Discriminating changes in intracellular NADH/NAD+ levels due to anoxicity and H2 supply in R. eutropha cells using the Frex fluorescence sensor.

The hydrogen-oxidizing "Knallgas" bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.

Seminal plasma and seminal plasma proteins added to bulk sorted sperm do not alter the mRNA expression of in vitro produced bovine embryos.

Although sex-sorted sperm have been used for AI and IVF for over a decade there is still need to improve the technology as the results are highly variable. The goal of the present study was to assess the effect of seminal plasma and seminal plasma proteins as a supplement to sorted sperm on subsequent embryonic development, as a beneficial effect of these substances has been reported. In vitro matured oocytes were fertilized in vitro with either unsorted sperm (n = 215; Group 1), bulk sorted sperm (n = 226; Group 2), bulk sorted sperm extended in the presence of 1% seminal plasma (n = 185; Group 3) or bulk sorted sperm supplemented with seminal plasma proteins (4 mg mL(-1); n = 254; Group 4). An additional group of oocytes (n = 307; Group 5) was fertilized with the semen of another bull routinely used for IVF and served as a laboratory standard control. Subsequently, the presumptive zygotes were cultured for 8 days under standard conditions (SOFaa, 39 °C, 5% CO(2), 5% N(2)). Cleavage rates were assessed on day 3 p.i. (post insemination; group 1: 30.5 ± 14.7%; group 2: 28.8 ± 9.8%; group 3: 20.8 ± 14.9%; group 4: 25.7 ± 8.2%; group 5: 54.8 ± 11.5%). Development rates were documented on days 7 p.i. (group 1: 7.3 ± 6.6%; group 2: 5.6 ± 3.1%, group 3: 6.2 ± 7.7%, group 4: 6.7 ± 5.9%, group 5: 20.2 ± 6.9%) and 8 p.i. (group 1: 8.9 ± 7.0%; group 2: 6.0 ± 2.9%; group 3: 8.6 ± 11.3%; group 4: 7.8 ± 6.2%; group 5: 23.3 ± 7.8%), respectively. Significant differences among cleavage and development rates could only be seen for Group 5 compared to all other groups. However, this difference between Groups 1-4 vs. Group 5 regarding the development rates on Day 8 could not be detected when assessing the development rates on base of the number of cleaved embryos instead of the number of oocytes fertilized (group 1: 31.4 ± 17.2%; group 2: 26.0 ± 21.0%; group 3: 33.3 ± 19.05%; group 4: 26.6 ± 17.8%; group 5: 42.6 ± 11.3%). The relative abundance of six different developmentally important gene transcripts (G6PD, HSP1A1, SLC2A3, BAX, BCL2L1, DNMT3A) was determined using single Day 8 expanded blastocysts of all five groups. No significant differences were seen among the embryos of the five groups. Our results show that neither the bulk sorting procedure nor the addition of seminal plasma or seminal plasma proteins, respectively, affected cleavage and development rates when sperm from a specific bull was used. Additionally, sorting and subsequent exposure of sperm to either seminal plasma or seminal plasma proteins did not influence mRNA expression in bovine IVP embryos.

Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level.

There are still immense differences in the quality of in vitro produced embryos compared to their in vivo generated counterparts. These differences include a higher sensitivity of in vitro produced embryos towards cryopreservation. The quality of such embryos has been evaluated by morphological examination as well as the assessment of total cell numbers, pregnancy rates and in few cases through the analysis of their gene expression. The aim of the present study was to determine whether different cryopreservation methods have an influence on the quality of in vitro produced embryos after thawing. Bovine blastocysts were produced in a standard culture system (SOFaa). Having reached the stage of an expanding blastocyst on day 7, embryos were randomly either vitrified (n = 106) or cryopreserved conventionally (n = 131). Reexpansion (24h post thawing; 86/106 [81.1 ± 20.3%] of the vitrified embryos, 104/131 [79.4 ± 16.5%] of the conventionally cryopreserved embryos respectively) and hatching rates (48 h post thawing; 67/106 [63.2 ± 24.5%] of the vitrified embryos, 80/131 [61.1 ± 29.3%] of the conventionally cryopreserved embryos respectively) were similar. No significant differences in total cell numbers as well as live-dead cell ratio could be seen in hatched blastocysts of either cryopreservation group as well as in a control group (embryos cultured up to the stage of a hatched blastocyst and not cryopreserved by either method). Additionally, RT-qPCR was used to assess the relative abundance of eight developmentally important genes (HSPA1A, SLC2A1, SLC2A3, DSC2, CDH1, TJP1, DNMT3A, IFNT2) in hatched embryos of all groups. The results revealed significant differences in 4 of 8 (HSPA1A, SLC2A1, TJP1, DSC2) gene transcripts when comparing vitrified embryos to embryos of the control group and in 6 of 8 gene transcripts (HSPA1A, SLC2A1, TJP1, SLC2A3, DNMT3A, IFNT2) when comparing slow frozen embryos to embryos of the control group. Furthermore, the comparison of vitrified embryos to those frozen conventionally solely showed significant differences in the relative abundance of IFNT2. These results indicate that although not visible in gross morphology, but in the relative amount of gene transcripts, vitrification may be the more suitable method to cryopreserve in vitro produced bovine embryos cultured in SOF media.

