Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.     

Metformin protects against doxorubicin-induced cardiotoxicity: involvement of the adiponectin cardiac system

Doxorubicin has cardiotoxic effects that limit its clinical benefit in cancer patients. Metformin exerts cardioprotective actions via AMP-activated protein kinase (AMPK) and increases the expression of adiponectin and its receptors (adipoR1 and adipoR2) in skeletal muscle and adipose tissue, but its effect on cardiac tissue is still unknown. This work aimed to study whether metformin exerts any protective action against the cardiotoxicity of doxorubicin and whether the cardiac system of adiponectin is involved in any such action. The addition of doxorubicin (5μM) to adult mouse cardiomyocytes (HL-1 cell line) induced apoptosis, which was characterized by a loss of cell viability, activation of caspases, and fragmentation of the genetic material. Doxorubicin treatment also caused a decrease in the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase. Pretreatment with metformin (4mM, 24h) provided protection against doxorubicin-induced damage. This pretreatment significantly increased cell viability, attenuated the activation of caspases and the fragmentation of genetic material, and restored the antioxidant activity. In addition, metformin up-regulated the expression of adiponectin and its receptors, adipoR1 and adipoR2, in cardiomyocytes. In contrast, silencing either adipoR1 or adipoR2 with siRNA inhibited the AMPK activation and the protective effects of metformin. Taken together, these results demonstrate that metformin protects cardiomyocytes from doxorubicin-induced damage and that the cardiac adiponectin system plays an important role in this protective action.     

Biofiltration of reduced sulphur compounds and community analysis of sulphur-oxidizing bacteria

The present work aims to use a two-stage biotrickling filters for simultaneous treatment of hydrogen sulphide (H₂S), methyl mercaptan (MM), dimethyl sulphide (DMS) and dimethyl disulphide (DMDS). The first biofilter was inoculated with Acidithiobacillus thiooxidans (BAT) and the second one with Thiobacillus thioparus (BTT). For separate feeds of reduced sulphur compounds (RSC), the elimination capacity (EC) order was DMDS > DMS > MM. The EC values were 9.8 g_MM-S/m³/h (BTT; 78% removal efficiency (RE); empty bed residence time (EBRT) 58 s), 36 g_DMDS-S/m³/h (BTT; 94.4% RE; EBRT 76 s) and 57.5 g_H2S-S/m³/h (BAT; 92% RE; EBRT 59 s). For the simultaneous removal of RSC in BTT, an increase in the H₂S concentration from 23 to 293 ppmv (EBRT of 59 s) inhibited the RE of DMS (97-84% RE), DMDS (86-76% RE) and MM (83-67% RE). In the two-stage biofiltration, the RE did not decrease on increasing the H₂S concentration from 75 to 432 ppmv.     

Viral genome segmentation can result from a trade-off between genetic content and particle stability

The evolutionary benefit of viral genome segmentation is a classical, yet unsolved question in evolutionary biology and RNA genetics. Theoretical studies anticipated that replication of shorter RNA segments could provide a replicative advantage over standard size genomes. However, this question has remained elusive to experimentalists because of the lack of a proper viral model system. Here we present a study with a stable segmented bipartite RNA virus and its ancestor non-segmented counterpart, in an identical genomic nucleotide sequence context. Results of RNA replication, protein expression, competition experiments, and inactivation of infectious particles point to a non-replicative trait, the particle stability, as the main driver of fitness gain of segmented genomes. Accordingly, measurements of the volume occupation of the genome inside viral capsids indicate that packaging shorter genomes involves a relaxation of the packaging density that is energetically favourable. The empirical observations are used to design a computational model that predicts the existence of a critical multiplicity of infection for domination of segmented over standard types. Our experiments suggest that viral segmented genomes may have arisen as a molecular solution for the trade-off between genome length and particle stability. Genome segmentation allows maximizing the genetic content without the detrimental effect in stability derived from incresing genome length.     

