Disease-specific induced pluripotent stem cells

Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.     

Ping Pong Dolls

This article examines ways in which music education advocacy efforts have become disconnected from the unified visions and declarations of music educators espoused in the Tanglewood and Housewright declarations and are thus reifying the disconnect between what we value and what we say we value. We first analyze the policies posited by the recently formed Music Education Policy Roundtable and consider several counterarguments. Second, we suggest new directions in music education advocacy by discussing ways to make our programs more culturally relevant and valuable to our schools and communities. Finally, we conclude with a call for our professional organization to take a leadership role in situating the arts as an important element of American public school education by reigniting national aims discussions that lead to liberal and humanistic education policies.     

Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer

Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC-N (gspC-N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5-751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC-N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse-chase analysis also suggested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild-type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.     

Direct Imaging of Cl‐ and Cu‐Induced Short‐Circuit Efficiency Changes in CdTe Solar Cells

To achieve high‐efficiency polycrystalline CdTe‐based thin‐film solar cells, the CdTe absorbers must go through a post‐deposition CdCl₂ heat treatment followed by a Cu diffusion step. To better understand the roles of each treatment with regard to improving grains, grain boundaries, and interfaces, CdTe solar cells with and without Cu diffusion and CdCl₂ heat treatments are investigated using cross‐sectional electron beam induced current, electron backscatter diffraction, and scanning transmission electron microscope techniques. The evolution of the cross‐sectional carrier collection profile due to these treatments that cause an increase in short‐circuit current and higher open‐circuit voltage are identified. Additionally, an increased carrier collection in grain boundaries after either/both of these treatments is revealed. The increased current at the grain boundaries is shown to be due to the presence of a space charge region with an intrinsic carrier collection profile width of ≈350 nm. Scanning transmission electron microscope electron‐energy loss spectroscopy shows a decreased Te and increased Cl concentration in grain boundaries after treatment, which causes the inversion. Each treatment improves the overall carrier collection efficiency of the cell separately, and, therefore, the benefits realized by each treatment are shown to be independent of each other.     

Microbial Biogeography of Six Salt Lakes in Inner Mongolia,China, and a Salt Lake in Argentina

