HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.     

Persistent damage induces mitochondrial DNA degradation

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6 h after induction of mutant uracil-N-glycosylase and by 12 h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144 h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.     

Expression,surface immobilization,and characterization of functional recombinant cannabinoid receptor CB2

Human peripheral cannabinoid receptor CB₂, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB₂ may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB₂ and immobilization at solid interfaces. Expression of CB₂ as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB₂ was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB₂ was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB₂ from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB₂ and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB₂.     

Resilience to Freezing in the Vegetative Cells of the Microalga Lobosphaera incisa (Trebouxiophyceae,Chlorophyta)

The chlorophyte microalga Lobosphaera incisa was isolated from the snowy slopes of Mt. Tateyama in Japan. This microalga stores exceptionally high amounts of the omega-6 LC-PUFA arachidonic acid in triacylglycerols, and therefore represents a potent photosynthetic source for this essential LC-PUFA. Assuming that freezing tolerance may play a role in adaptation of L. incisa to specific ecological niches, we examined the capability of L. incisa to tolerate extreme sub-zero temperatures. We report here, that the vegetative cells of L. incisa survived freezing at −20°C and −80°C (over 1 month), without cryoprotective agents or prior treatments. Cells successfully recovered upon thawing and proliferated under optimal growth conditions (25°C). However, cells frozen at −80°C showed better recovery and lower cellular ROS generation upon thawing, compared to those preserved at −20°C. Photosynthetic yield of PSII, estimated by F_v/F_m, temporarily decreased at day 1 post freezing and resumed to the original level at day 3. Interestingly, the thawed algal cultures produced a higher level of chlorophylls, exceeding the control culture. The polar metabolome of the vegetative cells comprised a range of compatible solutes, dominated by glutamate, sucrose, and proline. We posit that the presence of endogenous cryoprotectants, a rigid multilayer cell wall, the high LC-PUFA content in membrane lipids, and putative cold-responsive proteins may contribute to the retention of functionality upon recovery from the frozen state, and therefore for the survival under cryospheric conditions. From the applied perspective, this beneficial property holds promise for the cryopreservation of starter cultures for research and commercial purposes.     

The strengthening role of <Emphasis Type="Italic">D</Emphasis>:<Emphasis Type="Italic">D</Emphasis> interactions in fibrin self-assembly under oxidation

The effect on ozone-induced oxidation on the self-assembly of fibrin in the presence of fibrin-stabilizing factor FXIIIa of soluble cross-linked fibrin oligomers was studied in a medium containing moderate urea concentrations. It is established that fibrin oligomers were formed by the protofibrils cross-linked through γ-γ dimers and the fibrils additionally cross-linked by through α-polymers. The oxidation promoted both the accumulation of greater amounts of γ-γ dimers and the formation of protofibrils, fibrils, and their dissociation products emerging with increasing urea concentrations, which have a high molecular weight. It is concluded that the oxidation enhances the axial interactions between D-regions of fibrin molecules.     

Genetic system for maintaining the mitochondrial human genome in yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.     

Melanoma central nervous system metastases: An update to approaches,challenges, and opportunities

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.     

Establishing a Cultivable Cell Line of the Tick Dermacentor marginatus

Russian Journal of Genetics - Owing to the changes in the general ecological situation in Russia, livestock losses from ovine anaplasmosis have rapidly increased in recent years. The development of...