MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo

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Beta-Amyloid Toxicity in Embryonic Rat Astrocytes

The senile plaques of Alzheimer’s disease contain a high concentration of beta-amyloid (βA) protein, which may affect the glial population in the septal nucleus, an area of increased risk in AD. βA toxicity was measured in septal glia, via a dose-response experiment, by quantifying the effects of three different doses (0.1, 1, and 10 μM) of βA on cell survival. Astrocytes from embryonic day-16 rats were grown in serum-free media in a single layer culture. Cells were treated on day in vitro (DIV)1 and survival was determined on DIV3 to ascertain which concentration was most toxic. In a separate set of experiments, an attempt was made to protect glial cells from the degenerative effects of βA, with treatments of growth factors and estrogen. βA (10 μM) treatment was administered on DIV1, on DIV2 the cells were treated with estrogen (EST, 10 nM), insulin-like growth factors (IGF1 and IGF2, each 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml) or nerve growth factor (NGF, 100 ng/ml), and on DIV3 the cells were visualized and quantified by fluorescence microscopy with DAPI (4,6-diamidino-2-phenylindole). In addition to dose-response and glial protection, experiments were also conducted to determine whether toxic effects were due to apoptosis. Our results suggest that the survival of glial populations is significantly affected in all three concentrations (0.1, 1.0, and 10 μM) of βA. Glial protection was evident in the presence of NGF, for it showed the significantly highest survival rate relative to the βA treatment alone. Furthermore, toxic effects of βA appear to be due primarily to apoptosis. Significant reversal of βA-induced apoptosis was seen with bFGF and IGF1.     

Liposome-Antigen-Nucleic Acid Complexes Protect Mice from Lethal Challenge with Western and Eastern Equine Encephalitis Viruses

Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine.     

Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.     

Nucleotide binding and dimerization at the chloroplast pre‐protein import receptor,atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.     

Replication of long-bone length QTL in the F9-F10 LG,SM advanced intercross

Quantitative trait locus (QTL) mapping techniques are frequently used to identify genomic regions associated with variation in phenotypes of interest. However, the F₂ intercross and congenic strain populations usually employed have limited genetic resolution resulting in relatively large confidence intervals that greatly inhibit functional confirmation of statistical results. Here we use the increased resolution of the combined F₉ and F₁₀ generations (n = 1455) of the LG,SM advanced intercross to fine-map previously identified QTL associated with the lengths of the humerus, ulna, femur, and tibia. We detected 81 QTL affecting long-bone lengths. Of these, 49 were previously identified in the combined F₂-F₃ population of this intercross, while 32 represent novel contributors to trait variance. Pleiotropy analysis suggests that most QTL affect three to four long bones or serially homologous limb segments. We also identified 72 epistatic interactions involving 38 QTL and 88 novel regions. This analysis shows that using later generations of an advanced intercross greatly facilitates fine-mapping of confidence intervals, resolving three F₂-F₃ QTL into multiple linked loci and narrowing confidence intervals of other loci, as well as allowing identification of additional QTL. Further characterization of the biological bases of these QTL will help provide a better understanding of the genetics of small variations in long-bone length. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Impact of Silver(I) on the Metabolism of Shewanella oneidensis