Quantitative mRNA expression in ovine blastocysts produced from X- and Y-chromosome bearing sperm, both in vitro and in vivo.

Artificial insemination (AI) of sex-sorted sperm results in decreased fertility, compared with non-sorted sperm, in most species. However, this has not been the case in sheep, where the low-dose AI of sex-sorted ram sperm produced similar, if not superior, fertility to non-sorted controls. The aim of the present study was to determine the impact of sex-sorting technology on ovine embryo gene expression following embryo production in vivo and in vitro. After semen collection, ejaculates were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen. Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched sperm, or non-sorted control sperm, and collected by uterine flushing on Day 6 after AI. Embryos were produced in vitro using the same sperm treatments and cultured in vitro for 6 d. The relative abundance of selected gene transcripts was measured in high-grade blastocysts, defined by morphological assessment, using RT-qPCR. The mRNA expression of DNMT3A and SUV39H1 was upregulated in embryos cultured in vitro, compared to those cultured in vivo (DNMT3A: 3.61 ± 1.08 vs 1.99 ± 0.15; SUV39H1: 1.88 ± 0.11 vs 0.88 ± 0.07; mean ± SEM; P < 0.05). Both G6PD and SLC2A3 transcripts were reduced in embryos produced from sex-sorted sperm, in vivo (SLC2A3: 0.23 ± 0.03 vs 0.64 ± 0.10; G6PD: 0.32 ± 0.04 vs 1.01 ± 0.16; P < 0.05). The expression of DNMT3A was up-regulated in male (3.85 ± 0.31), compared to female embryos (2.34 ± 0.15; P < 0.05). This study contributes to the growing body of evidence citing aberrant patterns of gene expression resulting from in vitro culture. Whereas the process of sex-sorting altered the expression of several of the genes examined, no effect on embryo development was detected.

No association between MDM2 SNP309 promoter polymorphism and basal cell carcinoma of the skin.

BACKGROUND: The MDM2 oncoprotein promotes cell survival and cell cycle progression by inhibiting the p53 tumour suppressor protein. Further, overexpression of the MDM2 gene can inhibit DNA double-strand break repair in a p53-independent manner. Recent studies have shown that a single nucleotide polymorphism (SNP) in the intronic promoter region of MDM2 (called SNP309) can significantly change the expression of MDM2 and thereby suppress the p53 pathway. This SNP was also found to be associated with the onset and risk of different cancer types. Basal cell carcinoma of the skin (BCC) is one of the most common neoplasms in the world. BCC development is associated with environmental factors (especially sun exposure) as well as heritable factors. OBJECTIVES: The present case-control study investigated the association of the MDM2 SNP309 with the risk and the age at onset of BCC. Methods Data from 509 individuals affected by BCC and 513 healthy controls were genotyped with TaqMan polymerase chain reaction. RESULTS: Cases and controls showed a similar genotype distribution and the SNP did not modify the age at onset of BCC. CONCLUSIONS: These results suggest that the MDM2 SNP309 alone affects neither the risk nor the age at onset of BCC.

Case-control study in basal cell carcinoma of the skin: single nucleotide polymorphisms in three interleukin promoters pre-analysed in pooled DNA.

