Chronic graft-versus-host in Ig knockin transgenic mice abrogates B cell tolerance in anti-double-stranded DNA B cells

Anti-dsDNA Abs are specific diagnostic markers of systemic lupus erythematosus, and are also implicated in kidney pathology. Anti-dsDNA B cells have been shown to be tolerized in nonautoimmune mice. The immunodysregulation that causes these cells to break tolerance is presumably part of the fundamental defects in systemic lupus erythematosus. To explore these mechanisms, we used the chronic graft-versus-host model mediated by MHC class II differences. Induction of chronic graft-vs-host in anti-DNA H chain knockin (3H9.KI) transgenic mice on a nonautoimmune background resulted in specific activation of anti-dsDNA B cells, as evidenced by high titers of soluble Ab in sera and a high frequency (70%) of anti-dsDNA B cell clones recovered as hybridomas. In addition, the lambda(+)-anti-dsDNA B cells developed increased expression of cell surface activation markers, and concentrated in the T cell area of the follicle with an Ab-forming cell-compatible phenotype. Genetic analysis of the hybridoma clones showed strong evidence of secondary rearrangements of the L chain associated with anti-dsDNA reactivity. Thus, our study indicates that alloreactive T cell help can break tolerance in a complex manner, involving several events.     

Methanogenic potential of tailings samples from oil sands extraction plants

Approximately 20% of Canada's oil supply now comes from the extraction of bitumen from the oil sands deposits in northeastern Alberta. The oil sands are strip-mined, and the bitumen is typically separated from sand and clays by an alkaline hot water extraction process. The rapidly expanding oil sands industry has millions of cubic metres of tailings for disposal and large areas of land to reclaim. There are estimates that the consolidation of the mature fine tails (MFT) in the settling ponds will take about 150 years. Some of the settling ponds are now evolving microbially produced methane, a greenhouse gas. To hasten consolidation, gypsum (CaSO4 x 2H2O) is added to MFT, yielding materials called consolidated or composite tailings (CT). Sulfate from the gypsum has the potential to stimulate sulfate-reducing bacteria (SRB) to out-compete methanogens, thereby stopping methanogenesis. This investigation examined three MFT and four CT samples from three oil sands extractions companies. Each was found to contain methanogens and SRB. Serum bottle microcosm studies showed sulfate in the CT samples stopped methane production. However, if the microcosms were amended with readily utilizable electron donors, the sulfate was consumed, and when it reached approximately 20 mg/L, methane production began. Some unamended microcosms were incubated for 372 days, with no methane production detected. This work showed that each MFT and CT sample has the potential to become methanogenic, but in the absence of exogenous electron donors, the added sulfate can inhibit methanogenesis for a long time.     

A covalent modification of NADP+ revealed by the atomic resolution structure of FprA,a Mycobacterium tuberculosis oxidoreductase

FprA is a mycobacterial oxidoreductase that catalyzes the transfer of reducing equivalents from NADPH to a protein acceptor. We determined the atomic resolution structure of FprA in the oxidized (1.05 A resolution) and NADPH-reduced (1.25 A resolution) forms. The comparison of these FprA structures with that of bovine adrenodoxin reductase showed no significant overall differences. Hence, these enzymes, which belong to the structural family of the disulfide oxidoreductases, are structurally conserved in very distant organisms such as mycobacteria and mammals. Despite the conservation of the overall fold, the details of the active site of FprA show some peculiar features. In the oxidized enzyme complex, the bound NADP+ exhibits a covalent modification, which has been identified as an oxygen atom linked through a carbonylic bond to the reactive C4 atom of the nicotinamide ring. Mass spectrometry has confirmed this assignment. This NADP+ derivative is likely to form by oxidation of the NADP+ adduct resulting from nucleophilic attack by an active-site water molecule. A Glu-His pair is well positioned to activate the attacking water through a mechanism analogous to that of the catalytic triad in serine proteases. The NADP+ nicotinamide ring exhibits the unusual cis conformation, which may favor derivative formation. The physiological significance of this reaction is presently unknown. However, it could assist with drug-design studies in that the modified NADP+ could serve as a lead compound for the development of specific inhibitors.     

Tamplicon-7, a Novel T-Lymphotropic Vector Derived from Human Herpesvirus 7

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.     

