Development of a multiplex PCR assay for fine-scale population genetic analysis of the Komodo monitor Varanus komodoensis based on 18 polymorphic microsatellite loci

Multiplex PCR assays for the coamplification of microsatellite loci allow rapid and cost-effective genetic analyses and the production of efficient screening protocols for international breeding programs. We constructed a partial genomic library enriched for di-nucleotide repeats and characterized 14 new microsatellite loci for the Komodo monitor (or Komodo dragon, Varanus komodoensis). Using these novel microsatellites and four previously described loci, we developed multiplex PCR assays that may be loaded on a genetic analyser in three separate panels. We tested the novel set of microsatellites for polymorphism using 69 individuals from three island populations and evaluated the resolving power of the entire panel of 18 loci by conducting (i) a preliminary assignment test to determine population(s) of origin and (ii) a parentage analysis for 43 captive Komodo monitors. This panel of polymorphic loci proved useful for both purposes and thus can be exploited for fine-scale population genetic analyses and as part of international captive breeding programs directed at maintaining genetically viable ex situ populations and reintroductions.     

Control of myeloid-specific integrin alpha Mbeta 2 (CD11b/CD18) expression by cytokines is regulated by Stat3-dependent activation of PU.1

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in regulating multiple aspects of hematopoiesis. To elucidate the role of G-CSF in controlling hematopoietic cell migration capabilities, we studied inducible expression of the myeloid-specific marker, integrin alpha(M)beta(2) (CD11b/CD18, Mac-1), in the myeloid cell line, 32D. We found that G-CSF stimulates the synthesis and cell surface expression of alpha(M) and beta(2) integrin subunits. Induction of both alpha(M) and beta(2) is dependent on Stat3, a major G-CSF-responsive signaling protein. However, the kinetics of expression suggested the involvement of an intermediate protein regulated by Stat3. Our results demonstrate that Stat3 signaling stimulates the expression of PU.1, a critical regulator of myelopoiesis. Furthermore, we show that PU.1 is an essential intermediate for the inducible expression of alpha(M)beta(2) integrin. Thus, Stat3 promotes alpha(M)beta(2) integrin expression through its activation of PU.1. These findings indicate that G-CSF-dependent Stat3 signals stimulate the changes in cell adhesion and migration capabilities that occur during myeloid cell development. These data also demonstrate a link between Stat3 and PU.1, suggesting that Stat3 may play an instructive role in hematopoiesis.     

Molecular identification of small cetacean samples from Peruvian fish markets

In the last 60 years, incidental entanglement in fishing gears (so called by-catch) became the main cause of mortality worldwide for small cetaceans and is pushing several populations and species to the verge of extinction. Thus, monitoring and quantifying by-catches is an important step towards proper and sustainable management of cetacean populations. Continuous studies indicated that by-catches and directed takes of small cetaceans in Peru greatly increased since 1985. Legal measures banning cetacean takes, enforced in 1994 and 1996, ironically made monitoring highly problematic as fishers continue catching these animals but utilize or dispose of carcasses clandestinely. Hence, in locations where cetaceans are landed covertly or already butchered, molecular genetic methods can provide the only means of identification of the species, sex, and sometimes the population of each sample. Here, we generate and analyse a fragment of the mitochondrial DNA cytochrome b gene and 5 nuclear microsatellite markers from 182 meat and skin samples of unidentified small cetaceans collected at three Peruvian markets between July 2006 and April 2007. Our results, compared to past surveys, indicate that Lagenorhynchus obscurus, Phocoena spinipinnis, Tursiops truncatus, Delphinus capensis, and D. delphis continue to be caught and marketed, but that the relative incidence of P. spinipinnis is highly reduced, possibly because of population depletion. The small number of possible sampling duplicates demonstrates that a high monitoring frequency is required for a thorough evaluation of incidental catches in the area. A wide public debate on by-catch mitigation measures is greatly warranted in Peru.     

Nonlinear analysis of biomagnetic signals recorded from uterine myomas

Objective

To determine if there is any non-linearity in the biomagnetic recordings of uterine myomas and to find any differences that may be present in the mechanisms underlying their signal dynamics.

Methods

Twenty-four women were included in the study. Sixteen of them were characterised with large myomas and 8 with small ones. Uterine artery waveform measurements were evaluated by use of Pulsatility Index (PI) (normal value PI<1.45).

Results

Applying nonlinear analysis to the biomagnetic signals of the uterine myomas, we observed a clear saturation value for the group of large ones (mean = 11.35 ± 1.49) and no saturation for the small ones.

Conclusion

The comparison of the saturation values in the biomagnetic recordings of large and small myomas may be a valuable tool in the evaluation of functional changes in their dynamic behavior.

     

DNA Methylation Is Dispensable for Suppression of the Agouti viable yellow Controlling Element in Murine Embryonic Stem Cells

The agouti viable (A^vy) locus is considered a model to understand how retroelements function as controlling elements in mammals. Epigenetic factors, principally CpG methylation, are widely held to play a dominant regulatory role in controlling the locus'' activity. The purpose of this study was to examine its behavior in ES cells and determine if this locus could be exploited for use in screen-based investigations. We have derived multiple A^vy ES cell lines from the C57BL/6 strain and generated a cell line carrying a GFP-reporter gene (A^vy/A^GFP). Use of the DNA demethylating drug 5-azacitidine on various ES cell lines does not induce either agouti or GFP expression. Methylation analysis reveals that although most lines display normal methylation at IAP elements in general, the A^vy IAP element is essentially unmethylated. In addition, we find that different repeat compartments are epigenetically unstable in a number of derived cell lines.     

The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease

Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C⁶⁰⁹T variant genotypes was observed among MS patients compared to controls (P = 0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P = 0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P = 0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C⁶⁰⁹T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.     

Isolation of rat trachea interstitial fluid and demonstration of local cytokine production in lipopolysaccharide-induced systemic inflammation.

Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate this fluid in rats by exposing excised trachea to G forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer 51Cr-labeled EDTA in fluid isolated at 239 g and plasma averaged 0.94 +/- 0.03 (n = 14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83% of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n = 5) was 18.8 +/- 0.6 mmHg with a corresponding pressure in plasma of 22 +/- 1.5 mmHg, whereas after overhydration (n = 5) these pressures fell to 9.8 +/- 0.4 and 11.9 +/- 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial fluid, and bronchoalveolar lavage fluid in LPS-induced inflammation. In control animals, low levels of IL-1 beta, IL-6, and TNF-alpha in serum, trachea interstitial fluid, and bronchoalveolar lavage fluid were detected. LPS resulted in a significantly higher concentration in IL-1 beta and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding, e.g., inflammatory-induced airway obstruction.