Evidence of hybridization in the Argentinean lizards Liolaemus gracilis and Liolaemus bibronii (IGUANIA: LIOLAEMINI): an integrative approach based on genes and morphology

The lizard genus Liolaemus is endemic to temperate South America and includes more than 225 species. Liolaemus gracilis and L. bibronii are closely related species that have large and overlapping geographic distributions, and the objective of this work is to further investigate the L. bibronii–L. gracilis mtDNA paraphyletic pattern previously detected, using an integrative approach, based on mtDNA, nuclear DNA and morphological characters. We identified eight morphological L. bibronii introgressed with L. gracilis mtDNAs, and the reciprocal for one L. gracilis, from six localities in the region of sympatry overlap. The morphological identity of these introgressed individuals was confirmed by diagnostic nuclear markers, and this represents the first well-documented case of interspecific hybridization in the lizard genus Liolaemus. Of the three most likely hypotheses for these observed patterns, we suggest that asymmetrical mtDNA introgression as a result of recent or ongoing hybridization between L. bibronii and L. gracilis is the most likely. This may be due to size selection by L. gracilis female preference for the larger L. bibroni males in sympatry, but this requires experimental confirmation.     

Coupling in vitro and in vivo paradigm reveals a dose dependent inhibition of angiogenesis followed by initiation of autophagy by C6-ceramide

The activity of N-hexanoyl-D-erythro-sphingosine, a C6-ceramide against angiogenesis was tested in vitro and in vivo. The effect of ceramide in inhibiting MCF-7 cancer cells was also determined. The aim of this study was to potentiate the effect of ceramide as anti-angiogenic compound that can regulate tumor induced angiogenesis.C6-ceramide inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) tube formation in a dose-dependent manner within 24 hours. Ceramide at concentrations between 12.5 and 25 μM inhibited the viability of MCF-7 cells and reduced VEGF-induced cell migration in 24 hours. At 50 μM, ceramide induced MCF-7 cell death via autophagy as demonstrated by accumulation of MDC in ceramide-treated MCF-7 vacuoles. The expression of VEGF was reduced and the levels of cathepsin D in MCF-7 increased. In vivo, 50 μM ceramide caused a 40% reduction of new vessel formation in the CAM assay within 24 hours. Zebrafish exposed to 100 - 400 μM ceramide had a distinct disruption of blood vessel development at 48 hours post-fertilization. Ceramide-exposed embryos also had primary motoneurons exhibiting abnormal axonal trajectories and ectopic branching. Ceramide induced cell-death was not detected in the zebrafish assay. Collectively, these data indicate that ceramide is a potent anti-angiogenic compound and that the mechanism underlying its anti-angiogenic capabilities does not rely upon the induction of apoptosis.     

A subpopulation of mushroom body intrinsic neurons is generated by protocerebral neuroblasts in the tobacco hornworm moth, Manduca sexta (Sphingidae, Lepidoptera)

Subpopulations of Kenyon cells, the intrinsic neurons of the insect mushroom bodies, are typically sequentially generated by dedicated neuroblasts that begin proliferating during embryogenesis. When present, Class III Kenyon cells are thought to be the first born population of neurons by virtue of the location of their cell somata, farthest from the position of the mushroom body neuroblasts. In the adult tobacco hornworm moth Manduca sexta, the axons of Class III Kenyon cells form a separate Y tract and dorsal and ventral lobelet; surprisingly, these distinctive structures are absent from the larval Manduca mushroom bodies. BrdU labeling and immunohistochemical staining reveal that Class III Kenyon cells are in fact born in the mid-larval through adult stages. The peripheral position of their cell bodies is due to their genesis from two previously undescribed protocerebral neuroblasts distinct from the mushroom body neuroblasts that generate the other Kenyon cell types. These findings challenge the notion that all Kenyon cells are produced solely by the mushroom body neuroblasts, and may explain why Class III Kenyon cells are found sporadically across the insects, suggesting that when present, they may arise through de novo recruitment of neuroblasts outside of the mushroom bodies. In addition, lifelong neurogenesis by both the Class III neuroblasts and the mushroom body neuroblasts was observed, raising the possibility that adult neurogenesis may play a role in mushroom body function in Manduca.     

