Enhancement of suboptimal CD8 cytotoxic T cell effector function in vivo using antigen-specific CD80 defective T cells

T cell upregulation of B7 molecules CD80 and CD86 limits T cell expansion in immunodeficient hosts; however, the relative roles of CD80 separate from CD86 on CD4 versus CD8 T cells in a normal immune system are not clear. To address this question, we used the parent-into-F1 (P→F1) murine model of graft-versus-host disease and transferred optimal and suboptimal doses of CD80 and/or CD86 knockout (KO) T cells into normal F1 hosts. Enhanced elimination of host B cells by KO T cells was observed only at suboptimal donor cell doses and was greatest for CD80 KO→F1 mice. Wild-type donor cells exhibited peak CD80 upregulation at day 10; CD80 KO donor cells exhibited greater peak (day 10) donor T cell proliferation and CD8 T cell effector CTL numbers versus wild-type→F1 mice. Fas or programmed cell death-1 upregulation was normal as was homeostatic contraction of CD80 KO donor cells from days 12-14. Mixing studies demonstrated that maximal host cell elimination was seen when both CD4 and CD8 T cells were CD80 deficient. These results indicate an important role for CD80 upregulation on Ag-activated CD4 and CD8 T cells in limiting expansion of CD8 CTL effectors as part of a normal immune response. Our results support further studies of therapeutic targeting of CD80 in conditions characterized by suboptimal CD8 effector responses.     

Far-Red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy

Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or near-red fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.     

Genetic transformation of plastids of different Solanaceae species using tobacco cells as organelle hosts

The plastid genome of angiosperms represents an attractive target for genetic manipulations. However plastid transformation of higher plants, especially of agriculturally valuable crops is an extremely difficult problem. Transformation protocols developed for tobacco 15 years ago failed to produce similar results with more than a handful of other species so far. We have analyzed plastid transformability of remote cytoplasmic hybrids (cybrids) that combine nuclei of tobacco, an easily transformable species, and plastids of some other, recalcitrant Solanaceae species. Here, we demonstrate that the plastids of five species of Solanaceae family, representing two subfamilies and three tribes, can be easily transformed if the plastids of these species are transferred into a cell of a transformable species (tobacco). The results can be considered to be an alternative approach to the development of plastid transformation technologies for recalcitrant species using a transformable intermediary (“clipboard”) host.     

Reaction pathway and free energy profile determined for specific recognition of oligosaccharide moiety of carboxypeptidase Y

The interaction of mannose specific lectin (from Lens culinaris, LcL) with the carbohydrate moiety of carboxypeptidase Y (CaY) was studied using both atomic force microscope (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D). The AFM enables to determine the positions of energy barriers present in the energy landscape of the single complex undergoing dissociation. The QCM-D measurements allow the estimation of the quantitative parameters characterizing the kinetics of the studied molecular interaction (namely the association and dissociation rate constants and the association constant). The use of both methods not only delivers the complementary characterization of kinetic and thermodynamic parameters but also permits to investigate the mechanism of the binding and unbinding of the molecules. The results for LcL were compared with those obtained for concanavalin A i.e. lectin, which interacts with the carbohydrate moiety on a similar way.     

Genome Sequence of Candidatus Nitrososphaera evergladensis from Group I.1b Enriched from Everglades Soil Reveals Novel Genomic Features of the Ammonia-Oxidizing Archaea

The activity of ammonia-oxidizing archaea (AOA) leads to the loss of nitrogen from soil, pollution of water sources and elevated emissions of greenhouse gas. To date, eight AOA genomes are available in the public databases, seven are from the group I.1a of the Thaumarchaeota and only one is from the group I.1b, isolated from hot springs. Many soils are dominated by AOA from the group I.1b, but the genomes of soil representatives of this group have not been sequenced and functionally characterized. The lack of knowledge of metabolic pathways of soil AOA presents a critical gap in understanding their role in biogeochemical cycles. Here, we describe the first complete genome of soil archaeon Candidatus Nitrososphaera evergladensis, which has been reconstructed from metagenomic sequencing of a highly enriched culture obtained from an agricultural soil. The AOA enrichment was sequenced with the high throughput next generation sequencing platforms from Pacific Biosciences and Ion Torrent. The de novo assembly of sequences resulted in one 2.95 Mb contig. Annotation of the reconstructed genome revealed many similarities of the basic metabolism with the rest of sequenced AOA. Ca. N. evergladensis belongs to the group I.1b and shares only 40% of whole-genome homology with the closest sequenced relative Ca. N. gargensis. Detailed analysis of the genome revealed coding sequences that were completely absent from the group I.1a. These unique sequences code for proteins involved in control of DNA integrity, transporters, two-component systems and versatile CRISPR defense system. Notably, genomes from the group I.1b have more gene duplications compared to the genomes from the group I.1a. We suggest that the presence of these unique genes and gene duplications may be associated with the environmental versatility of this group.     

