Cutting Edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation

The killer cell Ig-like receptor (KIR) gene, KIR3DS1, has been implicated in slowing disease progression in HIV infection; however, little is known about its expression, function, or ligand specificity. Using retrovirally transduced NKL cells and peripheral blood NK cells from KIR3DS1-positive donors we assessed expression of this gene by flow cytometry and its function by in vitro assays measuring KIR3DS1-induced cell-mediated cytotoxicity and cytokine production. In the present study, we demonstrate that KIR3DS1 is expressed on peripheral blood NK cells and triggers both cytotoxicity and IFN-gamma production. Using cotransfection and coimmunoprecipitation, we found that KIR3DS1 associates with the ITAM-bearing adaptor, DAP12. Soluble KIR3DS1-Ig fusion proteins did not bind to EBV-transformed B lymphoid cell lines transfected with HLA-Bw4 80I or 80T allotypes, suggesting that if KIR3DS1 does recognize HLA-Bw4 ligands, this may be peptide dependent.     

A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation

The energy balance of Phaeodactylum tricornutum cells from photon to biomass have been analysed under nutrient-replete and N-limiting conditions in combination with fluctuating (FL) and non-fluctuating (SL) dynamic light. For this purpose, the amount of photons absorbed has been related to electrons transported by photosystem II, to gas exchange rates, and to the newly formed biomass differentially resolved into carbohydrates, proteins, and lipids measured by means of Fourier transform infrared (FTIR) spectroscopy. Under high nutrient conditions, the quantum efficiency of carbon-related biomass production (Phi(C)) and the metabolic costs of carbon (C) production were found to be strongly controlled by the light climate. Under N-limited conditions, the light climate was less important for the efficieny of primary production. Thus, the largest range of Phi(C) dependent on the nutrient status of the cells was observed under non-fluctuating light conditions which are comparable with stratified conditions in the natural environment. It is evident that N limitation induced pronounced changes in the composition of macromolecular compounds and, thus, influenced the degree of reduction of the biomass as well as the metabolic costs of C production. However, Phi(C) and the metabolic costs are not predictable from the photosynthesis rates. In consequence, the results clearly show that bio-optical methods as well as gas exchange measurements during the light phase can severely mismatch the true energy storage in the biomass especially under high nutrient in combination with non-fluctuating light conditions.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point

CD8(+) T cells are believed to play an important role in the control of human immunodeficiency virus type 1 (HIV-1) infection. However, despite intensive efforts, it has not been possible to consistently link the overall magnitude of the CD8(+) T-cell response with control of HIV-1. Here, we have investigated the association of different CD8(+) memory T-cell subsets responding to HIV-1 in early infection with future control of HIV-1 viremia. Our results demonstrate that both a larger proportion and an absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(+) effector memory T cells (T(EMRA) cells) were associated with a lower future viral load set point. In contrast, a larger absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(-) effector memory T cells (T(EM)) was not related to the viral load set point. Overall, the findings suggest that CD8(+) T(EMRA) cells have superior antiviral activity and indicate that both qualitative and quantitative aspects of the CD8(+) T-cell response need to be considered when defining the characteristics of protective immunity to HIV-1.     

Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.     

Changing hydrozoan bauplans by silencing Hox-like genes

Regulatory genes of the Antp class have been a major factor for the invention and radiation of animal bauplans. One of the most diverse animal phyla are the Cnidaria, which are close to the root of metazoan life and which often appear in two distinct generations and a remarkable variety of body forms. Hox-like genes have been known to be involved in axial patterning in the Cnidaria and have been suspected to play roles in the genetic control of many of the observed bauplan changes. Unfortunately RNAi mediated gene silencing studies have not been satisfactory for marine invertebrate organisms thus far. No direct evidence supporting Hox-like gene induced bauplan changes in cnidarians have been documented as of yet. Herein, we report a protocol for RNAi transfection of marine invertebrates and demonstrate that knock downs of Hox-like genes in Cnidaria create substantial bauplan alterations, including the formation of multiple oral poles ("heads") by Cnox-2 and Cnox-3 inhibition, deformation of the main body axis by Cnox-5 inhibition and duplication of tentacles by Cnox-1 inhibition. All phenotypes observed in the course of the RNAi studies were identical to those obtained by morpholino antisense oligo experiments and are reminiscent of macroevolutionary bauplan changes. The reported protocol will allow routine RNAi studies in marine invertebrates to be established.     

Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake

Neuromedin U (NMU) is a 25 amino acid peptide expressed and secreted in the brain and gastrointestinal tract. Data have shown that peripheral administration of human NMU decreases food intake and body weight and improves glucose tolerance in mice, suggesting that NMU receptors constitute a possible anti‐diabetic and anti‐obesity drug target. However, the clinical use of native NMU is hampered by a poor pharmacokinetic profile. In the current study, we report in vitro and in vivo data from a series of novel lipidated NMU analogs. In vitro plasma stability studies of native NMU were performed to investigate the proteolytic stability and cleavage sites using LC–MS. Native NMU was found to be rapidly cleaved at the C‐terminus between Arg²⁴ and Asn²⁵, followed by cleavage between Arg¹⁶ and Gly¹⁷. Lipidated NMU analogs were generated using solid‐phase peptide synthesis, and in vitro potency was investigated using a human embryonic kidney 293‐based inositol phosphate accumulation assay. All lipidated analogs had preserved in vitro activity on both NMU receptors with potency improving as the lipidation site was moved away from the receptor‐interacting C‐terminal octapeptide segment. In vivo efficacy was assessed in lean mice as reduction in food intake after acute subcutaneous administration of 1, 0.3, 0.1, and 0.03 µmol/kg. These lipidated NMU analogs prolonged the anorectic effect of NMU in a dose‐dependent manner. This was likely an effect of improved pharmacokinetic properties because of improved vitro plasma stability. Accordingly, the data demonstrate that lipidated NMU analogs may represent drug candidates for the treatment of obesity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A novel noninvasive procedure for high‐throughput screening of major seed traits

The large numbers of samples processed in breeding and biodiversity programmes require the development of efficient methods for the nondestructive evaluation of basic seed properties. Near‐infrared spectroscopy is the state‐of‐the‐art solution for this analytical demand, but it also has some limitations. Here, we present a novel, rapid, accurate procedure based on time domain‐nuclear magnetic resonance (TD‐NMR), designed to simultaneously quantify a number of basic seed traits without any seed destruction. Using a low‐field, benchtop ¹H‐NMR instrument, the procedure gives a high‐accuracy measurement of oil content (R² = 0.98), carbohydrate content (R² = 0.99), water content (R² = 0.98) and both fresh and dry weight of seeds/grains (R² = 0.99). The method requires a minimum of ~20 mg biomass per sample and thus enables to screen individual, intact seeds. When combined with an automated sample delivery system, a throughput of ~1400 samples per day is achievable. The procedure has been trialled as a proof of concept on cereal grains (collection of ~3000 accessions of Avena spp. curated at the IPK genebank). A mathematical multitrait selection approach has been designed to simplify the selection of outlying (most contrasting) accessions. To provide deeper insights into storage oil topology, some oat accessions were further analysed by three‐dimensional seed modelling and lipid imaging. We conclude that the novel TD‐NMR‐based screening tool opens perspectives for breeding and plant biology in general.     

Mitogenomic phylogeny of the common long-tailed macaque (Macaca fascicularis fascicularis)

Background

Long-tailed macaques (Macaca fascicularis) are an important model species in biomedical research and reliable knowledge about their evolutionary history is essential for biomedical inferences. Ten subspecies have been recognized, of which most are restricted to small islands of Southeast Asia. In contrast, the common long-tailed macaque (M. f. fascicularis) is distributed over large parts of the Southeast Asian mainland and the Sundaland region. To shed more light on the phylogeny of M. f. fascicularis, we sequenced complete mitochondrial (mtDNA) genomes of 40 individuals from all over the taxon’s range, either by classical PCR-amplification and Sanger sequencing or by DNA-capture and high-throughput sequencing.

Results

Both laboratory approaches yielded complete mtDNA genomes from M. f. fascicularis with high accuracy and/or coverage. According to our phylogenetic reconstructions, M. f. fascicularis initially diverged into two clades 1.70 million years ago (Ma), with one including haplotypes from mainland Southeast Asia, the Malay Peninsula and North Sumatra (Clade A) and the other, haplotypes from the islands of Bangka, Java, Borneo, Timor, and the Philippines (Clade B). The three geographical populations of Clade A appear as paraphyletic groups, while local populations of Clade B form monophyletic clades with the exception of a Philippine individual which is nested within the Borneo clade. Further, in Clade B the branching pattern among main clades/lineages remains largely unresolved, most likely due to their relatively rapid diversification 0.93-0.84 Ma.

Conclusions

Both laboratory methods have proven to be powerful to generate complete mtDNA genome data with similarly high accuracy, with the DNA-capture and high-throughput sequencing approach as the most promising and only practical option to obtain such data from highly degraded DNA, in time and with relatively low costs. The application of complete mtDNA genomes yields new insights into the evolutionary history of M. f. fascicularis by providing a more robust phylogeny and more reliable divergence age estimations than earlier studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1437-0) contains supplementary material, which is available to authorized users.