain, metallic taste, flatulency and nausea. Most of these patients were harboured one parasite, and only 2 of them simultaneously presented another faecal parasite associated to G. lamblia. Treatment with metronidazole was successful in all of them. Twenty nine patients were found to have B. hominis. The source of infection could not be inferred, this amoeboid was present as the only parasite in 25 patients. Predominant symptoms were flatulence, abdominal distention and colis. All patients suffered from chronic diarrhea, alternating, in some cases, with constipation. Good therapeutic results were obtained with metronidazole. Considering that one third of the patients examined presented faecal parasites associated to chronic disorders, it is important to insist on the detection of parasites to chronic disorders, it is important to insist on the detection of parasites using appropriate diagnostic techniques since the application of specific therapy made their eradication possible as well as relieving the patients' symptomatology.

[Parasites found in HIV-seropositive patients]. / Hallazgos de parásitos en pacientes HIV seropositivos.

For 41 months, the presence of parasites was investigated in a seropositive HIV population with the clinical characteristics of stages 3 and 4 according to the OMS classification; of 212 fecal samples belonging to 135 patients which were analyzed, 53.33% presented enteroparasites. A direct parasitological exam and a Ritchie concentration were performed on the feces collected in formol 10%. Two smears were stained with Safranine 1% and two with modified Ziehl-Nielsen to identify Cryptosporidium sp. The detected frequencies were: Cryptosporidium The detected frequencies were: Cryptosporidium sp. 11.11%; I. belli 2.96%; G. lamblia 11.85%; B. hominis 26.66%; A. lumbricoides 2.96%; E. vermicularis 1.48%; H. nana 0.74%; E. coli 13.33%; E. nana 5.93%; Ch. mesnilii 2.22% and I. butschlii 0.74%. There were 46 monoparasitized patients, 19 biparasitized, 5 triparasitized and 2 tetraparasitized. Furthermore, 17 bronchoalveolar lavages (BAL) and 194 sputa were processed, collected in formol 10% and centrifuged to exhaustion; 10 smears were prepared with sediment and were stained with toluidine blue. Groccot (Gomori) coloration was used to confirm doubtful cases. In 47% of the BAL and in 22,68% of the sputa P. carinii was diagnosed. This represents 34.68%. The percentage of positive cases was: 30.88% for those patients who sent a single sputum, 36.84% for those who sent more than one and 27.27% for BAL. Finally, in 7 patients who sent BAL and sputa, there were 2 positive and 2 negative cases in both materials, while P. carinii was diagnosed in 3 patients only in their BAL.

[Pneumocystis carinii pneumonia: laboratory diagnosis]. / Neumonía por Pneumocystis carinii: diagnóstico de laboratorio.

We worked with 51 samples, 7 bronchoalveolar lavages (BAL) and 44 sputa (S) of 31 AIDS patients with clinical and radiographic symptoms compatible with Pneumocystis pneumonia. With the aim of finding a specific sensitive methodology for the diagnosis of Pneumocystis carinii, we evaluated 4 coloration techniques (silver methenamine, its modification without gold chloride, toluidine blue and Giemsa). 35% of the patients studied were positive. P. carinii were observed in 18% of the 44 sputa. We observed that the analysis of a single sputum sample (S) has a very low sensitivity and that the processing of two or more samples is necessary since only one of the 14 patients who had sent a single sample was found P. carinii positive, while in the remaining ten who had sent more than one (S) sample, the microorganism was detected in 50%. 4 of the 7 BAL were positive. 4 BAL were preceded by the analysis of an (S) sample: in two cases the results were negative while BAL allowed us to make the diagnosis, thus demonstrating its greater efficacy. To enhance sensitivity each sample was centrifuged until exhaustion and 10 slides were prepared for coloration with the final sediment. The four techniques employed were specific and all the Pneumocystis pneumonia patients responded to the treatment. Silver methenamine, its modification without gold chloride, and toluidine blue were very sensitive, in contrast to of Giemsa. The stain to be chosen is either silver methenamine, or its modification, because both achieve the best contrast, allowing optimum P. carinii identification. We suggest the implementation of some of these techniques in laboratory routine.