A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)

<p>Abstract</p> <p>Background</p> <p>Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.</p> <p>Results</p> <p>A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size.</p> <p>Conclusions</p> <p>BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.</p

Anthracyclines, proteasome activity and multi-drug-resistance

BACKGROUND: P-glycoprotein is responsible for the ATP-dependent export of certain structurally unrelated compounds including many chemotherapeutic drugs. Amplification of P-glycoprotein activity can result in multi-drug resistance and is a common cause of chemotherapy treatment failure. Therefore, there is an ongoing search for inhibitors of P-glycoprotein. Observations that cyclosporin A, and certain other substances, inhibit both the proteasome and P-glycoprotein led us to investigate whether anthracyclines, well known substrates of P-gp, also inhibit the function of the proteasome. METHODS: Proteasome function was measured in cell lysates from ECV304 cells incubated with different doses of verapamil, doxorubicin, daunorubicin, idarubicin, epirubicin, topotecan, mitomycin C, and gemcitabine using a fluorogenic peptide assay. Proteasome function in living cells was monitored using ECV304 cells stably transfected with the gene for an ubiquitin/green fluorescent protein fusion protein. The ability of the proteasome inhibitor MG-132 to affect P-glycoprotein function was monitored by fluorescence due to accumulation of daunorubicin in P-glycoprotein overexpressing KB 8-5 cells. RESULTS: Verapamil, daunorubicin, doxorubicin, idarubicin, and epirubicin inhibited 26S chymotrypsin-like function in ECV304 extracts in a dose-dependent fashion. With the exception of daunorubicin, 20S proteasome function was also suppressed. The proteasome inhibitor MG-132 caused a dose-dependent accumulation of daunorubicin in KB 8-5 cells that overexpress P-glycoprotein, suggesting that it blocked P-glycoprotein function. CONCLUSION: Our data indicate that anthracyclines inhibit the 26S proteasome as well as P-glycoprotein. Use of inhibitors of either pathway in cancer therapy should take this into consideration and perhaps use it to advantage, for example during chemosensitization by proteasome inhibitors

Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis

Leptospirosis is a bacterial disease that is mainly spread by rodents and other small mammals. Transmission frequently occurs in (sub-) tropical countries, where environmental circumstances are most favourable. Severe leptospirosis can cause bleeding and vital organ dysfunction. An exaggerated immune response is thought to play an important role in the pathophysiology of leptospirosis. Soluble ST2 (sST2) is thought to inhibit negative regulatory pathways of this response. Soluble ST2 is produced by cells that surround, for example, blood vessels, and several of these blood cells play an important part in the host immune response. In an observational study, we measured the extent of sST2 release in patients suffering from severe leptospirosis. We found that patients that died from leptospirosis displayed higher levels of sST2. Moreover, from this study we have seen that sST2 levels were associated with bleeding, whereas other markers of infection were not. In an experiment, we showed that (white) blood cells did not seem to be the source of sST2 production. Damage to blood vessels is likely to cause bleeding in leptospirosis patients, exposing sST2 producing cells like fibroblasts to the blood stream. Hence, we believe that sST2 may be used as a marker for tissue damage in patients suffering from severe leptospirosis

Interannual environmental-soil thawing rate variation and its control on transpiration from Larix cajanderi, Central Yakutia, Eastern Siberia

Sapflow measurements were carried out in a larch forest in eastern Siberia, an area of wide permafrost distribution. Canopy transpiration and canopy conductance were scaled up from these values. The objective was to analyze the relationship between environmental variables, mainly vapour pressure deficit (D), soil moisture and soil thawing rate with canopy transpiration and canopy conductance. Maximum sapflow rate was 42.4 kg d−1 tree−1 with bigger trees showing a more accentuated response to environmental changes. Canopy transpiration (Ec) showed inter-annual variability, with a maximum value of 1.7 mm d−1 in 2003 and 1.2 mm d−1 in 2004. Soil moisture was higher in 2003 because of higher precipitation (230 mm in 2003 compared to 110 mm in 2004 for the total growing season). Maximum soil thawing rate in 2003 and 2004 was 140 cm and 120 cm, respectively, because of different air temperature, soil water content and precipitation regime among other factors. Canopy conductance (gc) was positively correlated with D during fine weather and well-watered days in both years. On the other hand, canopy conductance was well correlated with soil moisture (R2 = 0.83) in the upper layers (20–30 cm depth) during 2003 (wet year) but not in 2004 (dry year), representing its strong but limited control over water fluxes from the forest. By comparison with other studies in this region, canopy transpiration is estimated to contribute to almost 50% of the total forest evaporation, highlighting the important role of understorey transpiration in permafrost regions. Our results show that it is not only the impermeability of permafrost with the property of keeping soil moisture in the thin active layer but it is also the slow soil thawing rate that plays the important role of controlling the amount of water available for trees roots in the upper soil layers during dry years

Transient Freeze-Thaw Deformation Responses to the 2018 and 2019 Fires Near Batagaika Megaslump, Northeast Siberia

Wildfires in Arctic regions impact landforms via permafrost degradation and subsequent deformation that can last for many years. However, it remains uncertain on if and how much deformations occur, and what controls their magnitude, particularly during the first couple of years. Here, we examine the transient post-fire deformation responses near the Batagaika megaslump, which is the world's largest retrogressive thaw slump at Batagay, Sakha Republic. There were wildfires in the summers of 2018 and 2019 on the same slope, which could trigger the formation of another megaslump; many fires occurred nearby in 2019. We use interferometric synthetic aperture radar (InSAR) to measure surface displacements, including both post-fire and span-fire images. We also perform onsite measurements of temperature and thaw depth around the two scars near Batagaika megaslump in 2019, 2020, and 2021 and around the 2014 scar in 2019. At the three fire scars formed in 2018 and 2019, we demonstrate year-to-year and location-specific changes in the amplitude of subsidence, heave, and duration. The 2018 scar shows cumulative subsidences of up to 10 cm by March 2021, more clearly than the nearby 2019 scar. On the other hand, another 2019 scar adjacent to the 2014 scar shows up to 13 cm net subsidence during the first span-fire year, although the subsiding area is limited. These diverse transient post-fire responses demonstrate that under the yedoma area the spatial heterogeneities of the active layer depth and the timing of fires will control subsequent thermokarst processes