Anaerobic cultures of Shewanella oneidensis MR-1 reduced toxic Ag(I), forming nanoparticles of elemental Ag(0), as confirmed by X-ray diffraction analyses. The addition of 1 to 50 μM Ag(I) had a limited impact on growth, while 100 μM Ag(I) reduced both the doubling time and cell yields. At this higher Ag(I) concentration transmission electron microscopy showed the accumulation of elemental silver particles within the cell, while at lower concentrations the metal was exclusively reduced and precipitated outside the cell wall. Whole organism metabolite fingerprinting, using the method of Fourier transform infrared spectroscopy analysis of cells grown in a range of silver concentrations, confirmed that there were significant physiological changes at 100 μM silver. Principal component-discriminant function analysis scores and loading plots highlighted changes in certain functional groups, notably, lipids, amides I and II, and nucleic acids, as being discriminatory. Molecular analyses confirmed a dramatic drop in cellular yields of both the phospholipid fatty acids and their precursor molecules at high concentrations of silver, suggesting that the structural integrity of the cellular membrane was compromised at high silver concentrations, which was a result of intracellular accumulation of the toxic metal.Silver is an element that has been used widely in industrial processes as diverse as photographic processing, catalysis, mirror production, electroplating, alkaline battery production, and jewelry making (18). It has been known for some time that silver ions and silver-based compounds can be highly toxic to microorganisms, and with increasing concern about pathogenic “superbugs” with high resistance to conventional antibiotics, silver is attracting much interest as a potential biocide (11, 18, 36, 42). Silver has no known physiological functions and can exist in several oxidation states, although it is most commonly encountered in its elemental [Ag(0)] and monovalent [Ag(I)] forms. Although use of nanoscale elemental Ag(0) as a biocide has been increasing, for example, in wound dressings and as an antimicrobial coating on consumer products, little is known about its mode of toxicity. This is despite the surprising ability of actively growing Fe(III)-reducing bacteria such as Geobacter sulfurreducens to precipitate nanoscale Ag(0) particles within and around the cell surface via reduction of Ag(I) (18). Ionic Ag(I), in contrast, has been the focus of more studies on the mode of metal toxicity. Previous research showed that silver ions have antimicrobial activities against a wide diversity of bacteria (19). They have been shown to disrupt the respiratory chain of Escherichia coli (3) and inhibit the exchange of phosphate and its uptake (34). Ag(I) has also been linked to copper metabolism in E. coli, potentially competing with copper binding sites on the cell surface and subsequent copper transport into the cell (8). However, the toxicity of silver is not limited to prokaryotes, as long-term exposure in humans can cause argyria, impaired night vision, and abdominal pain (31, 32, 36). The detailed mechanism of toxicity in prokaryotes or eukaryotes remains to be identified, although it has been proposed that silver ions react with cellular proteins via SH groups (16), leading to the disruption of cellular metabolism.Microbial cells have evolved an extremely diverse range of mechanisms to survive high concentrations of toxic metals. The mechanisms invoked include biosorption, bioaccumulation, special efflux systems, alteration of solubility and toxicity via reduction or oxidation, extracellular complexation or precipitation of metals, and lack of specific metal transport systems (1). For example, for silver ions the bacterial cell wall can be an efficient permeability barrier to block the uptake of metal (21), with additional complexation in the periplasm by specific silver-binding proteins (35). Redox transformations also offer the potential to detoxify Ag(I) ions, e.g., through the reduction to insoluble elemental Ag(0) (30). In addition, the energy-dependent efflux of toxic Ag(I) is perhaps the best-studied resistance mechanism for silver, mediated via ATPases and chemiosmotic cation/protons antiporters (9).Shewanella spp., Gram-negative, dissimilatory metal-reducing bacteria, can use a wide variety of terminal electron acceptors for growth (23, 39), including high oxidation state metals such as Fe(III), Mn(IV), Cr(VI), U(VI), and Au(III) (5, 17, 26, 28, 41). Shewanella species also have the potential to reduce Ag(I), given their similar activities against Au(III), and the reduction of Ag(I) to form nanoscale deposits of Ag(0) within the cell has been documented for other Fe(III)-reducing bacteria (18). This metabolic versatility offers considerable potential for bioremediation applications, for example, via reduction of U(VI) to insoluble U(IV) (5, 17, 26, 28, 41), and the recovery of precious metals such as silver and gold via reductive precipitation. It also offers an interesting model organism to study the metabolism of toxic metals such as silver, including the physiological impact of ionic Ag(I) and nanoscale Ag(0).This paper describes interactions of Shewanella oneidensis MR-1 with various concentrations of Ag(I), including demonstrations of the reduction and deposition of silver nanoparticles under anaerobic conditions. A range of techniques, including X-ray diffraction (XRD) and analytical transmission electron microscopy (TEM), were used to investigate the nature and cellular localization of the precipitates, while Fourier transform infrared (FT-IR) spectroscopy metabolic profiling techniques were used to identify the impact of toxic metal accumulation on the cell. The disruption of membrane integrity was implied by these investigations and confirmed by fatty acid methyl ester (FAME) analysis, which showed a dramatic decrease in the quantities of membrane lipid components.     