We used cultivation-independent methods to investigate the prokaryotic biogeography of the water column in six salt lakes in Inner Mongolia, China, and a salt lake in Argentina. These lakes had different salt compositions and pH values and were at variable geographic distances, on both local and intercontinental scales, which allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance. Fourteen 16S rRNA gene clone libraries were constructed, and over 200 16S rRNA gene sequences were obtained. These sequences were used to construct biotic similarity matrices, which were used in combination with environmental similarity matrices and a distance matrix in the Mantel test to discover which factors significantly influenced biotic similarity. We showed that archaeal biogeography was influenced by contemporary environmental factors alone (Na⁺, CO₃²⁻, and HCO₃⁻ ion concentrations; pH; and temperature). Bacterial biogeography was influenced both by contemporary environmental factors (Na⁺, Mg²⁺, and HCO₃⁻ ion concentrations and pH) and by geographic distance.Biogeography aims to explain spatial patterns of diversity in the context of evolutionary events such as speciation, dispersal, extinction, and species interactions (42). Macroecologists have long studied the biogeography of higher plants and animals in various habitats (9, 13). In contrast, there is very little information available on the biogeography of prokaryotes. This stemmed from the difficulty of assessing microbial communities by cultivation methods, which only sampled 0.1 to 10% of the microbial community (30). However, with the advent of cultivation-independent sequencing techniques, microbial communities of many environments have been characterized, including soil (43), the Arctic and Antarctic Oceans (5), and the Sargasso Sea (61). This in turn facilitated prokaryotic biogeography studies in a number of environments on scales ranging from 20,000 km to 0.002 km (42).A study of the biogeography of soil bacteria across the Americas showed that differences were largely attributed to soil pH, with higher diversity observed in neutral soils (20). Bacterial communities in an estuary in Massachusetts were found to vary with the salinity gradient (14). Such studies demonstrated that environmental parameters influenced biogeographical patterns in microbial diversity. Further studies demonstrated that biogeography of hot spring cyanobacteria, hyperthermophilic archaea, and Pseudomonas strains was influenced by geographic distance, which led to isolation of disparate populations and subsequent genetic divergence (12, 51, 63). The apparent allopatric speciation demonstrated in these studies therefore contested the idea that prokaryotes were not affected by limits to dispersal due to their small size, abundance, and metabolic plasticity (i.e., “everything is everywhere” [see below]) (21).A simple framework was suggested to distinguish between the effects of evolutionary events and contemporary environmental conditions on the spatial variation of microbial diversity (42). At the center of this framework were four hypotheses. The null hypothesis stated that microorganisms were distributed randomly over space. Upon rejection of the null hypothesis, the second hypothesis stated that spatial variation reflected the influence of contemporary environmental variation. It assumed that geographic distance did not affect diversity due to the wide dispersal of microorganisms. This hypothesis represents the famously quoted “everything is everywhere; the milieu selects” by Baas-Becking (4, 6). The third hypothesis stated that variation was shaped by evolutionary events (geographic distance) that limited dispersal and that past environmental conditions led to genetic divergence between different microbial assemblages. The fourth hypothesis stated that the biogeography of microorganisms was determined by both contemporary environmental conditions and past evolutionary events (geographic distance). It is important to note here the possibility that evolutionary events can be represented by geographic distances. (For more details on this framework, see reference 42.)Many studies have been carried out on salt lakes and salterns around the world (28), but few have tried to explain variations in microbial community composition. Those that did identified salinity, altitude, redox and ionic concentration, pO₂, and seasonal events as relevant factors (7, 11, 16, 17, 34, 35, 38, 65). To our knowledge, only two studies have looked at the effect of intercontinental geographic distances on microbial community composition in salt lakes. Foti and colleagues looked specifically at the biogeography of Thioalkalivibrio in soda lakes across Mongolia, Kenya, California, Egypt, and Siberia and found that these bacteria showed a tendency for endemism; hence, geographic distance was a significant factor in influencing community composition (22). A further study looked at the biogeography of Salinibacter ruber strains from salterns in the Mediterranean, Atlantic, and Peruvian regions using a metabolomic approach. Geographically distinct strains were distinguished by characteristic metabolites (58).We examined the prokaryotic community composition in several salt lakes using ribosomal DNA methods. Six of the salt lakes in this study were situated on the Inner Mongolian steppe, northwest of Beijing, which had an average elevation of 1,000 to 2,000 m above sea level. The lakes were mostly several hundred kilometers apart (0.147 to 395.2 km) and were in different climate and vegetation zones: from typical grassland steppe in the north and east to desert steppe bordering the Gobi desert in the south and west (70). The lakes were Bagaejinnor, Chagannor, Ejinnor, Erliannor, Shangmatala, and an unnamed lake near Xilinhot. Lakes Ejinnor and Erliannor were extensively developed into salterns. Salar Guayatayoc Lake was situated in the same basin as the Salinas Grandes in the Argentine Altiplano at an elevation of 3,432 m, north-west of the city Salta, ∼18,000 km from the other lakes. All salt lakes were athalassohaline, located in arid climates, and subjected to high solar radiation and wide ranges of temperature. The lakes had different salt compositions and allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance.Here we describe the microbial diversity of six salt lakes in Inner Mongolia and one salt lake in Argentina. Using the framework previously described, we present evidence that biogeography of Archaea in these salt lakes was significantly influenced (P < 0.05) by environmental factors (Na⁺, CO₃²⁻, and HCO₃⁻ ion concentrations, pH, and temperature), but not geographic distance, consistent with the previously stated hypothesis 2. We also show that the biogeography of Bacteria was significantly influenced (P < 0.05) by both environmental factors (Na⁺, Mg²⁺, and HCO₃⁻ ion concentrations and pH) and geographic distance, consistent with the previously stated hypothesis 4.     

Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe–S cluster scaffold protein

Frataxin is an iron binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron-sulfur cluster and heme biosynthesis. There is a high degree of structural homology for frataxin proteins from diverse sources, and all possess an anionic surface defined by acidic residues. In the human protein these residues principally lie on a surface defined by the alpha1 helix and beta1 sheet and the impact of multiple substitutions of these carboxylate residues on iron binding is described. Full-length human frataxin has previously been shown to undergo self-cleavage to produce a truncated form both in vitro and in vivo. This truncated protein has been shown to bind approximately seven iron centers that are presumably associated with the acidic patch. Relative to this native protein, the stoichiometry decreases according to the number and sites of mutations. Nevertheless, the iron-dependent binding affinity of each frataxin derivative to the iron-sulfur cluster scaffold protein ISU is found to be similar to that of native frataxin, as defined by isothermal titration calorimetry experiments, requiring only one iron center to promote nanomolar binding. While frataxins from various cell types appear to bind differing numbers of iron centers, the physiologically relevant number of bound irons appears to be small, with significantly higher binding affinity following complex formation with partner proteins (micromolar compared with nanomolar binding). By contrast, in reconstitution assays for frataxin-promoted [2Fe-2S](2+) cluster assembly on ISU, one derivative does display a modestly lower reconstitution rate. The overall consensus from these data is to consider a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets.     

