Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm

The mammalian blastocyst must hatch from its extracellular coat, or zona pellucida, to implant in the uterus and continue development normally. Results of experiments described here strongly suggest that a proteinase (74K Mr), called "strypsin," is directly involved in hatching of isolated mouse blastocysts in vitro. Strypsin is a trypsin-like proteinase, based on its substrate specificity and sensitivity to inhibitors, that is present in mouse blastocysts and exhibits certain properties characteristic of membrane-associated enzymes. Histochemical and autoradiographic evidence suggests that, prior to hatching of blastocysts, strypsin is found with cells of mural trophectoderm; not with polar trophectoderm or inner cell mass. Following hatching, strypsin is also found associated with empty zonae pellucidae, specifically at the opening through which the embryo emerged. These and other observations suggest that hatching of mouse blastocysts in vitro is initiated by limited proteolysis of the region of zona pellucida overlying mural trophectoderm.     

Laser Doppler blood flow measurements of common cutaneous donor sites for reconstructive surgery

The purpose of this study was to evaluate cutaneous blood flow in regions commonly used as donor sites in reconstructive surgery in order to better establish normal flow ranges. Flow was measured with the TSI Laserflo BPM 403 in 27 healthy volunteers and compared to the flow in uncomplicated postoperative autologous tissue transplants. The forehead produced the highest flow, with an average value of 6.50 +/- 0.31 (mean +/- SE), and the dorsalis pedis had the lowest flow, with an average value of 0.60 +/- 0.04. Gender differences were noted in the latissimus dorsi, pectoralis major, and rectus abdominis areas. There were no significant differences between smokers and nonsmokers, hand dominance, musculocutaneous and fasciocutaneous tissues, or supine and sitting body positions. Flow levels in volunteers were similar to those in postoperative surviving autologous tissue transplants. The site-specific flow and flow changes over long time periods (hours) have helped clinical monitoring of 77 patients in the last 24 months. In every case identified by the flowmeter as decreased perfusion, a definite etiology for low reduction was documented. Complications occurred in 12 patients, and the rate of salvaging compromised tissue has increased from 50 percent using temperature monitoring and clinical observation to 83 percent with the computerized laser Doppler flowmeter.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

Desulfurization of dibenzothiophene using the 4S enzymatic route: Influence of operational conditions on initial reaction rates

The influence of operational conditions (pH, temperature and oxygen transfer rate) on the initial reaction rates of the four reactions involved in the 4S biodesulfurization route of dibenzothiophenes (DBT) has been studied. The bioprocess was carried out using a genetically modified organism, Pseudomonas putida CECT 5279. The rates of the four reactions were calculated from the rates of production of different compounds involved in the 4S pathway, by matrix manipulation. The initial (zero time) reaction rates showed a slight dependence on oxygen transfer rate. Temperature and pH were optimal at 30°C and 9, respectively, temperature being the most important variable. This study also identifies the last reaction as the limiting step in the pathway.     

Structural and Compositional Changes in Erythrocyte Membrane of Obese Compared to Normal-Weight Adolescents

Unhealthy dietary habits are key determinants of obesity in adolescents. Assuming that dietary fat profile influences membrane lipid composition, the aim of this study was to analyze structural changes in the erythrocyte membrane of obese compared to normal-weight adolescents. The study was conducted in a group of 11 obese and 11 normal-weight adolescent subjects. The lipid profile, lipid peroxidation and acetylcholinesterase enzyme (AChE) activity were analyzed by conventional methods. The structural properties of reconstituted erythrocyte membrane were characterized by X-ray diffraction. Erythrocyte membrane from obese adolescents had a lipid profile characterized by a higher cholesterol/phospholipid ratio, an increase in saturated fatty acid and a decrease in monounsaturated and n-6 polyunsaturated fatty acid concentrations. Differences in lipid content were associated with changes in the structural properties of reconstituted membranes and the oxidative damage of erythrocyte membrane. The lower oxidative level shown in the obese group (0.15 ± 0.04 vs. 0.20 ± 0.06 nmol/mg for conjugated diene concentrations and 2.43 ± 0.25 vs. 2.83 ± 0.31 nmol/mg protein for malondialdehyde levels) was related to a lower unsaturation index. These changes in membrane structural properties were accompanied by a lower AChE activity (1.64 ± 0.13 vs. 1.91 ± 0.24 nmol AChE/[min mg protein]) in the obese group. The consequences of unhealthy dietary habits in adolescents are reflected in the membrane structural properties and may influence membrane-associated protein activities and functions.     

Exome Sequencing of Index Patients with Retinal Dystrophies as a Tool for Molecular Diagnosis

Background

Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.

Methodology/Principal Findings

We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.

Conclusions/Significance

Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.     

Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies

Background

Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics.

Objective

To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias.

Design, Setting, and Participants

A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler.

Outcome Measurements and Statistical Analysis

Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer.

Results and Limitations

We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics.

Conclusion

Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.     

An engineered class I transfer RNA with a class II tertiary fold

Structure-based engineering of the tertiary fold of Escherichia coli tRNA(Gln)2 has enabled conversion of this transfer RNA to a class II structure while retaining recognition properties of a class I glutamine tRNA. The new tRNA possesses the 20-nt variable stem-loop of Thermus thermophilus tRNA(Ser). Enlargement of the D-loop appears essential to maintaining a stable tertiary structure in this species, while rearrangement of a base triple in the augmented D-stem is critical for efficient glutaminylation. These data provide new insight into structural determinants distinguishing the class I and class II tRNA folds, and demonstrate a marked sensitivity of glutaminyl-tRNA synthetase to alteration of tRNA tertiary structure.