Mutant loxP vectors for selectable marker recycle and conditional knock-outs

BACKGROUND: Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. RESULTS: We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. CONCLUSIONS: Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved β-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines

Hydrocarbons content in soils of the northernmost taiga ecosystem of Komi Republic (North-East of Russia)

The background concentrations of hydrocarbons (HCs) were estimated for soils of the northernmost and northern taiga ecosystem of the Komi Republic. It was shown that accumulation and distribution of hydrocarbons in soil cover is regulated by following pedological factors: texture class, parent material and landform type and the type of soil forming process. In all studied soils of accumulative positions showed more pronounces accumulation of hydrocarbons than the soils of well-drained eluvial positions. Interprofile differentiation of hydrocarbons content is more expressed in clay-textured soils than sandy ones

Further investigations of the deep double donor magnesium in silicon

The deep double donor levels of substitutional chalcogen impurities in silicon have unique optical properties which may enable a spin/photonic quantum technology. The interstitial magnesium impurity (Mg$_i$) in silicon is also a deep double donor but has not yet been studied in the same detail as have the chalcogens. In this study we look at the neutral and singly ionized Mg$_i$ absorption spectra in natural silicon and isotopically enriched 28-silicon in more detail. The 1s(A$_1$) to 1s(T$_2$) transitions, which are very strong for the chalcogens and are central to the proposed spin/photonic quantum technology, could not be detected. We observe the presence of another double donor (Mg$_{i*}$) that may result from Mg$_i$ in a reduced symmetry configuration, most likely due to complexing with another impurity. The neutral species of Mg$_{i*}$ reveal unusual low lying ground state levels detected through temperature dependence studies. We also observe a shallow donor which we identify as a magnesium-boron pair

A Mg-pair isoelectronic bound exciton identified by its isotopic fingerprint in 28^{28}Si

We use the greatly improved optical linewidths provided by highly enriched $^{28}$Si to study a photoluminescence line near 1017 meV previously observed in the luminescence spectrum of natural Si diffused with Mg, and suggested to result from the recombination of an isoelectronic bound exciton localized at a Mg-pair center. In $^{28}$Si this no-phonon line is found to be comprised of five components whose relative intensities closely match the relative abundances of Mg-pairs formed by random combinations of the three stable isotopes of Mg, thus confirming the Mg-pair hypothesis. We further present the results of temperature dependence studies of this center that reveal unusual and as yet unexplained behaviour.Comment: 5 pages, 4 figure

The content of polyarenes in soils of antarctica: Variability across landscapes

Soil cover of the Earth is faced with intensive polychemical contamination. The pathways of the key pollutants are not investigated enough. Thus, the occasional transportation of soils to remote regions could serve as an informative tool for the elaboration of threshold levels of hazardous materials concentration. One of the most striking examples of such transboundary impact was the transfer of soils and grounds to the Antarctic stations Russkaya and Leningradskaya (before the implementation of the Madrid Protocol in 1991). Thus, the complex investigation of qualitative and quantitative composition of polycyclic aromatic hydrocarbons (PAHs) in soils of various genesis (transported from Eurasia and pristine) of Antarctic have been conducted by the method of high-performance liquid chromatography (HPLC) in a gradient elution mode. The variability of PAHs content was evaluated across landscapes: pristine (the Hudson Mountains and the Haswell Archipelago), contaminated soils (stations Myrniy, Druzhnaya 4 and Bellinshausen) and unique samples of former agrosoils transported by fly in-fly of polar staff from St. Petersburg to Antarctic for local polar greenhouses in the Soviet times, when it was not strictly prohibited. The selected objects of study allow us to not only estimate the degree of contamination of Antarctic soils and grounds, but to also make it possible to assess the rate of PAH degradation in Antarctic conditions. Both high molecular and low molecular PAHs are accumulated following intense anthropogenic activity (fossil organic fuel combustion). The PAHs pool is dominated by low molecular weight representatives (naphthalene, phenanthrene, fluoranthene, pyrene). In most cases, the highest concentrations of benz(a)pyrene does not exceed the Russian Threshold Standard rate, which is the strictest one in the world. The statistical analysis of raw data allowed us to conclude that the contamination of pristine soils of Antarctica across variable landscapes is at the very initial stage. However, we recorded extremely high levels of PAHs in the transported former agrosoils. We can assume that our data could be used as background levels for the elaboration of threshold concentrations of the PAHs for such an internationally managed region as Antarctica

Decreased Expression of 15-hydroxyprostaglandin Dehydrogenase in Gastric Carcinomas

prostaglandin E2 (PGE2) when compared to non-neoplastic mucosa, and cyclooxygenase-2 (COX-2), which is the ratelimiting enzyme in prostaglandin (PG) biosynthesis, is often overexpressed in gastric carcinomas and during gastric carcinogenesis. However, little is known about the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme responsible for the biological inactivation of PG, in gastric carcinomas. Materials and Methods: We investigated the expression of 15-PGDH in 28 cases of advanced gastric carcinomas by Western blot analysis and also the relation between its expression and the gene promoter methylation. Results: 15-PGDH expression was significantly decreased in gastric carcinomas compared to corresponding non-neoplastic tissues and inversely correlated with the expression of proliferating cell nuclear antigen in gastric carcinomas. However, there was no correlation between 15-PGDH expression and pathological findings such as nodal metastasis and vascular invasion. Promoter hypermethylation of 15-PGDH gene was not detected in carcinomas, with only a negligible expression of the enzyme. Conclusion: Our results suggested that 15-PGDH has tumor suppressor activity in gastric carcinomas. Key Words: 15-hydroxyprostaglandin dehydrogenase, gastric carcinoma, methylatio

High-field impurity magneto-optics of Si:Se

Contains fulltext : 130582.pdf (publisher's version ) (Open Access

Polymer selection for microencapsulation of probiotics: impact on viability, stability, and delivery in functional foods for improved manufacturing and product development in the food industry

Probiotics have won considerable interest in the food industry because of their health benefits. However, ensuring probiotics' viability, stability, and effective delivery in functional ingredients constitute a major concern. Microencapsulation is a promising method to ensure probiotic viability and stability. The best polymer for microencapsulation of probiotics is a determining factor. This paper presents an overview of the impact of polymer selection on probiotic viability, stability, and delivery in functional foods. It discusses numerous microencapsulation techniques and factors influencing polymer selection. It further explores the consequences of various polymers on probiotic viability, highlighting their protecting mechanisms. Additionally, it examines the role of polymer selection in enhancing probiotic stability during delivery, launch kinetics, storage and processing. The business packages of microencapsulated probiotics in foods and case studies on precise polymer choices for probiotic product improvement are also presented. Finally, we present challenges and future directions in using polymers for probiotic microencapsulation in the food industry. This review thus presents insights to enhance manufacturing tactics and product development within the food industry

Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

<p>Abstract</p> <p>Background</p> <p>14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown.</p> <p>Methods</p> <p>We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome.</p> <p>Results</p> <p>In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (<it>p </it>= 0.001, <it>p </it>= 0.002 and <it>p </it>= 0.001, respectively) and deep invasion (<it>p </it>= 0.01, <it>p </it>= 0.001 and <it>p </it>= 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival.</p> <p>Conclusion</p> <p>Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis.</p