The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58C1Rifr::pGSFR1161 in the presence of acetosyringone

Summary Explants of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) were co-cultivated with Agrobacterium tumefaciens C58C1Rif^r::pGSFR1161 in the presence of 20 (M acetosyringone). Transformed root clones were selected on kanamycin medium and the presence of the nptII gene in the plant DNA confirmed by the polymerase chain reaction. Root clones derived from acetosyringone treatment grew more vigorously in the presence of kanamycin and synthesized a greater amount of NPT-II enzyme. The conclusion is that acetosyringone treatment enhances the transformation process, possibly by stimulating multiple insertions of the T-DNA into the host genome.Abbreviations AS acetosyringone - CTAB hexadecyltrimethylammonium bromide - EDTA ethylenediaminetetraacetic acid - ELISA enzyme-linked immunosorbant assay - MS Murashige and Skoog (1962) medium - PCR polymerase chain reaction     

Plenty of rooftops with few neighbours occupied by young breeding Yellow-legged Gulls (Larus michahellis): Does this occur at the expense of their health condition?

Gulls, as largely flexible opportunistic individuals, have been increasingly breeding in many cities around the world, but it is still unclear whether urban habitats are of equal or higher quality than traditional natural habitats or represent an ecological trap with immediate reproductive benefits but longer-term detrimental consequences to health. Here we present a study of breeding parameters (nest density, egg dimensions, clutch size, hatching success and adult body condition) and physiological parameters (erythrocyte sedimentation rate, heterophil/lymphocyte ratio, haemoglobin concentration and measurements of oxidative stress) as indicators of the general health condition of Yellow-legged Gull Larus michahellis adults and chicks from natural and urban colonies. Yellow-legged Gulls in the largest urban area (Porto) laid smaller eggs and clutches, showed a significantly lower occurrence of inflammatory processes in chicks, and showed a slower early chick growth than in the natural colony of Deserta. This suggests that urban gulls might be facing important trade-offs between the advantages of breeding in lower density urban colonies, with fewer intraspecific interactions and a lower disease transmission probability, and the disadvantages of having an anthropogenic diet usually lower in nutritional value.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Identifying Mozambique's most critical areas for plant conservation: An evaluation of protected areas and Important Plant Areas

Successful protected area networks must represent biodiversity across taxonomic groups. However, too often plant species are overlooked in conservation planning, and the resulting protected areas may, as a result, fail to encompass the most important sites for plant diversity. The Mozambique Tropical Important Plant Areas project sought to promote the conservation of Mozambique's flora through the identification of Important Plant Areas (IPAs). Here, we use the Weighted Endemism including Global Endangerment (WEGE) index to identify the richest areas for rare and endemic plants in Mozambique and subsequently evaluate how well represented these hotspots are within the current protected area and IPA networks. We also examine the congruence between IPA and protected areas to identify opportunities for strengthening the conservation of plants in Mozambique. We found that high WEGE scores, representing areas rich in endemic/near-endemic and threatened species, predict the presence of IPAs in Mozambique, but do not predict the presence of protected areas. We also find that there is limited overlap between IPAs and protected areas in Mozambique. We demonstrate how IPAs could be an important tool for ensuring priority sites for plant diversity are included within protected area network expansions, particularly following the adoption of the “30 by 30” target agreed within the post-2020 Convention on Biological Diversity framework, with great potential for this method to be replicated elsewhere in the global tropics.     

The gene coding for the α1 subunit of the skeletal dihydropyridine receptor (Cchl1a3=mdg) maps to mouse Chromosome 1 and human 1q32

Using both chromosomal in situ hybridization and molecular techniques, we report the genetic localization of the gene coding for the alpha 1 subunit of the skeletal slow Ca²⁺ current channel/DHP receptor gene (Cchl1a3) on human Chromosome (Chr) 1 (1q31–1q32 region) and on mouse Chr 1 region (F-G). On the basis of single-strand conformation polymorphism (SSCP-PCR) analysis in an interspecific backcross, we have determined that the Cchl1a3=mdg (muscular dysgenesis) locus is very closely linked to the myogenin (Myog) locus.     

Piper longum Constituents Induce PANC-1 Human Pancreatic Cancer Cell Death under Nutrition Starvation

Pancreatic cancer is a highly aggressive form of cancer with a poor prognosis, partly due to ‘austerity’, a phenomenon of tolerance to nutrient deprivation and survival in its hypovascular tumor microenvironment. Anti-austerity agents which preferentially diminish the survival of cancer cells under nutrition starvation is regarded as new generation anti-cancer agents. This study investigated the potential of Piper longum constituents as anti-austerity agents. The ethanolic extract of Piper longum was found to have preferential cytotoxicity towards PANC-1 human pancreatic cancer cells in a nutrient-deprived medium (NDM). Further investigation led to the identification of pipernonaline ( 3 ) as the lead compound with the strongest anti-austerity activity, inducing cell death and inhibiting migration in a normal nutrient medium, as well as strongly inhibiting the Akt/mTOR/autophagy pathway. Therefore, pipernonaline ( 3 ) holds promise as a novel antiausterity agent for the treatment of pancreatic cancer.