A highly sensitive quantitative real-time PCR assay for determination of mutant JAK2 exon 12 allele burden

Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.     

Spermatogenesis in Atlantic cod (Gadus morhua): a novel model of cystic germ cell development

Precocious male puberty significantly compromises sustainability aspects of aquaculture in a number of finfish species. As part of a program aiming to understand and eventually control testis maturation in farmed Atlantic cod, we studied the first reproductive cycle. The gonadosomatic index shows a 41-fold increase from immature (August) to mature (March) stages, reaching almost 10% of the total body weight. The paired cod testes are composed of several lobes arranged around a central collecting duct. In each individual lobe, spermatogenesis occurs in a marked gradient of development, with undifferentiated spermatogonia in the periphery of the lobe and the most advanced germ cells in the vicinity of the collecting duct, suggesting a tight spatiotemporal organization of spermatogenesis in the testis lobes of this species. Spermatogonial proliferation starts in August and continues for about 6 mo. Meiosis and spermiogenesis are first observed in October and are completed in all cysts by February, when a 2-mo-long spawning season starts. Spermatogonia go through 11 mitotic divisions before differentiating to primary spermatocytes. Apoptosis is rare, but when observed it occurs mainly during the last spermatogonial generations. Our observations suggest a model in which a maturational wave progresses through each growing lobe that is first driven by appositional growth from the lobe's periphery, reflecting spermatogonial proliferation and cyst formation which, when ceasing, is terminated by completing spermiogenesis and spermiation that progress toward the lobe's periphery.     

Acclimation of subsurface microbial communities to mercury

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.     

The structure of bovine complement component 3 reveals the basis for thioester function

The third component of complement (C3) is a 190 kDa glycoprotein essential for eliciting the complement response. The protein consists of two polypeptide chains (alpha and beta) held together with a single disulfide bridge. The beta-chain is composed of six MG domains, one of which is shared with the alpha-chain. The disulfide bridge connecting the chains is positioned in the shared MG domain. The alpha-chain consists of the anaphylatoxin domain, three MG domains, a CUB domain, an alpha(6)/alpha(6)-barrel domain and the C-terminal C345c domain. An internal thioester in the alpha-chain of C3 (present in C4 but not in C5) is cleaved during complement activation. This mediates covalent attachment of the activated C3b to immune complexes and invading microorganisms, thereby opsonizing the target. We present the structure of bovine C3 determined at 3 Angstroms resolution. The structure shows that the ester is buried deeply between the thioester domain and the properdin binding domain, in agreement with the human structure. This domain interface is broken upon activation, allowing nucleophile access. The structure of bovine C3 clearly demonstrates that the main chain around the thioester undergoes a helical transition upon activation. This rearrangement is proposed to be the basis for the high level of reactivity of the thioester group. A strictly conserved glutamate residue is suggested to function catalytically in thioester proteins. Structure-based design of inhibitors of C3 activation may target a conserved pocket between the alpha-chain and the beta-chain of C3, which appears essential for conformational changes in C3.     

A simple bioluminescence procedure for early warning detection of coliform bacteria in drinking water

Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18–24 h to complete. Bioluminescence-based enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking water. The bioluminescence procedure targeting β-d-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-β-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6–8 h in selective media containing isopropyl-β-d-thiogalactopyranoside (IPTG) to induce β-d-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of β-d-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure. Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water within one working day. The results suggest the bioluminescence assays targeting β-d-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated drinking water wells.     

Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs.     

Seasonal dynamics and bioavailability of dissolved organic matter in two contrasting temperate estuaries

Production and bioavailability of dissolved organic matter (DOM) were followed during a year in the nutrient-rich estuary, Roskilde Fjord (RF), and the more oligotrophic strait, Great Belt (GB), in Denmark. Bioavailability of dissolved organic carbon (DOC), nitrogen (DON), and phosphorous (DOP) was determined during incubations over six months. Overall, RF had three to five times larger pools of total nitrogen (TN) and total phosphorous (TP) and five to eight times higher concentrations of inorganic nutrients compared to GB. However, the allocation of carbon, nitrogen, and phosphorous into different pools were remarkably similar between the two systems. DON and DOP contributed with about equal relative fractions in the two systems: 72 ± 13% of total nitrogen and 21 ± 12% of total phosphorous. The average bioavailability of DOM was 25 ± 15, 17 ± 5.5, and 49 ± 29% for carbon, nitrogen, and phosphorous, respectively. The observed release of DIN from degradation of DON amounted to between 0.1 (RF winter) and 14 times (GB summer) the loadings from land and contributed with half of the total input of bioavailable nitrogen during summer. Hence, this study shows that nitrogen in DOM is important for the nitrogen cycling, especially during summer. The sum of inorganic nutrients, particulate organic matter, and bioavailable DOM (the dynamic pools of nutrients) accounted for 42 and 92% of nitrogen, and phosphorous, respectively, and was remarkably similar between the two systems compared to the difference in nutrient richness. It is hypothesized that the pelagic metabolism of nutrients in marine systems dictates a rather uniform distribution of the different fractions of nitrogen and phosphorous containing compounds regardless of eutrophication level.