Deeplasmid: deep learning accurately separates plasmids from bacterial chromosomes

Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid''s ability to detect novel plasmids.     

Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells

Background

Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions.

Methodology/Principal Findings

Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls.

Conclusions/Significance

Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted “cardiosomes”, can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.     

Calcium and pancreatic beta-cell function. The mechanism of insulin secretion studied with the aid of lanthanum.

La3+ was used to study the involvement of Ca2+ in insulin secretion in beta-cell-rich pancreatic islets micro-dissected from non-inbred ob/ob mice. Ultrastructural studies revealed that the localization of La3+ was entirely restricted to the exterior of the cells. Consistent with a membrane action, exposure to La3+ failed to affect glucose oxidation and either the sucrose space or the general ultrastructure of the islets. In contrast, La3+ had marked effects on insulin release and 45Ca fluxes. Exposure to La3+ resulted in pronounced inhibition of insulin release irrespective of the presence or absence of Ca2+, 3-isobutyl-1-methylxanthine or glucose. Perifusion experiments revealed that the inhibitory action was prompt, sustained and readily reversible. Removal of La3+ was associated with a subsequent prolonged stimulatory phase of insulin release even in medium deficient in Ca2+. This action could not be attributed to an increase in cyclic AMP, but was potentiated by 3-isobutyl-1-methylxanthine and abolished by L-adrenaline. La3+ displaced 45Ca from superficially located binding sites and inhibited the uptake and efflux of 45Ca. The stimulatory and inhibitory actions of glucose on 45Ca efflux were also abolished in the presence of 2 mM-La3+ Removal of La3+ was associated with the preferential mobilization of 45Ca incorporated in response to glucose. The results indicate that binding of La3+ to superficial sites in the plasma membrane leads to inhibition of insulin release by suppression of transmembrane Ca2+ fluxes. It is suggested that accumulation of Ca2+ in the cytoplasm accounts for the stimulation of insulin release seen after removal of La3+ from inhibitory binding sites in the beta-cell plasma membrane.     

Rate of incorporation of [3H]thymidine in various tissues of the mouse. A basis for the evaluation of genotoxic effects of chemicals

[3H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible. The present paper deals with some practical aspects on the use of [3H]thymidine in vivo. [6-3H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and DNA-associated radioactivities. A rapid increase of the [3H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities associated with DNA closely correlated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [3H]thymidine incorporation into DNA.     

Two trypanosome-specific proteins are essential factors for 5S rRNA abundance and ribosomal assembly in Trypanosoma brucei

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.     

Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins

The white-rot basidiomycete Phanerochaete chrysosporium employs extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose, and lignin. Analysis of a total of 10,048 v2.1 gene models predicts 769 secreted proteins, a substantial increase over the 268 models identified in the earlier database (v1.0). Within the v2.1 'computational secretome,' 43% showed no significant similarity to known proteins, but were structurally related to other hypothetical protein sequences. In contrast, 53% showed significant similarity to known protein sequences including 87 models assigned to 33 glycoside hydrolase families and 52 sequences distributed among 13 peptidase families. When grown under standard ligninolytic conditions, peptides corresponding to 11 peptidase genes were identified in culture filtrates by mass spectrometry (LS-MS/MS). Five peptidases were members of a large family of aspartyl proteases, many of which were localized to gene clusters. Consistent with a role in dephosphorylation of lignin peroxidase, a mannose-6-phosphatase (M6Pase) was also identified in carbon-starved cultures. Beyond proteases and M6Pase, 28 specific gene products were identified including several representatives of gene families. These included 4 lignin peroxidases, 3 lipases, 2 carboxylesterases, and 8 glycosyl hydrolases. The results underscore the rich genetic diversity and complexity of P. chrysosporium's extracellular enzyme systems.     

Stem cell regulation via dynamic interactions of the nervous and immune systems with the microenvironment

Physiological interactions between the nervous and immune systems with components of the local microenvironment are needed to maintain homeostasis throughout the body. Dynamic regulation of bone remodeling, hematopoietic stem cells, and their evolving niches via neurotransmitter signaling are part of the host defense and repair mechanisms. This crosstalk links activated leukocytes, neuronal, and stromal cells, which combine to directly and indirectly regulate hematopoietic stem cells. Together, interactions between diverse systems create a regulatory "brain-bone-blood triad," contributing an additional dimension to the concept of the hematopoietic stem cell niche.