The early life history of two sympatric New Zealand octopuses: eggs and paralarvae of Octopus huttoni and Pinnoctopus cordiformis

This study combined morphological and morphometric information on egg clutches, egg capsules and paralarvae of two sympatric coastal octopuses from New Zealand waters, Octopus huttoni and Pinnoctopus cordiformis, to provide species-specific traits to identify their early life stages obtained from field surveys. Eggs of O. huttoni (2.5 mm length; 1 mm width) were entwined with one another forming strings that ranged from 11 to 25.8 mm in length. Eggs of P. cordiformis (6.4 mm length; 1.5 mm width) were significantly bigger than those of O. huttoni and were grouped in small clusters of about seven eggs. Paralarvae O. huttoni and P. cordiformis differed in hatching size (1.4 mm versus 3.1 mm mantle length), number of suckers per arm (four versus eight), number of lamellae per outer demibranch (five versus ten) and arrangements of chromatophores in the body surface (29 to 59 versus 91 to 179), respectively. The morphological traits described in hatchlings from the laboratory allowed comparisons with field-collected paralarvae, suggesting that such characters were reliable species-specific patterns to enable a consistent differentiation between the early life stages of these two sympatric species, even in the absence of the brooding female.     

Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with ¹⁴C-thymidine and the newly synthesized strands - with bromodeoxyu ridine and ³H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15–20% newly replica ted DNA Analysis of the distribution of ¹⁴C-labeled parental DNA fragments complementary to the ³H-nascent strand has shown that the ¹⁴C-labeled fragments prevail in the region of 30–50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5′-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems tobe induced by interactions in the chromatin.     

Polymer gene delivery vectors encapsulated in thermally sensitive bioreducible shell

Stable, nanosized polyelectrolyte complexes between rationally designed thermally sensitive block copolymers and plasmid DNA (polyplexes) were formed and their in vitro transfection efficiency was tested. The polyplexes were further stabilized through encapsulation into a biodegradable polymer shell. Although reduced as compared to that of the corresponding polyplexes, the encapsulated systems still show acceptable transfection efficiency. That opens the possibility to tune the balance between the safe transport and efficient delivery of DNA into the cells.     

Design,synthesis and structure–activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.     

Suboptimal growth temperatures enhance the biological activity of cultured cyanobacterium Gloeocapsa sp.

The cytotoxic, antibacterial, and antifungal activities of cyanobacterium Gloeocapsa sp. strain Gacheva 2007/R-06/1 were investigated and the possibility for an enhancement of these activities by changing the culture conditions evaluated. Fatty acids of this cyanobacterium were found to be active against Streptococcus pyogenes. Exopolysaccharides inhibited the growth of both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. Both exopolysaccharides and fatty acid mixtures also significantly decreased the viability of human cervical carcinoma cells, HeLa. Greater biological activities were exhibited by Gloeocapsa sp., cultured at suboptimal temperatures (15–26°C) than at optimal and supraoptimal ones. In comparison with higher light intensity, the low-light cultivation stimulated the cytotoxicity of the fatty acids. In general, low temperatures decreased the growth of Gloeocapsa sp., but promoted its biological activity. Prolonged cultivation also had a beneficial impact on the bioactivity. Compared to 4 days, the 17-day cultivation resulted in fourfold higher antibacterial and antifungal activities of exopolysaccharides and more than twice increases in their cytotoxicity. The study revealed that this cyanobacterial isolate is a new source of natural products with potential for pharmacological and medical applications.     

Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

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rAAV-Mediated Subcellular Targeting of Optogenetic Tools in Retinal Ganglion Cells In Vivo

Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration. One potential approach for restoring retinal light sensitivity after photoreceptor degeneration is to express optogenetic tools in retinal ganglion cells (RGCs). For this approach, restoration of ON and OFF center-surround receptive fields in RGCs, a key feature of visual information processing, may be important. A possible solution is to differentially express depolarizing and hyperpolarizing optogenetic tools, such as channelrhodopsin-2 and halorhodopsin, to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs. Recombinant adeno-associated virus (rAAV) vectors have proven to be a powerful vehicle for in vitro and in vivo gene delivery, including in the retina. Therefore, the search for protein targeting motifs that can achieve rAAV-mediated subcellular targeted expression would be particularly valuable for developing therapeutic applications. In this study, we identified two protein motifs that are suitable for rAAV-mediated subcellular targeting for generating center-surround receptive fields while reducing the axonal expression in RGCs. Resulting morphological dendritic field and physiological response field by center-targeting were significantly smaller than those produced by surround-targeting. rAAV motif-mediated protein targeting could also be a valuable tool for studying physiological function and clinical applications in other areas of the central nervous system.     

