Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

Increased isolation of two Biosphere Reserves and surrounding protected areas (WAP ecological complex, West Africa)

Protected areas such as nature reserves have been found to be effective in preventing habitat destruction and protecting ecosystems within their borders. Recent studies however found extensive loss of tropical forest habitat around protected areas, vastly contributing to increase the levels of ecological isolation. Using high-resolution satellite data we investigated the isolation trend occurring in the W-Arly-Pendjari (WAP) ecological complex in West Africa. A land-cover change analysis was performed for the period 1984–2002: savanna vegetation extension and loss were derived within the complex and in a 30 km peripheral buffer. Sample regions in the buffer were also analysed using selected spatial indicators to quantify temporal trends in habitat fragmentation. Implications for change in relative capacity to conserve biodiversity were discussed through the calculation of the species richness capacity (SRC). More than 14.5% of savanna habitat was lost in the WAP peripheral areas, while 0.3% was converted inside the complex. The degree of fragmentation of remnant savanna habitat has also drastically increased. Despite the effectiveness of the park conservation programme, we found through the SRC approach that the WAP complex is decreasing its potential capacity to conserve species richness. This process is mainly due to the rapid and extended agricultural expansion taking place around the complex. A better understanding of the ecological dynamics occurring in the peripheral regions of reserves and the consideration of development needs are key variables to achieve conservation goals in protected areas.     

Seeing 'where' through the ears: effects of learning-by-doing and long-term sensory deprivation on localization based on image-to-sound substitution

Background

Sensory substitution devices for the blind translate inaccessible visual information into a format that intact sensory pathways can process. We here tested image-to-sound conversion-based localization of visual stimuli (LEDs and objects) in 13 blindfolded participants.

Methods and Findings

Subjects were assigned to different roles as a function of two variables: visual deprivation (blindfolded continuously (Bc) for 24 hours per day for 21 days; blindfolded for the tests only (Bt)) and system use (system not used (Sn); system used for tests only (St); system used continuously for 21 days (Sc)). The effect of learning-by-doing was assessed by comparing the performance of eight subjects (BtSt) who only used the mobile substitution device for the tests, to that of three subjects who, in addition, practiced with it for four hours daily in their normal life (BtSc and BcSc); two subjects who did not use the device at all (BtSn and BcSn) allowed assessment of its use in the tasks we employed. The impact of long-term sensory deprivation was investigated by blindfolding three of those participants throughout the three week-long experiment (BcSn, BcSn/c, and BcSc); the other ten subjects were only blindfolded during the tests (BtSn, BtSc, and the eight BtSt subjects). Expectedly, the two subjects who never used the substitution device, while fast in finding the targets, had chance accuracy, whereas subjects who used the device were markedly slower, but showed much better accuracy which improved significantly across our four testing sessions. The three subjects who freely used the device daily as well as during tests were faster and more accurate than those who used it during tests only; however, long-term blindfolding did not notably influence performance.

Conclusions

Together, the results demonstrate that the device allowed blindfolded subjects to increasingly know where something was by listening, and indicate that practice in naturalistic conditions effectively improved “visual” localization performance.     

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. ‘paramerae’) and one crustose (semi‐vagrant, Circinaria sp. ‘oromediterranea’) lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. ‘oromediterranea’ and Circinaria sp. ‘paramerae’ formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high‐throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.     

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

Anaerobic Cometabolic Conversion of Benzothiophene by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [¹³C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the ¹³C content of the carboxyl group were formed, indicating the addition of a C₁ unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.     

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.