Pomalidomide Shows Significant Therapeutic Activity against CNS Lymphoma with a Major Impact on the Tumor Microenvironment in Murine Models

Primary CNS lymphoma carries a poor prognosis. Novel therapeutic agents are urgently needed. Pomalidomide (POM) is a novel immunomodulatory drug with anti-lymphoma activity. CNS pharmacokinetic analysis was performed in rats to assess the CNS penetration of POM. Preclinical evaluation of POM was performed in two murine models to assess its therapeutic activity against CNS lymphoma. The impact of POM on the CNS lymphoma immune microenvironment was evaluated by immunohistochemistry and immunofluorescence. In vitro cell culture experiments were carried out to further investigate the impact of POM on the biology of macrophages. POM crosses the blood brain barrier with CNS penetration of ~ 39%. Preclinical evaluations showed that it had significant therapeutic activity against CNS lymphoma with significant reduction in tumor growth rate and prolongation of survival, that it had a major impact on the tumor microenvironment with an increase in macrophages and natural killer cells, and that it decreased M2-polarized tumor-associated macrophages and increased M1-polarized macrophages when macrophages were evaluated based on polarization status. In vitro studies using various macrophage models showed that POM converted the polarization status of IL4-stimulated macrophages from M2 to M1, that M2 to M1 conversion by POM in the polarization status of lymphoma-associated macrophages is dependent on the presence of NK cells, that POM induced M2 to M1 conversion in the polarization of macrophages by inactivating STAT6 signaling and activating STAT1 signaling, and that POM functionally increased the phagocytic activity of macrophages. Based on our findings, POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment. It can induce significant biological changes in tumor-associated macrophages, which likely play a major role in its therapeutic activity against CNS lymphoma. POM should be further evaluated in clinical trials.     

Spatially resolved characterization of biogenic manganese oxide production within a bacterial biofilm

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn+2 [Mn+2(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn+2(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.     

High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.     

Predicting the response of the deep-ocean microbiome to geochemical perturbations by hydrothermal vents

Submarine hydrothermal vents perturb the deep-ocean microbiome by injecting reduced chemical species into the water column that act as an energy source for chemosynthetic organisms. These systems thus provide excellent natural laboratories for studying the response of microbial communities to shifts in marine geochemistry. The present study explores the processes that regulate coupled microbial-geochemical dynamics in hydrothermal plumes by means of a novel mathematical model, which combines thermodynamics, growth and reaction kinetics, and transport processes derived from a fluid dynamics model. Simulations of a plume located in the ABE vent field of the Lau basin were able to reproduce metagenomic observations well and demonstrated that the magnitude of primary production and rate of autotrophic growth are largely regulated by the energetics of metabolisms and the availability of electron donors, as opposed to kinetic parameters. Ambient seawater was the dominant source of microbes to the plume and sulphur oxidisers constituted almost 90% of the modelled community in the neutrally-buoyant plume. Data from drifters deployed in the region allowed the different time scales of metabolisms to be cast in a spatial context, which demonstrated spatial succession in the microbial community. While growth was shown to occur over distances of tens of kilometers, microbes persisted over hundreds of kilometers. Given that high-temperature hydrothermal systems are found less than 100 km apart on average, plumes may act as important vectors between different vent fields and other environments that are hospitable to similar organisms, such as oil spills and oxygen minimum zones.     

Changing Museum Visitors’ Conceptions of Evolution

We examined whether a single visit to an evolution exhibition contributed to conceptual change in adult (n?=?30), youth, and child (n?=?34) museum visitors?? reasoning about evolution. The exhibition included seven current research projects in evolutionary science, each focused on a different organism. To frame this study, we integrated a developmental model of visitors?? understanding of evolution, which incorporates visitors?? intuitive beliefs, with a model of free-choice learning that includes personal, sociocultural, and contextual variables. Using pre- and post-measures, we assessed how visitors?? causal explanations about biological change, drawn from three reasoning patterns (evolutionary, intuitive, and creationist), were modified as a result of visiting the exhibition. Whatever their age, background beliefs, or prior intuitive reasoning patterns, visitors significantly increased their use of explanations from the evolutionary reasoning pattern across all measures and extended this reasoning across diverse organisms. Visitors also increased their use of one intuitive reasoning pattern, need-based (goal-directed) explanations, which, we argue, may be a step toward evolutionary reasoning. Nonetheless, visitors continued to use mixed reasoning (endorsing all three reasoning patterns) in explaining biological change. The personal, socio-cultural, and contextual variables were found to be related to these reasoning patterns in predictable ways. These findings are used to examine the structure of visitors?? reasoning patterns and those aspects of the exhibition that may have contributed to the gains in museum visitors?? understanding of evolution.     

Creating porcine biomedical models through recombineering

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to 'forward genetics', in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the 'reverse genetics' approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a 'genetic model' organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide 'clones' of genetically modified animals.     

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.     

Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through PI3-kinase-dependent mechanism

Bile acids are cytoprotective in hepatocytes by activating phosphatidylinositol-3-kinase (PI3-K) and its downstream signal AKT. Our aim was to determine whether feeding taurocholate to CCl(4)-treated rats reduces cholangiocyte apoptosis and whether this cytoprotective effect is dependent on PI3-K. Cholangiocyte proliferation, secretion, and apoptosis were determined in cholangiocytes from bile duct ligation (BDL), CCl(4)-treated BDL rats, and CCl(4)-treated taurocholate-fed rats. In vitro, we tested whether CCl(4) induces apoptosis and whether loss of cholangiocyte proliferation and secretion is dependent on PI3-K. The CCl(4)-induced cholangiocyte apoptosis and loss of cholangiocyte proliferation and secretion were reduced in CCl(4)-treated rats fed taurocholate. CCl(4)-induced cholangiocyte apoptosis, loss of cholangiocytes secretion, and proliferation were prevented by preincubation with taurocholate. Taurocholate cytoprotective effects were ablated by wortmannin. Taurocholate prevented, in vitro, CCl(4)-induced decrease of phosphorylated AKT protein expression in cholangiocytes. The cytoprotective effects of taurocholate on CCl(4) effects on cholangiocyte proliferation and secretion were abolished by wortmannin. Taurocholate protects cholangiocytes from CCl(4)-induced apoptosis by a PI3-K-dependent mechanism. Bile acids are important in the prevention of drug-induced ductopenia in cholangiopathies.