Social Aggregation in Pea Aphids: Experiment and Random Walk Modeling

From bird flocks to fish schools and ungulate herds to insect swarms, social biological aggregations are found across the natural world. An ongoing challenge in the mathematical modeling of aggregations is to strengthen the connection between models and biological data by quantifying the rules that individuals follow. We model aggregation of the pea aphid, Acyrthosiphon pisum. Specifically, we conduct experiments to track the motion of aphids walking in a featureless circular arena in order to deduce individual-level rules. We observe that each aphid transitions stochastically between a moving and a stationary state. Moving aphids follow a correlated random walk. The probabilities of motion state transitions, as well as the random walk parameters, depend strongly on distance to an aphid''s nearest neighbor. For large nearest neighbor distances, when an aphid is essentially isolated, its motion is ballistic with aphids moving faster, turning less, and being less likely to stop. In contrast, for short nearest neighbor distances, aphids move more slowly, turn more, and are more likely to become stationary; this behavior constitutes an aggregation mechanism. From the experimental data, we estimate the state transition probabilities and correlated random walk parameters as a function of nearest neighbor distance. With the individual-level model established, we assess whether it reproduces the macroscopic patterns of movement at the group level. To do so, we consider three distributions, namely distance to nearest neighbor, angle to nearest neighbor, and percentage of population moving at any given time. For each of these three distributions, we compare our experimental data to the output of numerical simulations of our nearest neighbor model, and of a control model in which aphids do not interact socially. Our stochastic, social nearest neighbor model reproduces salient features of the experimental data that are not captured by the control.     

Extracellular Vesicles Secreted from Cancer Cell Lines Stimulate Secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.     

Dehydroepiandrosterone stimulates phosphorylation of FoxO1 in vascular endothelial cells via phosphatidylinositol 3-kinase- and protein kinase A-dependent signaling pathways to regulate ET-1 synthesis and secretion

Dehydroepiandrosterone (DHEA) is an endogenous adrenal steroid hormone with controversial actions in humans. We previously reported that DHEA has opposing actions in endothelial cells to stimulate phosphatidylinositol (PI) 3-kinase/Akt/endothelial nitric-oxide synthase leading to increased production of nitric oxide while simultaneously stimulating MAPK-dependent secretion of the vasoconstrictor ET-1. In the present study we hypothesized that DHEA may stimulate PI 3-kinase-dependent phosphorylation of FoxO1 in endothelial cells to help regulate endothelial function. In bovine or human aortic endothelial cells (BAEC and HAEC), treatment with DHEA (100 nM) acutely enhanced phosphorylation of FoxO1. DHEA-stimulated phosphorylation of FoxO1 was inhibited by pretreatment of cells with wortmannin (PI 3-kinase inhibitor) or H89 (protein kinase A (PKA) inhibitor) but not ICI182780 (estrogen receptor blocker), or PD98059 (MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor). Small interfering RNA knockdown of PKA inhibited DHEA-stimulated phosphorylation of FoxO1. DHEA promoted nuclear exclusion of FoxO1 that was blocked by pretreatment of cells with wortmannin, H89, or by small interfering RNA knockdown of PKA. DHEA treatment of endothelial cells increased PKA activity and intracellular cAMP concentrations. Transfection of BAEC with a constitutively nuclear FoxO1 mutant transactivated a co-transfected ET-1 promoter luciferase reporter. Treatment of BAEC with DHEA inhibited transactivation of the ET-1 promoter reporter in cells overexpressing FoxO1. ET-1 promoter activity and secretion in response to DHEA treatment was augmented by PI 3-kinase blockade and inhibited by MAPK blockade. We conclude that DHEA stimulates phosphorylation of FoxO1 via PI 3-kinase- and PKA-dependent pathways in endothelial cells that negatively regulates ET-1 promoter activity and secretion. Balance between PI 3-kinase-dependent inhibition and MAPK-dependent stimulation of ET-1 secretion in response to DHEA may determine whether DHEA supplementation improves or worsens cardiovascular and metabolic function.     

Using Lamm-Equation modeling of sedimentation velocity data to determine the kinetic and thermodynamic properties of macromolecular interactions

The interaction of macromolecules with themselves and with other macromolecules is fundamental to the functioning of living systems. Recent advances in the analysis of sedimentation velocity (SV) data obtained by analytical ultracentrifugation allow the experimenter to determine important features of such interactions, including the equilibrium association constant and information about the kinetic off-rate of the interaction. The determination of these parameters is made possible by the ability of modern software to fit numerical solutions of the Lamm Equation with kinetic considerations directly to SV data. Herein, the SV analytical advances implemented in the software package SEDPHAT are summarized. Detailed analyses of SV data using these strategies are presented. Finally, a few highlights of recent literature reports that feature this type of SV data analysis are surveyed.