Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.     

Core histones H2B and H4 are mobilized during infection with herpes simplex virus 1

The infecting genomes of herpes simplex virus 1 (HSV-1) are assembled into unstable nucleosomes soon after nuclear entry. The source of the histones that bind to these genomes has yet to be addressed. However, infection inhibits histone synthesis. The histones that bind to HSV-1 genomes are therefore most likely those previously bound in cellular chromatin. In order for preexisting cellular histones to associate with HSV-1 genomes, however, they must first disassociate from cellular chromatin. Consistently, we have shown that linker histones are mobilized during HSV-1 infection. Chromatinization of HSV-1 genomes would also require the association of core histones. We therefore evaluated the mobility of the core histones H2B and H4 as measures of the mobilization of H2A-H2B dimers and the more stable H3-H4 core tetramer. H2B and H4 were mobilized during infection. Their mobilization increased the levels of H2B and H4 in the free pools and decreased the rate of H2B fast chromatin exchange. The histones in the free pools would then be available to bind to HSV-1 genomes. The mobilization of H2B occurred independently from HSV-1 protein expression or DNA replication although expression of HSV-1 immediate-early (IE) or early (E) proteins enhanced it. The mobilization of core histones H2B and H4 supports a model in which the histones that associate with HSV-1 genomes are those that were previously bound in cellular chromatin. Moreover, this mobilization is consistent with the assembly of H2A-H2B and H3-H4 dimers into unstable nucleosomes with HSV-1 genomes.     

Invertebrate DNA metabarcoding reveals changes in communities across mine site restoration chronosequences

Invertebrate biomonitoring can reveal crucial information about the status of restoration projects; however, it is routinely underused because of the high level of taxonomic expertise and resources required. Invertebrate DNA metabarcoding has been used to characterize invertebrate biodiversity but its application in restoration remains untested. We use DNA metabarcoding, a new approach for restoration assessment, to explore the invertebrate composition from pitfall traps at two mine site restoration chronosequences in southwestern Australia. Invertebrates were profiled using two cytochrome oxidase subunit 1 assays to investigate invertebrate biodiversity. The data revealed differences between invertebrate communities at the two mines and between the different age plots of the chronosequences. Several characteristic taxa were identified for each age within the chronosequence, including springtails within the youngest sites (Order: Collembola) and millipedes within the oldest and reference sites (Order: Julida). This study facilitates development of a molecular “toolkit” for the monitoring of ecological restoration projects. We suggest that a metabarcoding approach shows promise in complementing current monitoring practices that rely on alpha taxonomy.     

Wide range of viral load in healthy african green monkeys naturally infected with simian immunodeficiency virus

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (Chlorocebus pygerythrus) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.     

Methylene amine substituted arylindenopyrimidines as potent adenosine A2A/A1 antagonists

A novel series of arylindenopyrimidines were identified as A_2A and A₁ receptor antagonists. The series was optimized for in vitro activity by substituting the 8- and 9-positions with methylene amine substituents. The compounds show excellent activity in mouse models of Parkinson’s disease when dosed orally.     

MWC allosteric model explains unusual hemoglobin-oxygen binding curves from sickle cell drug binding

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.