Genetic and epigenetic relationship in rye, Secale cereale L., somaclonal variation within somatic embryo-derived plants

In vitro regenerated plants of rye, Secale cereale L., Ailés and Merced cultivars, were studied to verify if genetic and/or epigenetic changes were promoted by in vitro conditions. Inter-simple sequence repeat (ISSR) fingerprints on HpaII/MspI-digested and uncut DNA were generated. DNA digested with methylation-sensitive isoschizomers revealed epigenetic modifications, while modification of ISSR patterns obtained with undigested DNA indicated genetic changes. With this technique, it was possible to study both genetic and/or epigenetic changes within the same DNA sequences. The frequency of plants with at least one variation was high: 73% and 30% of rye plants showed at least one genetic change, and 50% and 73% carried at least one methylation change, in the Ailés and Merced cultivars, respectively. Further analyses revealed that a considerable number of variable markers showed both types of modifications, indicative of both genetic and epigenetic changes. Moreover, genetic variation was related to the presence of the CCGG target in the analyzed bands. These results indicate the possible existence of a common mechanism connecting both types of variation.     

Thermodynamic concepts in the study of microbial populations: age structure in Plasmodium falciparum infected red blood cells

Variability is a hallmark of microbial systems. On the one hand, microbes are subject to environmental heterogeneity and undergo changeable conditions in their immediate surroundings. On the other hand, microbial populations exhibit high cellular diversity. The relation between microbial diversity and variability of population dynamics is difficult to assess. This connection can be quantitatively studied from a perspective that combines in silico models and thermodynamic methods and interpretations. The infection process of Plasmodium falciparum parasitizing human red blood cells under laboratory cultivation conditions is used to illustrate the potential of Individual-based models in the context of predictive microbiology and parasitology. Experimental data from several in vitro cultures are compared to the outcome of an individual-based model and analysed from a thermodynamic perspective. This approach allows distinguishing between intrinsic and external constraints that give rise to the diversity in the infection forms, and it provides a criterion to quantitatively define transient and stationary regimes in the culture. Increasing the ability of models to discriminate between different states of microbial populations enhances their predictive capability which finally leads to a better the control over culture systems. The strategy here presented is of general application and it can substantially improve modelling of other types of microbial communities.     

A rare fraction of human hematopoietic stem cells with large telomeres

The lack of specific markers for stem cells makes the physical identification of this compartment difficult. Hematopoietic stem cells differ in their repopulating and self-renewal potential. Our study shows that multiple classes of human hematopoietic CD34+ greatly differ in telomere length. Flow-cytometry-based fluorescent in situ hybridization and confocal microscopy of CD34+ cells has revealed remarkable telomere length heterogeneity, with a hybridization pattern consistent with different classes of human hematopoietic progenitor cells. These results also point to the existence of a significant clonal heterogeneity among primitive hematopoietic cells and provide the first evidence of a rare fraction of CD34+ cells with large telomeres in humans. Marta García-Escarp and Vanessa Martinez-Muñoz contributed equally to this work.This work was supported by a grant to J.P. from the Spanish Ministry of Science and Technology (SAF2002-02618) and by a grant to V.M.-M. from DakoCytomation.     

Carbohydrate Depletion in Roots and Leaves of Salt-Stressed Potted <Emphasis Type="Italic">Citrus clementina</Emphasis> L.

In citrus, damage produced by salinity is mostly due to toxic ion accumulation, since this salt-sensitive crop adjusts osmotically with high efficiency. In spite of this observation, the putative role of sugars as osmolites under salinity remains unknown. In this work, we have studied carbohydrate contents (total hexoses, sucrose and starch) in leaves and roots of citrus grown under increasing salinity. The experimental system was characterized through the analyses of several parameters known to be strongly affected by salinity in citrus, such as chloride accumulation, photosynthetic rate, ethylene production and leaf abscission. Three-year-old plants of the Clementina de Nules cultivar grafted on Carrizo citrange rootstock were watered with three different levels of salinity (NaCl was added to the watering solutions to achieve final concentrations of 30, 60 and 90 mM). Data indicate that salt stress caused an accumulation of chloride ions in a way proportional to the external increase in NaCl. The adverse conditions reduced CO₂ assimilation, increased ethylene production and triggered abscission of the injured leaves. Data also show that salinity induced progressive depletions of carbohydrates in leaves and roots of citrus plants. This observation clearly indicates that sugar accumulation is not a main component of the osmotic adjustment machinery in citrus.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.