ProDeGe: a computational protocol for fully automated decontamination of genomes

Single amplified genomes and genomes assembled from metagenomes have enabled the exploration of uncultured microorganisms at an unprecedented scale. However, both these types of products are plagued by contamination. Since these genomes are now being generated in a high-throughput manner and sequences from them are propagating into public databases to drive novel scientific discoveries, rigorous quality controls and decontamination protocols are urgently needed. Here, we present ProDeGe (Protocol for fully automated Decontamination of Genomes), the first computational protocol for fully automated decontamination of draft genomes. ProDeGe classifies sequences into two classes—clean and contaminant—using a combination of homology and feature-based methodologies. On average, 84% of sequence from the non-target organism is removed from the data set (specificity) and 84% of the sequence from the target organism is retained (sensitivity). The procedure operates successfully at a rate of ~0.30 CPU core hours per megabase of sequence and can be applied to any type of genome sequence.Recent technological advancements have enabled the large-scale sampling of genomes from uncultured microbial taxa, through the high-throughput sequencing of single amplified genomes (SAGs; Rinke et al., 2013; Swan et al., 2013) and assembly and binning of genomes from metagenomes (GMGs; Cuvelier et al., 2010; Sharon and Banfield, 2013). The importance of these products in assessing community structure and function has been established beyond doubt (Kalisky and Quake, 2011). Multiple Displacement Amplification (MDA) and sequencing of single cells has been immensely successful in capturing rare and novel phyla, generating valuable references for phylogenetic anchoring. However, efforts to conduct MDA and sequencing in a high-throughput manner have been heavily impaired by contamination from DNA introduced by the environmental sample, as well as introduced during the MDA or sequencing process (Woyke et al., 2011; Engel et al., 2014; Field et al., 2014). Similarly, metagenome binning and assembly often carries various errors and artifacts depending on the methods used (Nielsen et al., 2014). Even cultured isolate genomes have been shown to lack immunity to contamination with other species (Parks et al., 2014; Mukherjee et al., 2015). As sequencing of these genome product types rapidly increases, contaminant sequences are finding their way into public databases as reference sequences. It is therefore extremely important to define standardized and automated protocols for quality control and decontamination, which would go a long way towards establishing quality standards for all microbial genome product types.Current procedures for decontamination and quality control of genome sequences in single cells and metagenome bins are heavily manual and can consume hours/megabase when performed by expert biologists. Supervised decontamination typically involves homology-based inspection of ribosomal RNA sequences and protein coding genes, as well as visual analysis of k-mer frequency plots and guanine–cytosine content (Clingenpeel, 2015). Manual decontamination is also possible through the software SmashCell (Harrington et al., 2010), which contains a tool for visual identification of contaminants from a self-organizing map and corresponding U-matrix. Another existing software tool, DeconSeq (Schmieder and Edwards, 2011), automatically removes contaminant sequences, however, the contaminant databases are required input. The former lacks automation, whereas the latter requires prior knowledge of contaminants, rendering both applications impractical for high-throughput decontamination.Here, we introduce ProDeGe, the first fully automated computational protocol for decontamination of genomes. ProDeGe uses a combination of homology-based and sequence composition-based approaches to separate contaminant sequences from the target genome draft. It has been pre-calibrated to discard at least 84% of the contaminant sequence, which results in retention of a median 84% of the target sequence. The standalone software is freely available at http://prodege.jgi-psf.org//downloads/src and can be run on any system that has Perl, R (R Core Team, 2014), Prodigal (Hyatt et al., 2010) and NCBI Blast (Camacho et al., 2009) installed. A graphical viewer allowing further exploration of data sets and exporting of contigs accompanies the web application for ProDeGe at http://prodege.jgi-psf.org, which is open to the wider scientific community as a decontamination service (Supplementary Figure S1).The assembly and corresponding NCBI taxonomy of the data set to be decontaminated are required inputs to ProDeGe (Figure 1a). Contigs are annotated with genes following