Disentangling stocking introgression and natural migration in brown trout: survival success and recruitment failure in populations with semi‐supportive breeding

1. Introgression into natural salmonid populations from stocked conspecifics has been widely studied. Outcomes vary from no effect even after decades of stocking, to population replacement after only a couple of generations. Potential introgression caused by semi‐supportive breeding (i.e. using a mixture of local strains as brood stock) is, however, less well studied. 2. We investigated population structure of brown trout (Salmo trutta) in a regulated alpine lake with three natural, environmentally contrasting tributaries used as spawning and rearing habitat. Massive semi‐supportive breeding of admixed local strains has been implemented for decades. Stocked trout represented c. 17% of the total lake population, and a substantial post‐release survival reflects a considerable potential for introgression. However, the mark‐recapture studies indicate no spawning runs of stocked fish. 3. Using 13 polymorphic microsatellite loci, we found natural straying and non‐native reproduction, especially among wild populations inhabiting environmentally unstable habitat. Retained genetic structure across tributaries indicated low reproductive success of wild‐born non‐natives. Moreover, the genetic structure among tributaries has probably not been influenced by semi‐supportive breeding, because of recruitment failure of stocked trout.     

DNA damage in zebrafish larvae induced by exposure to low-dose rate gamma-radiation: detection by the alkaline comet assay

This study has determined the sensitivity of the alkaline comet assay for the detection of strand breaks in the DNA of cells taken from a whole organism rather than a single cell type as in previously reported studies. The assay has been performed on cells from whole zebrafish larvae irradiated for 1 or 24 h at dose rates of 0.4, 1.2 or 7.2 mGy/h. Zebrafish larvae exposed to only 1.2 mGy/h of gamma-radiation for 1h showed a statistically significant increase in DNA damage compared to controls. This represents a high sensitivity of this animal model for DNA damage and of the comet assay protocol used for detecting such damage. Increasing the exposure time from 1 to 24 h caused significant increases in DNA damage in zebrafish larvae, although the modest size of these increases in damage for the relatively large increases (24 times) in total absorbed dose indicates that dose rate may be the major factor in determining the level of DNA damage observed under the conditions of these experiments.     

Decreased brain volume in adults with childhood lead exposure

Background

Although environmental lead exposure is associated with significant deficits in cognition, executive functions, social behaviors, and motor abilities, the neuroanatomical basis for these impairments remains poorly understood. In this study, we examined the relationship between childhood lead exposure and adult brain volume using magnetic resonance imaging (MRI). We also explored how volume changes correlate with historic neuropsychological assessments.

Methods and Findings

Volumetric analyses of whole brain MRI data revealed significant decreases in brain volume associated with childhood blood lead concentrations. Using conservative, minimum contiguous cluster size and statistical criteria (700 voxels, unadjusted p < 0.001), approximately 1.2% of the total gray matter was significantly and inversely associated with mean childhood blood lead concentration. The most affected regions included frontal gray matter, specifically the anterior cingulate cortex (ACC). Areas of lead-associated gray matter volume loss were much larger and more significant in men than women. We found that fine motor factor scores positively correlated with gray matter volume in the cerebellar hemispheres; adding blood lead concentrations as a variable to the model attenuated this correlation.

Conclusions

Childhood lead exposure is associated with region-specific reductions in adult gray matter volume. Affected regions include the portions of the prefrontal cortex and ACC responsible for executive functions, mood regulation, and decision-making. These neuroanatomical findings were more pronounced for males, suggesting that lead-related atrophic changes have a disparate impact across sexes. This analysis suggests that adverse cognitive and behavioral outcomes may be related to lead''s effect on brain development producing persistent alterations in structure. Using a simple model, we found that blood lead concentration mediates brain volume and fine motor function.