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the “T-vector” can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3′-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5′-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.     

Mammalian Male Mutation Bias: Impacts of Generation Time and Regional Variation in Substitution Rates

In mammals, males undergo a greater number of germline cell divisions compared with females. Thus, the male germline accumulates more DNA replication errors, which result in male mutation bias—a higher mutation rate for males than for females. The phenomenon of male mutation bias has been investigated mostly for rodents and primates, however, it has not been studied in detail for other mammalian orders. Here we sequenced and analyzed five introns of three genes (DBX/DBY, UTX/UTY, and ZFX/ZFY) homologous between X and Y chromosomes in several species of perissodactyls (horses and rhinos) and of primates. Male mutation bias was evident: substitution rate was higher for a Y chromosome intron than for its X chromosome homologue for all five intron pairs studied. Substitution rates varied regionally among introns sequenced on the same chromosome and this variation influenced male mutation bias inferred from each intron pair. Interestingly, we observed a positive correlation in substitution rates between homologous X and homologous Y introns as well as between orthologous primate and perissodactyl introns. The male-to-female mutation rate ratio estimated from concatenated sequences of five perissodactyl introns was 3.88 (95% CI = 2.90–6.07). Using the data generated here and estimates available in the literature, we compared male mutation bias among several mammalian orders. We conclude that male mutation bias is significantly higher for organisms with long generation times (primates, perissodactyls, and felids) than for organisms with short generation times (e.g., rodents) since the former undergo a greater number of male germline cell divisions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Deborah Charlesworth]     

Evolution and Survival on Eutherian Sex Chromosomes

Since the two eutherian sex chromosomes diverged from an ancestral autosomal pair, the X has remained relatively gene-rich, while the Y has lost most of its genes through the accumulation of deleterious mutations in nonrecombining regions. Presently, it is unclear what is distinctive about genes that remain on the Y chromosome, when the sex chromosomes acquired their unique evolutionary rates, and whether X-Y gene divergence paralleled that of paralogs located on autosomes. To tackle these questions, here we juxtaposed the evolution of X and Y homologous genes (gametologs) in eutherian mammals with their autosomal orthologs in marsupial and monotreme mammals. We discovered that genes on the X and Y acquired distinct evolutionary rates immediately following the suppression of recombination between the two sex chromosomes. The Y-linked genes evolved at higher rates, while the X-linked genes maintained the lower evolutionary rates of the ancestral autosomal genes. These distinct rates have been maintained throughout the evolution of X and Y. Specifically, in humans, most X gametologs and, curiously, also most Y gametologs evolved under stronger purifying selection than similarly aged autosomal paralogs. Finally, after evaluating the current experimental data from the literature, we concluded that unique mRNA/protein expression patterns and functions acquired by Y (versus X) gametologs likely contributed to their retention. Our results also suggest that either the boundary between sex chromosome strata 3 and 4 should be shifted or that stratum 3 should be divided into two strata.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Probiotic Composition and Chondroitin Sulfate Regulate TLR-2/4-Mediated NF-κB Inflammatory Pathway and Cartilage Metabolism in Experimental Osteoarthritis

The therapeutic potential of using probiotics to treat osteoarthritis (OA) has only recently been recognized, with a small number of animal and human studies having been undertaken. The aim of this study was to describe the effect of a probiotic composition (PB) and chondroitin sulfate (CS), administered separately or in combination, on Tlr2, Tlr4, Nfkb1, and Comp gene expression in cartilage and levels of cytokines (IL-6, IL-8, TGF-β1, IGF-1) and COMP, ACAN, CHI3L1, CTSK, and TLR-2 in serum during monoiodoacetate (MIA)-induced OA in rats. Expression of Tlr2, Tlr4, Nfkb1, and Comp in cartilage was analyzed using one-step SYBR Green real-time RT-PCR. The levels of IL-6, IL-8, TGF-β1, IGF-1, COMP, ACAN, CHI3L1, CTSK, and TLR-2 were measured in serum by enzyme-linked immunosorbent assay. Experimental OA caused an upregulation in Tlr2, Tlr4, Nfkb1, and downregulation of Comp expression in the cartilage. MIA-OA caused a significant increase of TLR-2 soluble form and IL-6, IL-8, TGF-β1, COMP, ACAN, CHI3L1, and CTSK levels in the blood serum; the level of IGF-1, on contrary, decreased. Separate administration of PB and CS raised expression of Comp and reduced Tlr2, Tlr4, and Nfkb1 expressions in cartilage. The levels of the studied markers of cartilage metabolism in serum were decreased or increased (IGF-1). The combined use of PB and CS was more effective than separate application approaching above-mentioned parameters to control. The outcomes of our research prove that multistrain live probiotic composition amplifies the positive action of CS in osteoarthritis attenuation and necessitates further investigation with large-scale randomized controlled trial.