Phylogeny of the endemic Baikalian Sergentia (Chironomidae,Diptera)

Fragments of two mitochondrial genes, cytochrome b (CytB) and Cytochrome c oxidase subunit I (COI) have been used as phylogenetic markers in Sergentia (Chironomidae, Diptera). The concatenated (1241 bp) sequences from both genes were used to infer the phylogenetic relationships among seven Sergentia species. Five of the species belong to the endemic fauna of Lake Baikal. Alignments of the nucleotide sequences were used for the construction of trees using Neighbor-Joining and maximum parsimony methods. Both methods yielded similar results. Monophyly of both Sergentia and the Baikalian endemic species was well supported. The date of origin of the endemic group of Sergentia was estimated as 25.7 MYA which closely coincides with the start of geological changes in the Baikal area. A cytological tree, based on 12 chromosomal characteristics, for the same set of Sergentia species showed a great similarity to the molecular phylogeny.     

Regeneration processes of a boreal forest in Kamchatka with special reference to the contribution of sprouting to population maintenance

We studied regeneration patterns of three tree species Picea ajanensis, Betula platyphylla and Populus tremula from 1998 to 2000 in the Central Depression of the Kamchatka Peninsula. We paid special attention to the contribution of sprouting to their regeneration. P. ajanensis was the only species that regenerated by seedling. In a 40 × 40 m study plot, the density of P. ajanensis saplings < 2.0 cm in diameter at basal area (DBH) was 1132, and this was the highest among the three species studied. The number of saplings 2 cm in DBH declined sharply with size class. The spatial distribution of P. ajanensis saplings (< 2 cm in DBH) showed a significant positive correlation with that of adult trees and a negative correlation with that of gaps. These trends were not changed after re-measurement in 2000, although nearly half of the juveniles had died or been injured during the two years. These results suggest that small Picea saplings prefer habitats under the canopy of adult trees rather than in gaps for establishment. Most small individuals of B. platyphylla were produced from sprouts. The number of saplings in the smallest size class (< 2 cm in DBH) was much less than that of P. ajanensis, although the number of larger individuals did not decrease remarkably. The spatial distribution of B. platyphylla saplings showed a positive correlation with that of adult trunks and a negative correlation with that of canopy trees of P. ajanensis. These results suggest an effective contribution of sprouts to the regeneration of B. platyphylla. P. tremula was the only species that could invade big gaps and produce many root suckers efficiently. There were 181 suckers of P. tremula in the smallest size class (< 2 cm in DBH) in the study plot, although the number of saplings 2 cm in DBH declined abruptly. The spatial distribution of saplings of this species showed a slight positive correlation with that of gaps, and negative correlation with that of adult trees of B. platyphylla, P. ajanensis, and P. tremula. The root suckering strategy of P. tremula might be adaptive under severe conditions in high-latitude regions. Our data suggest, however, that it does not necessarily contribute to regeneration in mature forests. The three component species in this forest did not seem to utilize canopy gaps for regeneration; we suggest that gap dynamics do not work in this forest. The sparse canopy, which is a typical character of forests in high-latitude regions, might be a consequence of high mortalities of seedlings and root suckers inside gaps.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture-independent methods.
Materials and methods:  Samples of stomach, duodenum, ileum, cecum, colon, pancreas, liver, and bile from six dogs were evaluated for Helicobacter spp. by genus, gastric, and enterohepatic Helicobacter spp. Polymerase chain reaction, 16S rRNA gene sequence analysis, immunohistochemistry, and fluorescence in situ hybridization (FISH).
Results:  In the stomach, Helicobacter spp. DNA was detected in all six dogs, with H. bizzozeronii and H. felis identified by specific polymerase chain reaction. Helicobacter organisms were localized within the surface mucus, the lumen of gastric glands, and inside parietal cells. The small intestine harbored gastric and enterohepatic Helicobacter spp. DNA/antigen in low amounts. In the cecum and colon, Helicobacter spp. DNA, with highest similarity to H. bilis /flexispira taxon 8, H. cinaedi , and H. canis, was detected in all six dogs. Helicobacter organisms were localized at the mucosal surface and within the crypts. Gastric Helicobacter spp. DNA was detected occasionally in the large intestine, but no gastric Helicobacter spp. were present in clone libraries or detected by FISH.
Conclusions:  This study demonstrates that in addition to the stomach, the large intestine of dogs is also abundantly colonized by Helicobacter spp. Additional studies are necessary to investigate the association between enterohepatic Helicobacter spp. and presence of intestinal inflammatory or proliferative disorders in dogs.     