which, eukaryotic contamination is removed based on homology of genes at the nucleotide level using the eukaryotic subset of NCBI''s Nucleotide database as the reference. For detecting prokaryotic contamination, a curated database of reference contigs from the set of high-quality genomes within the Integrated Microbial Genomes (IMG; Markowitz et al., 2014) system is used as the reference. This ensures that errors in public reference databases due to poor quality of sequencing, assembly and annotation do not negatively impact the decontamination process. Contigs determined as belonging to the target organism based on nucleotide level homology to sequences in the above database are defined as ‘Clean'', whereas those aligned to other organisms are defined as ‘Contaminant''. Contigs whose origin cannot be determined based on alignment are classified as ‘Undecided''. Classified clean and contaminated contigs are used to calibrate the separation in the subsequent 5-mer based binning module, which classifies undecided contigs as ‘Clean'' or ‘Contaminant'' using principal components analysis (PCA) of 5-mer frequencies. This parameter can also be specified by the user. When data sets do not have taxonomy deeper than phylum level, or a single confident taxonomic bin cannot be detected using sequence alignment, solely 9-mer based binning is used due to more accurate overall classification. In the absence of a user-defined cutoff, a pre-calibrated cutoff for 80% or more specificity separates the clean contigs from contaminated sequences in the resulting PCA of the 9-mer frequency matrix. Details on ProDeGe''s custom database, evaluation of the performance of the system and exploration of the parameter space to calibrate ProDeGe for a high accurate classification rate are provided in the Supplementary Material.Open in a separate windowFigure 1(a) Schematic overview of the ProDeGe engine. (b) Features of data sets used to validate ProDeGe: SAGs from the Arabidopsis endophyte sequencing project, MDM project, public data sets found in IMG but not sequenced at the JGI, as well as genomes from metagenomes. All the data and results can be found in Supplementary Table S3.The performance of ProDeGe was evaluated using 182 manually screened SAGs (Figure 1b,Supplementary Table S1) from two studies whose data sets are publicly available within the IMG system: genomes of 107 SAGs from an Arabidopsis endophyte sequencing project and 75 SAGs from the Microbial Dark Matter (MDM) project* (only 75/201 SAGs from the MDM project had 1:1 mapping between contigs in the unscreened and the manually screened versions, hence these were used; Rinke et al., 2013). Manual curation of these SAGs demonstrated that the use of ProDeGe prevented 5311 potentially contaminated contigs in these data sets from entering public databases. Figure 2a demonstrates the sensitivity vs specificity plot of ProDeGe results for the above data sets. Most of the data points in Figure 2a cluster in the top right of the box reflecting a median retention of 89% of the clean sequence (sensitivity) and a median rejection of 100% of the sequence of contaminant origin (specificity). In addition, on average, 84% of the bases of a data set are accurately classified. ProDeGe performs best when the target organism has sequenced homologs at the class level or deeper in its high-quality prokaryotic nucleotide reference database. If the target organism''s taxonomy is unknown or not deeper than domain level, or there are few contigs with taxonomic assignments, a target bin cannot be assessed and thus ProDeGe removes contaminant contigs using sequence composition only. The few samples in Figure 2a that demonstrate a higher rate of false positives (lower specificity) and/or reduced sensitivity typically occur when the data set contains few contaminant contigs or ProDeGe incorrectly assumes that the largest bin is the target bin. Some data sets contain a higher proportion of contamination than target sequence and ProDeGe''s performance can suffer under this condition. However, under all other conditions, ProDeGe demonstrates high speed, specificity and sensitivity (Figure 2). In addition, ProDeGe demonstrates better performance in overall classification when nucleotides are considered than when contigs are considered, illustrating that longer contigs are more accurately classified (Supplementary Table S1).Open in a separate windowFigure 2ProDeGe accuracy and performance scatterplots of 182 manually curated single amplified genomes (SAGs), where each symbol represents one SAG data set. (a) Accuracy shown by sensitivity (proportion of bases confirmed ‘Clean'') vs specificity (proportion of bases confirmed ‘Contaminant'') from the Endophyte and Microbial Dark Matter (MDM) data sets. Symbol size reflects input data set size in megabases. Most points cluster in the top