Application of Quantitative Real-Time Polymerase Chain Reaction for Noninvasive Genetic Monitoring

ABSTRACT Noninvasive genetic monitoring of animal populations has become a widely used method in animal conservation and wildlife management due to its known advantages in sample availability of endangered or elusive species. A variety of methods have been suggested to overcome the difficulties of collecting reliable genetic data despite poor DNA quality and quantity of samples. We used quantitative real-time polymerase chain reaction (qPCR) to quantify DNA contents and preselect extracts suitable for microsatellite genotyping of noninvasive samples from 2 carnivore species, wolf (Canis lupus) and Eurasian otter (Lutra lutra). We tested 2 concentration thresholds for DNA extracts containing either 5 pg/μL or 25 pg/μL at minimum and evaluated the effect of excluding samples from genotyping falling below either of these DNA concentrations. Depending on species and threshold concentration applied, we reduced the genotyping effort by 21% to 47% and genotyping errors by 7% to 45%, yet we could still detect 82% to 99% of available genotypes. Thus, qPCR may potentially reduce genotyping effort and enhance data reliability in noninvasive genetic studies. Genetic laboratories working on noninvasive population genetic studies could transfer this approach to other species, streamline genetic analyses and, thus, more efficiently provide wildlife managers with reliable genetic data of wild populations.     

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4 ^−/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4 ^−/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.     

Bullatantriol,a sesquiterpene from annona bullata

Bullatantriol has been isolated from Annona bullata. Its constitution and relative configuration have been established by X-ray analysis. Its absolute configuration has been assigned by chiroptical investigation of the corresponding 7-ketone.     

TNT transformation products are affected by the growth conditions of Raoultella terrigena

High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [¹⁴C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.     

Chemotypes in Achillea collina based on sesquiterpene lactone profile

The lactone profile of six origins of Achillea collina growing in Bulgaria was studied and significant variability was observed. The reasons for the differences in the lactone composition are discussed. Twenty-five components in total were isolated and identified, while the presence of ten lactones was proved by intensive TLC analysis in comparison with reference compounds. The structures of the components 17, 20, 25-30 were established by spectroscopic methods. The structure of 7, a cyclization product of 6, was also discussed. The anti-inflammatory activity of some extracts, fractions and individual compounds was tested in vitro by determining the inhibitory effects on induced human neutrophils.     

Phylogeography of halophytes from European coastal and inland habitats

The repeated advance and retreat of glaciers during the Pleistocene ice ages have played a major role in shaping the present patterns of genetic variation within and among plant and animal populations of the temperate zone. In Europe, the geographic ranges of many species were confined to a few, mostly southern refugia during periods of full glaciation. Distribution ranges then reexpanded, and uninhabited northern areas were recolonized during the interglacials. These contraction–expansion cycles were repeated at least four times. Paleontological and molecular phylogeographic studies during the last decade have greatly increased our knowledge of refugial areas and postglacial recolonization patterns of European trees, shrubs and Alpine plants since the last glacial maximum about 20,000 years ago. Much less is known yet about non-Alpine herbaceous plants. In the present review, we summarize recent phylogeographic work on halophytic (salt-adapted) plants from coastal and inland habitats in Europe. Major refugial areas for these plants have been identified along the Mediterranean coasts, but some species could also have survived in saline inland localities. In general, recolonization of N and NW Europe occurred in a stepwise fashion along the Atlantic coastline. For a number of species, molecular studies revealed concordant genetic discontinuities on the background of an essentially continuous geographic distribution. Such congruency could be explained by the preferential seed dispersal through sea currents. However, phylogeographic patterns of halophytes also proved to be influenced by other factors like sea-level fluctuations during the Pleistocene, secondary contact between divergent lineages, long-distance dispersal, clonal growth, and special habitat and temperature requirements.     

Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.