An optimized, chemically regulated gene expression system for Chlamydomonas

Background

Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system.

Methodology

Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 µM) TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 µM). Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient “wave” in luciferase activity, which can be repeated in subsequent growth cycles.

Conclusions

We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled “waves” in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas.     

Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.     

Jaguar (Panthera onca) feeding ecology: distribution of predator and prey through time and space

Jaguars Panthera onca inhabiting tropical or subtropical evergreen moist forest have often been classified as opportunistic predators because they consume prey relative to its availability. However, these studies failed to address simultaneously the distribution of predator and prey through time and space, which may lead to an incomplete or erroneous understanding of jaguar foraging strategies. In this study, we reconstructed jaguar diet from scat, and used camera traps to investigate jaguar prey availability and the distribution of jaguar and its prey through space and time. We focused our examination of predator–prey temporal and spatial relations on forest infrastructure comprising man-made paths, small mammal trails, tapir Tapirus bairdi trail and trail-less, forested areas as they represent distinct habitats for prey selection. Overall, we observed high overlap between the prey used and available, suggestive of opportunistic foraging. However, jaguars exhibited selective tendencies in discriminating between larger prey. Jaguars used collared peccary Tayassu tajacu greater than its availability, while preying upon the equally abundant and similarly distributed white-lipped peccary Tayassu pecari and tapir less than predicted based upon availability. Armadillo Dasypus novemcinctus and paca Agouti paca , 56.6% of total consumption, were consumed relative to availability but exhibited low spatial overlap with jaguar. Armadillo and paca used trail-less, forested areas and small mammal trails not used by jaguar and were photographed more frequently at greater distances from man-made paths, major thoroughfares for jaguars. This study suggests that although forest jaguars use prey relative to its abundance, jaguars may rely on foraging strategies other than chance encounters for exploiting prey.     

Maternal Antiretroviral Therapy for the Prevention of Mother-To-Child Transmission of HIV in Malawi: Maternal and Infant Outcomes Two Years after Delivery

Background

Optimized preventive strategies are needed to reach the objective of eliminating pediatric AIDS. This study aimed to define the determinants of residual HIV transmission in the context of maternal antiretroviral therapy (ART) administration to pregnant women, to assess infant safety of this strategy, and to evaluate its impact on maternal disease.

Methodology/Principal Findings

A total of 311 HIV-infected pregnant women were enrolled in Malawi in an observational study and received a nevirapine-based regimen from week 25 of gestation until 6 months after delivery (end of breastfeeding period) if their CD4+ count was > 350/mm³ at baseline (n = 147), or indefinitely if they met the criteria for treatment (n. 164). Mother/child pairs were followed until 2 years after delivery. The Kaplan-Meier method was used to estimate HIV transmission, maternal disease progression, and survival at 24 months. The rate of HIV infant infection was 3.2% [95% confidence intervals (CI) 1.0-5.4]. Six of the 8 transmissions occurred among mothers with baseline CD4+ count > 350/mm³. HIV-free survival of children was 85.8% (95% CI 81.4-90.1). Children born to mothers with baseline CD4+ count < 350/mm³ were at increased risk of death (hazard ratio 2.6, 95% CI 1.1-6.1). Among women who had stopped treatment the risk of progression to CD4+ count < 350/mm³ was 20.6% (95% CI 9.2-31.9) by 18 months of drug discontinuation.

Conclusions

HIV transmission in this cohort was rare however, it occurred in a significative proportion among women with high CD4+ counts. Strategies to improve treatment adherence should be implemented to further reduce HIV transmission. Mortality in the uninfected exposed children was the major determinant of HIV-free survival and was associated to maternal disease stage. Given the considerable proportion of women reaching the criteria for treatment within 18 months of drug discontinuation, life-long ART administration to HIV-infected women should be considered.     

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar–parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation. Accepted: 16 September 1999     

Pendrin overexpression affects cell volume recovery, intracellular pH and chloride concentration after hypotonicity-induced cell swelling

The pendrin (SLC26A4 or PDS) gene is responsible, when mutated, for the Pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid dysfunctions. Pendrin protein is an anion exchanger and we focused on a still unexplored function that it might play in view of its importance in the inner ear: Cl(-) fluxes regulation during cellular volume control. We challenged HEK-293 Phoenix cells over-expressing wild type pendrin (PDS HEK cells) together with the EYFP (Enhanced Yellow Fluorescent Protein) or over-expressing the EYFP alone (control HEK cells) with hypo-osmolar solutions. Taking advantage of the confocal optical sectioning we measured the cell volume. In addition, we determined the intracellular pH and chloride concentration with fluorescent probes (EYFP and seminaphthorhodafluor-5F, SNARF-5F). Consequently, we could estimate simultaneously Cl(-) fluxes, cellular volume and intracellular pH variations. Cl(-) movements markedly differed between PDS and control HEK cells upon hypotonic shock and are accompanied by an attenuation of the swelling induced pH drop in PDS HEK cells. The contemporary measurements of the three variables not yet reported in living cells, allowed to assess a possible influence of pendrin upregulation in volume homeostasis and evidenced its participation to Cl(-) fluxes.     

Thionine increases electricity generation from microbial fuel cell using Saccharomyces cerevisiae and exoelectrogenic mixed culture

Microbial fuel cells (MFCs) have been shown to be capable of clean energy production through the oxidation of biodegradable organic waste using various bacterial species as biocatalysts. In this study we found Saccharomyces cerevisiae, previously known electrochemcially inactive or less active species, can be acclimated with an electron mediator thionine for electrogenic biofilm formation in MFC, and electricity production is improved with facilitation of electron transfer. Power generation of MFC was also significantly increased by thionine with both aerated and non-aerated cathode. With electrochemically active biofilm enriched with swine wastewater, MFC power increased more significantly by addition of thionine. The optimum mediator concentration was 500 mM of thionine with S. cerevisae in MFC with the maximum voltage and current generation in the microbial fuel cell were 420 mV and 700 mA/m(2), respectively. Cyclic voltametry shows that thionine improves oxidizing and reducing capability in both pure culture and acclimated biofilm as compared to non-mediated cell. The results obtained indicated that thionine has great potential to enhance power generation from unmediated yeast or electrochemically active biofilm in MFC.     

芦鹀种群中的文化、遗传和形态进化的同时比较

我们比较了芦 (Emberizaschoeniclus)两个亚种组 ,即北部的薄喙亚种组和南部的厚喙亚种组的 10个种群中的文化、遗传和形态变异。使用了四个不同的变异标记物 ,其中两个用来测量文化分化 ,一个用来测量遗传分化 ,即微卫星等位基因的频次 ,一个用来测量种群的形态分化 ,即喙的高度。将遗传分化作为进化时间的尺度 ,我们计算了亚种组间和组内分化指标与所估计的进化率之间的相关性 ,发现只有文化定量指标和遗传分化与种群的形态分化相关 ,而两个文化分化指标之间没有关系 ,文化分化与遗传分化之间也没有关系。使用文化 -定量分化指标 ,发现亚种组间的文化进化率高于亚种内的文化进化率 ,提示鸣唱在防止杂交方面只有微弱的、也许是次要的作用。鸣唱定量特征的变异与微卫星频次相同 ,实际上在自然界中更可能是遗传决定的 ,这可以解释由于分析两个文化变异指标所得出的结果的不一致性。鸣唱的声学特性可能由于栖息地的差异或形态上的限制而发生了演变 ,而文化传播单位 (Meme)的特性可能由于学习鸣唱和文化传播而受到了影响