right of the plot, showing ProDeGe''s high accuracy. Median and average overall results are shown in Supplementary Table S1. (b) ProDeGe completion time in central processing unit (CPU) core hours for the 182 SAGs. ProDeGe operates successfully at an average rate of 0.30 CPU core hours per megabase of sequence. Principal components analysis (PCA) of a 9-mer frequency matrix costs more computationally than PCA of a 5-mer frequency matrix used with blast-binning. The lack of known taxonomy for the MDM data sets prevents blast-binning, thus showing longer finishing times than the endophyte data sets, which have known taxonomy for use in blast-binning.All SAGs used in the evaluation of ProDeGe were assembled using SPAdes (Bankevich et al., 2012). In-house testing has shown that reads assembled with SPAdes from different strains or even slightly divergent species of the same genera may be combined into the same contig (Personal communications, KT and Robert Bowers). Ideally, the DNA in a well that gets sequenced belongs to a single cell. In the best case, contaminant sequences need to be at least from a different species to be recognized as such by the homology-based screening stage. In the absence of closely related sequenced organisms, contaminant sequences need to be at least from a different genus to be recognized as such by the composition-based screening stage (Supplementary Material). Thus, there is little risk of ProDeGe separating sequences from clonal populations or strains. We have found species- and genus-level contamination in MDA samples to be rare.To evaluate the quality of publicly available uncultured genomes, ProDeGe was used to screen 185 SAGs and 14 GMGs (Figure 1b). Compared with CheckM (Parks et al., 2014), a tool which calculates an estimate of genome sequence contamination using marker genes, ProDeGe generally marks a higher proportion of sequence as ‘Contaminant'' (Supplementary Table S2). This is because ProDeGe has been calibrated to perform at high specificity levels. The command line version of ProDeGe allows users to conduct their own calibration and specify a user-defined distance cutoff. Further, CheckM only outputs the proportion of contamination, but ProDeGe actually labels each contig as ‘Clean'' or ‘Contaminant'' during the process of automated removal.The web application for ProDeGe allows users to export clean and contaminant contigs, examine contig gene calls with their corresponding taxonomies, and discover contig clusters in the first three components of their k-dimensional space. Non-linear approaches for dimensionality reduction of k-mer vectors are gaining popularity (van der Maaten and Hinton, 2008), but we observed no systematic advantage of using t-Distributed Stochastic Neighbor Embedding over PCA (Supplementary Figure S2).ProDeGe is the first step towards establishing a standard for quality control of genomes from both cultured and uncultured microorganisms. It is valuable for preventing the dissemination of contaminated sequence data into public databases, avoiding resulting misleading analyses. The fully automated nature of the pipeline relieves scientists of hours of manual screening, producing reliably clean data sets and enabling the high-throughput screening of data sets for the first time. ProDeGe, therefore, represents a critical component in our toolkit during an era of next-generation DNA sequencing and cultivation-independent microbial genomics.     

The effect of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> red pigment on the expression of cloned human α-synuclein

Two transgenic yeast strains expressing human α-synuclein were used to study the impact of yeast red pigment exhibiting antiamyloid properties. It has been demonstrated that the endogenous red pigment produced under special conditions in strains carrying an ade1 mutation inhibits the expression of the hybrid protein α-synuclein-GFP. This was evident from the reduced mean value of GFP fluorescence and diminished number of cells accumulating cytoplasmic inclusions of α-synuclein-GFP. Exogenous forms of the purified red pigment (natural, synthetic and hydrolyzed derivatives) differ from the endogenous red pigment by their effect on α-synuclein. Exogenous red pigments increased the number of both cells expressing GFP fluorescence and those containing cytoplasmic inclusions. However, both endogenous and exogenous red pigments reduced the cloned α-synuclein toxicity and resulted in redistribution of the α-synuclein in cells. α-Synuclein content decreased in cell lysate pellets and increased in supernatants.     

Detection of microwave radiation of cytochrome CYP102 A1 solution during the enzyme reaction

Microwave radiation at 3.4–4.2 GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10^?8 and 10^?9 М. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.