Uremic toxins inhibit transport by breast cancer resistance protein and multidrug resistance protein 4 at clinically relevant concentrations

During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [(3)H]-methotrexate ([(3)H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 μM, respectively) and BCRP-mediated [(3)H]-estrone sulfate ([(3)H]-E1S) uptake (Ki values: 4 mM, 500 μM and 50 μM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [(3)H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC(50) value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 μM, 4 μM, 129 μM, 1 μM and 18 μM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations.     

Novel analysis of oceanic surface water metagenomes suggests importance of polyphosphate metabolism in oligotrophic environments

Polyphosphate is a ubiquitous linear homopolymer of phosphate residues linked by high-energy bonds similar to those found in ATP. It has been associated with many processes including pathogenicity, DNA uptake and multiple stress responses across all domains. Bacteria have also been shown to use polyphosphate as a way to store phosphate when transferred from phosphate-limited to phosphate-rich media--a process exploited in wastewater treatment and other environmental contaminant remediation. Despite this, there has, to date, been little research into the role of polyphosphate in the survival of marine bacterioplankton in oligotrophic environments. The three main proteins involved in polyphosphate metabolism, Ppk1, Ppk2 and Ppx are multi-domain and have differential inter-domain and inter-gene conservation, making unbiased analysis of relative abundance in metagenomic datasets difficult. This paper describes the development of a novel Isofunctional Homolog Annotation Tool (IHAT) to detect homologs of genes with a broad range of conservation without bias of traditional expect-value cutoffs. IHAT analysis of the Global Ocean Sampling (GOS) dataset revealed that genes associated with polyphosphate metabolism are more abundant in environments where available phosphate is limited, suggesting an important role for polyphosphate metabolism in marine oligotrophs.     

International stem cell collaboration: how disparate policies between the United States and the United Kingdom impact research

As the scientific community globalizes, it is increasingly important to understand the effects of international collaboration on the quality and quantity of research produced. While it is generally assumed that international collaboration enhances the quality of research, this phenomenon is not well examined. Stem cell research is unique in that it is both politically charged and a research area that often generates international collaborations, making it an ideal case through which to examine international collaborations. Furthermore, with promising medical applications, the research area is dynamic and responsive to a globalizing science environment. Thus, studying international collaborations in stem cell research elucidates the role of existing international networks in promoting quality research, as well as the effects that disparate national policies might have on research. This study examined the impact of collaboration on publication significance in the United States and the United Kingdom, world leaders in stem cell research with disparate policies. We reviewed publications by US and UK authors from 2008, along with their citation rates and the political factors that may have contributed to the number of international collaborations. The data demonstrated that international collaborations significantly increased an article's impact for UK and US investigators. While this applied to UK authors whether they were corresponding or secondary, this effect was most significant for US authors who were corresponding authors. While the UK exhibited a higher proportion of international publications than the US, this difference was consistent with overall trends in international scientific collaboration. The findings suggested that national stem cell policy differences and regulatory mechanisms driving international stem cell research in the US and UK did not affect the frequency of international collaborations, or even the countries with which the US and UK most often collaborated. Geographical and traditional collaborative relationships were the predominate considerations in establishing international collaborations.     

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.     

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

Review and application of group theory to molecular systems biology

In this paper we provide a review of selected mathematical ideas that can help us better understand the boundary between living and non-living systems. We focus on group theory and abstract algebra applied to molecular systems biology. Throughout this paper we briefly describe possible open problems. In connection with the genetic code we propose that it may be possible to use perturbation theory to explore the adjacent possibilities in the 64-dimensional space-time manifold of the evolving genome. With regards to algebraic graph theory, there are several minor open problems we discuss. In relation to network dynamics and groupoid formalism we suggest that the network graph might not be the main focus for understanding the phenotype but rather the phase space of the network dynamics. We show a simple case of a C ₆ network and its phase space network. We envision that the molecular network of a cell is actually a complex network of hypercycles and feedback circuits that could be better represented in a higher-dimensional space. We conjecture that targeting nodes in the molecular network that have key roles in the phase space, as revealed by analysis of the automorphism decomposition, might be a better way to drug discovery and treatment of cancer.