BACKGROUND: Basal cell carcinoma (BCC) is one of the most common neoplasms in the world. Development of BCC is associated with environmental factors (especially sun exposure) as well as heritable factors. OBJECTIVES: To analyse three single nucleotide polymorphisms (SNPs) in the promoter regions of interleukin (IL) genes in genomic DNA from 527 cases of BCC and 530 matched controls and to examine if DNA pooling is a useful method on which to base decisions regarding further SNP analysis. METHODS: The SNPs analysed were IL6-597, IL10-1082 and IL1B-511. The SNPs were first analysed from pooled DNA and afterwards from individual samples. The DNA pools resulted from a division of the samples into cases and controls, female and male, and three age groups. In these pools the allele frequencies were estimated by two methods, real-time polymerase chain reaction with allele-specific primers, and quantitative sequencing. RESULTS: No significant association was found when the allele frequencies in cases and controls were compared. However, by analysis of the individual genotypes we found SNP IL6-597 G/A to be significantly associated with BCC risk (P =0.007). Hereby the heterozygous genotype 'GA' had a protective effect (odds ratio 0.64, 95% confidence interval 0.49-0.84). No significant association was found for IL10-1082 and IL1B-511. CONCLUSIONS: The association of SNP IL6-597 with BCC could be found only by individual genotyping, but would have been missed if only data from the pooling analysis had been known.

The expression of HMGA genes is regulated by their 3'UTR.

Many benign mesenchymal tumors are characterized by chromosomal abnormalities of the regions 12q15 or 6p21.3 leading to aberrant expression of either HMGA2 (formerly HMGIC) or HMGA1 (formerly HMGIY). The proteins of both genes belong to the HMGA (formerly HMGI(Y)) family of architectural transcription factors. As a rule, aberrant HMGA transcripts found in a variety of benign tumors have intact coding regions at least for the DNA binding domains with a truncation of their 3' untranslated regions. Adding this to the finding that an altered HMGA protein level is not always correlated with an increased amount of corresponding mRNA indicates a posttranscriptional expression control mediated by regulatory elements within the 3'UTR. To check if HMGA expression is under control of such elements we performed luciferase assays with several HMGA2 and HMGA1 3'UTRs of different length. Experiments showed that an up to 12-fold increase in luciferase activity is obtained by the truncation of the 3'UTRs suggesting that the expression of HMGA2 and HMGA1 is controlled by negatively acting regulatory elements within their 3'UTR. Chromosomal aberrations affecting the HMGA genes may therefore influence their expression by an altered stability of the truncated transcripts as a result of the cytogenetic aberrations.

Spinal cord genes enriched in rat dorsal horn and induced by noxious stimulation identified by subtraction cloning and differential hybridization.

Persistent nociceptive input increases neuronal excitability and induces a program of gene expression in the dorsal spinal cord. The alteration in gene expression commences with phosphorylation and induction of immediate early genes and proceeds to target genes. Only a few target genes have been identified as yet. The present report uses a polymerase chain reaction-based subtraction cloning procedure to obtain an "anatomically focused" complementary DNA library enriched in transcripts related to sensory spinal cord (rat dorsal horn minus ventral horn). A subset of clones from this library (n=158) was screened to verify dorsal horn enrichment and to identify those regulated by carrageenan-induced peripheral inflammation. Molecular classes which displayed enriched expression included a proto-oncogene not previously associated with sensory processes, two regulators of the Rho/Rac pathway which controls cell shape, and three genes involved in cytoskeletal regulation and scaffolding. Additional transcripts coded for proteins involved in intercellular communication or intracellular function. Within the set of 158 transcripts, one known and two unknown genes were induced by persistent noxious input. The known gene codes for the secreted cysteine proteinase inhibitor, cystatin C, suggesting that modulation of extracellular proteolytic activity occurs. Since it is secreted, cystatin C may also provide a cerebrospinal fluid bio-marker for persistent pain states. Using a combined anatomical and functional approach, we have extended the molecular repertoire of genes expressed and induced in second-order neurons or supporting glial cells in several new directions, with particular emphasis on regulation of cell morphology and plasma membrane dynamics. Some of these proteins reveal new pathways for information signaling in the sensory half of the spinal cord and require further research to understand their role in the adult spinal cord. The induced genes may provide new molecular targets for therapeutic development and provide new probes for investigating the dynamic state of cellular activity that occurs during persistent pain states.