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

Background  

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.     

Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like cells to myocardial scar formation

Adenosine levels increase in ischemic hearts and contribute to the modulation of that pathological environment. We previously showed that A_2B adenosine receptors on mouse cardiac Sca1⁺CD31⁻ mesenchymal stromal cells upregulate secretion of paracrine factors that may contribute to the improvement in cardiac recovery seen when these cells are transplanted in infarcted hearts. In this study, we tested the hypothesis that A_2B receptor signaling regulates the transition of Sca1⁺CD31⁻ cells, which occurs after myocardial injury, into a myofibroblast phenotype that promotes myocardial repair and remodeling. In vitro, TGFβ1 induced the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and increased collagen I generation in Sca1⁺CD31⁻ cells. Stimulation of A_2B receptors attenuated TGFβ1-induced collagen I secretion but had no effect on αSMA expression. In vivo, myocardial infarction resulted in a rapid increase in the numbers of αSMA-positive cardiac stromal cells by day 5 followed by a gradual decline. Genetic deletion of A_2B receptors had no effect on the initial accumulation of αSMA-expressing stromal cells but hastened their subsequent decline; the numbers of αSMA-positive cells including Sca1⁺CD31⁻ cells remained significantly higher in wild type compared with A_2B knockout hearts. Thus, our study revealed a significant contribution of cardiac Sca1⁺CD31⁻ cells to the accumulation of αSMA-expressing cells after infarction and implicated A_2B receptor signaling in regulation of myocardial repair and remodeling by delaying deactivation of these cells. It is plausible that this phenomenon may contribute to the beneficial effects of transplantation of these cells to the injured heart.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9410-y) contains supplementary material, which is available to authorized users.     

Exploring the link between ceramide and ionizing radiation

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.     

Heavy metal distribution in superficial sedimenta ta Saco, Gulf of Cariaco, Sucre, Venezuela

The Gulf of Cariaco is a marine ecosystem with high primary productivity, which gives it an ecological and socioeconomic importance. Nevertheless, anthropogenic activities around the Gulf produce wastes that are deposited directly or by runoff into the sediments, and consequently, increases concentrations of metals in this ecosystem. The objective of this study was to determine the distribution of cadmium, copper, lead, manganese, nickel and zinc in geochemical fractions of surface sediments, using modified BCR sequential extraction procedure. The concentrations were measured using flame atomic absorption spectroscopy. In addition, the contents of soluble and exchangeable metals associated to carbonate fractions, determined by BCR, were compared with those determined by the method of Campanella. Samples were collected in 12 stations during June 2007. The applied methodologies were evaluated with a certified reference material of marine sediments (HISS-1) and the results indicated that these methods provide adequate accuracy and precision for the extraction of metals. The total metal concentrations (microg g(-1)) were, Cd: < limit of detection (LD)-5.0; Pb: 1.79-60.41; Cu: no detected (ND)-42.18; Zn: 25.13-104.57; Mn: 66.31-80.29 and Ni: 3.29-24.58. Cd, Cu, Ni and Pb at several stations, exceeded the Canadian Sediment Quality Guidelines of the Lowest Effect Levels (LEL). Cadmium was identified as being the most mobile of the elements, having the highest concentrations in soluble and exchangeable cations and carbonates. However, Pb, Cu, Mn and Zn levels were found highly associated to organic matter and sulfide fractions. The methods did not show significant statistical differences for the extraction of soluble and exchangeable cations and the metals associated to carbonate fraction. There are several significant correlations between heavy metals, which suggest their common origin.     

Review of prevalence of Simian Virus 40 (SV40) genomic infection in healthy subjects

Because of the carcinogenicity of SV40 in rodents, and its possible distribution through the polio vaccine, many studies have been conducted to determine if there is an association between SV40 genomic infection and different types of cancer; sometimes, these studies included data on the prevalence of genomic infection in healthy subjects as secondary information. We reviewed all the studies that reported the prevalence of SV40 genomic infection in healthy subjects, tested by PCR based methods. The 20 articles considered here included 1103 samples from healthy subjects, with a prevalence of infection ranging from 0 to 25.6%, with high heterogeneity, and no association with the type of sample analyzed (Mantel-Haenszel OR: 0.74; 95% CI: 0.44-1.23). The wide variation in frequency pose problems in terms of study design; in fact, the representativeness of the samples used as controls in the published studies may be very limited. Larger studies on healthy subjects, tested for SV40 genomic infection at various genomic regions, conducted in different geographic areas, are needed.