NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction

Background  

The major histocompatibility complex (MHC) molecule plays a central role in controlling the adaptive immune response to infections. MHC class I molecules present peptides derived from intracellular proteins to cytotoxic T cells, whereas MHC class II molecules stimulate cellular and humoral immunity through presentation of extracellularly derived peptides to helper T cells. Identification of which peptides will bind a given MHC molecule is thus of great importance for the understanding of host-pathogen interactions, and large efforts have been placed in developing algorithms capable of predicting this binding event.     

NetMHCpan,a method for MHC class I binding prediction beyond humans

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide–MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method’s ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at . Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA from formalin-fixed tissue: extraction or repair? That is the question

Establishing effective DNA-based protocols for use on archival material fixed in formaldehyde (formalin) is a particularly challenging task. Formalin fixation induces cross-linking with nucleic acids and proteins, thereby reducing the amount and quality of the extracted DNA. Previous attempts have primarily focused on optimizing DNA extraction protocols. Here we focus on the use of enzymes capable of in vitro repair of DNA extracts prior to amplification of the nucleic acids by the polymerase chain reaction (PCR). The amplification success of mitochondrial DNA was greater using the repair enzyme assay (56%) than with the regular PCR assay (20%), and even more convincing results were obtained with the amplified nuclear ribosomal region (91% versus 21%). These results indicate that in vitro repair of DNA damage (depurinated sites, strand nicks and base modifications) increases the number of samples that amplify, amplify to a greater extent and amplify fewer ancillary bands and that DNA repair has been overlooked as a way of improving the efficiency of molecular methods used on formalin-fixed samples. Fidelity has not been specifically investigated, but preliminary results indicate that misincorporation is not a major problem.     

Annual variation in maternal age and calving date generate cohort effects in moose (<Emphasis Type="Italic">Alces alces</Emphasis>) body mass

A general feature of the demography of large ungulates is that many demographic traits are dependent on female body mass at early ages. Thus, identifying the factors affecting body mass variation can give important mechanistic understanding of demographic processes. Here we relate individual variation in autumn and winter body mass of moose calves living at low density on an island in northern Norway to characteristics of their mother, and examine how these relationships are affected by annual variation in population density and climate. Body mass increased with increasing age of their mother, was lower for calves born late in the spring, decreased with litter size and was larger for males than for female calves. No residual effects of variation in density and climate were present after controlling for annual variation in mother age and calving date. The annual variation in adult female age structure and calving date explained a large part (71–75%) of the temporal variation in calf body mass. These results support the hypotheses that (a) body mass of moose calves are affected by qualities associated with mother age (e.g. body condition, calving date); and (b) populations living at low densities are partly buffered against temporal fluctuations in the environment.     

Absence of Gup1p in Saccharomyces cerevisiae results in defective cell wall composition, assembly, stability and morphology

Saccharomyces cerevisiae Gup1p and its homologue Gup2p, members of the superfamily of membrane-bound O-acyl transferases, were previously associated with glycerol-mediated salt-stress recovery and glycerol symporter activity. Several other phenotypes suggested Gup1p involvement in processes connected with cell structure organization and biogenesis. The gup1Delta mutant is also thermosensitive and exhibits an altered plasma membrane lipid composition. The present work shows that the thermosensitivity is independent of glycerol production and retention. Furthermore, the mutant grows poorly on salt, ethanol and weak carboxylic acids, suggestive of a malfunctioning membrane potential. Additionally, gup1Delta is sensitive to cell wall-perturbing agents, such as Calcofluor white, Zymolyase, lyticase and sodium dodecyl sulphate and exhibits a sedimentation/aggregation phenotype. Quantitative analysis of cell wall components yielded increased contents of chitin and beta-1,3-glucans and lower amounts of mannoproteins. Consistently, scanning electron microscopy showed a strikingly rough surface morphology of the mutant cells. These results suggest that the gup1Delta is affected in cell wall assembly and stability, although the Slt2p/MAP kinase from the PKC pathway was phosphorylated during hypo-osmotic shock to a normal extent. Results emphasize the pleiotropic nature of gup1Delta, and are consistent with a role of Gulp1p in connection with several pathways for cell maintenance and construction/remodelling.     

Inactivation as a new regulatory mechanism for neuronal Kv7 channels

Voltage-gated K(+) channels of the Kv7 (KCNQ) family have important physiological functions in both excitable and nonexcitable tissue. The family encompasses five genes encoding the channel subunits Kv7.1-5. Kv7.1 is found in epithelial and cardiac tissue. Kv7.2-5 channels are predominantly neuronal channels and are important for controlling excitability. Kv7.1 channels have been considered the only Kv7 channels to undergo inactivation upon depolarization. However, here we demonstrate that inactivation is also an intrinsic property of Kv7.4 and Kv7.5 channels, which inactivate to a larger extent than Kv7.1 channels at all potentials. We demonstrate that at least 30% of these channels are inactivated at physiologically relevant potentials. The onset of inactivation is voltage dependent and occurs on the order of seconds. Both time- and voltage-dependent recovery from inactivation was investigated for Kv7.4 channels. A time constant of 1.47 +/- 0.21 s and a voltage constant of 54.9 +/- 3.4 mV were determined. It was further demonstrated that heteromeric Kv7.3/Kv7.4 channels had inactivation properties different from homomeric Kv7.4 channels. Finally, the Kv7 channel activator BMS-204352 was in contrast to retigabine found to abolish inactivation of Kv7.4. In conclusion, this work demonstrates that inactivation is a key regulatory mechanism of Kv7.4 and Kv7.5 channels.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

Monitoring viral-induced cell death using electric cell-substrate impedance sensing

Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.     

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over-expression can compensate dystrophin's absence. Utrophin is enriched at neuromuscular junctions due to heregulin-mediated utrophin-A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK-dependent manner. MSK1/2 inhibition suppressed heregulin-mediated utrophin-A activation. MSK1 over-expression potentiated heregulin-mediated utrophin-A activation and chromatin remodeling at the utrophin-A promoter. These results identify MSK1/2 as key effectors modulating utrophin-A expression as well as identify novel targets for DMD therapy.