Human-Mediated Admixture and Selection Shape the Diversity on the Modern Swine (Sus scrofa) Y Chromosomes

Throughout its distribution across Eurasia, domestic pig (Sus scrofa) populations have acquired differences through natural and artificial selection, and have often interbred. We resequenced 80 Eurasian pigs from nine different Asian and European breeds; we identify 42,288 reliable SNPs on the Y chromosome in a panel of 103 males, among which 96.1% are newly detected. Based on these new data, we elucidate the evolutionary history of pigs through the lens of the Y chromosome. We identify two highly divergent haplogroups: one present only in Asia and one fixed in Europe but present in some Asian populations. Analyzing the European haplotypes present in Asian populations, we find evidence of three independent waves of introgression from Europe to Asia in last 200 years, agreeing well with the literature and historical records. The diverse European lineages were brought in China by humans and left significant imprints not only on the autosomes but also on the Y chromosome of geographically and genetically distinct Chinese pig breeds. We also find a general excess of European ancestry on Y chromosomes relative to autosomes in Chinese pigs, an observation that cannot be explained solely by sex-biased migration and genetic drift. The European Y haplotype is associated with leaner meat production, and we hypothesize that the European Y chromosome increased in frequency in Chinese populations due to artificial selection. We find evidence of Y chromosomal gene flow between Sumatran wild boar and Chinese pigs. Our results demonstrate how human-mediated admixture and selection shaped the distribution of modern swine Y chromosomes.     

Increase of catalytic activity of lipase towards olive oil by Langmuir-film immobilization of lipase

Proteins represent versatile building blocks for realization of nanostructured materials to be applied in nanobiotechnology. In the present work, the Langmuir–Blodgett technique was utilized to develop nanobiodevices based on protein molecules. Particularly, lipase thin films were fabricated and characterized, with characterization performed in order to optimize the working parameters. As the first step the protein films were studied at the air–water interface and then transferred onto a solid support for further characterization. The films were characterized by different techniques such as UV–Vis spectroscopy, nanogravimetry, atomic force microscopy, and biochemical assays. Catalytic activity of lipase characterized by the maximal reaction rate found to increase over 10 times as a result of inclusion into LB films, while the substrate binding characterized by the Michaelis constant remain unchanged. Catalytic activity per mole of enzyme was found to increase with the increased number of LB layers up to five, and then decrease at 10, while the surface coverage ranged from 70% to 100% from 1 to 10 layers of lipase. This study exploits the possibility to employ LB based protein structures to use in biocatalysis, exemplified by lipase, which is known as an interfacially-activated enzyme, with olive oil as substrate, when lipase should already be in the maximally active state even without a film. We show, however, that it was possible to form even more active lipase nanostructures by the Langmuir–Blodgett technique at the air–water interface, proving that Langmuir-film provides a better catalytic effect in lipase than a mere oil–water boundary.     

Analysis of the center of pressure displacement, ground reaction force and muscular activity during step exercises

Anterior Knee Pain (AKP) is considered as one of the most common, yet misunderstood, knee pathologies. The aim of this study was to evaluate the displacement area of the center of pressure, Ground Reaction Force (GRF), and the electromyography activity of the hip and the quadriceps muscles in healthy and AKP individuals during the step-up and step-down exercises. Both groups (Control group and AKP group) were composed of 15 volunteers submitted to the exercises on a force plate. The AKP group presented greater displacement area of the center of pressure for all the situations evaluated than the Control group (p < 0.05), as well as a lesser magnitude of the GRF during the step-down exercise. The AKP group presented lower electromyography activity than the Control group in all situations evaluated. AKP individuals do not have muscle imbalances; they present a lower electromyography activity of the stabilizing muscles of the patella and hip and show greater instability in activities such as step up and down compared to normal subjects.     

A mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma

Neurocutaneous syndromes represent a vast, largely heterogeneous group of disorders characterized by neurological and dermatological manifestations, reflecting the common embryonic origin of epidermal and neural tissues. In the present report, we describe a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK syndrome). Using homozygosity mapping in two large families, we localized the disease gene to 22q11.2 and identified, in all patients, a 1-bp deletion in SNAP29, which codes for a SNARE protein involved in vesicle fusion. SNAP29 expression was decreased in the skin of the patients, resulting in abnormal maturation of lamellar granules and, as a consequence, in mislocation of epidermal lipids and proteases. These data underscore the importance of vesicle trafficking regulatory mechanisms for proper neuroectodermal differentiation.