Total and regional body adiposity increases during menopause—evidence from a follow‐up study

For women, menopausal transition is a time of significant hormonal changes, which may contribute to altered body composition and regional adipose tissue accumulation. Excess adiposity, and especially adipose tissue accumulation in the central body region, increases women''s risk of cardiovascular and metabolic conditions and affects physical functioning. We investigated the associations between menopausal progression and total and regional body adiposity measured with dual‐energy X‐ray absorptiometry and computed tomography in two longitudinal cohort studies of women aged 47–55 (n = 230 and 148, mean follow‐up times 1.3 ± 0.7 and 3.9 ± 0.2 years, mean baseline BMI 25.5 kg/m²). We also examined associations between menopausal progression and skeletal muscle fiber characteristics, as well as adipose tissue‐derived adipokines. Relative increases of 2%–14% were observed in regional and total body adiposity measures, with a pronounced fat mass increase in the android area (4% and 14% during short‐ and long‐term follow‐ups). Muscle fiber oxidative and glycolytic capacities and intracellular adiposity were not affected by menopause, but were differentially correlated with total and regional body adiposity at different menopausal stages. Menopausal progression and regional adipose tissue masses were positively associated with serum adiponectin and leptin, and negatively associated with resistin levels. Higher diet quality and physical activity level were also inversely associated with several body adiposity measures. Therefore, healthy lifestyle habits before and during menopause might delay the onset of severe metabolic conditions in women.     

Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies

The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.     

Cooperative cleavage of the R peptide in the Env trimer of Moloney murine leukemia virus facilitates its maturation for fusion competence

The spike protein of murine leukemia virus, MLV, is made as a trimer of the Env precursor. This is primed for receptor-induced activation of its membrane fusion function first by cellular furin cleavage in the ectodomain and then by viral protease cleavage in the endodomain. The first cleavage separates the peripheral surface (SU) subunit from the transmembrane (TM) subunit, and the latter releases a 16-residue-long peptide (R) from the TM endodomain. Here, we have studied the distribution of R peptide cleavages in the spike TM subunits of Moloney MLV preparations with partially R-peptide-processed spikes. The spikes were solubilized as trimers and separated with an R peptide antibody. This showed that the spikes were either uncleaved or cleaved in all of its TM subunits. Further studies showed that R peptide cleavage-inhibited Env mutants, L(649)V and L(649)I, were rescued by wild-type (wt) Env in heterotrimeric spikes. These findings suggested that the R peptide cleavages in the spike are facilitated through positive allosteric cooperativity; i.e., the cleavage of the TM subunit in one Env promoted the cleavages of the TMs in the other Envs. The mechanism ensures that protease cleavage in newly released virus will generate R-peptide-cleaved homotrimers rather than heterotrimeric intermediates. However, using a cleavage site Env mutant, L(649)R, which was not rescued by wt Env, it was possible to produce virus with heterotrimers. These were shown to be less fusion active than the R-peptide-cleaved homotrimers. Therefore, the cooperative cleavage will speed up the maturation of released virus for fusion competence.     

The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish

The histaminergic and hypocretin/orexin (hcrt) neurotransmitter systems play crucial roles in alertness/wakefulness in rodents. We elucidated the role of histamine in wakefulness and the interaction of the histamine and hcrt systems in larval zebrafish. Translation inhibition of histidine decarboxylase (hdc) with morpholino oligonucleotides (MOs) led to a behaviorally measurable decline in light-associated activity, which was partially rescued by hdc mRNA injections and mimicked by histamine receptor H1 (Hrh1) antagonist pyrilamine treatment. Histamine-immunoreactive fibers targeted the dorsal telencephalon, an area that expresses histamine receptors hrh1 and hrh3 and contains predominantly glutamatergic neurons. Tract tracing with DiI revealed that projections from dorsal telencephalon innervate the hcrt and histaminergic neurons. Translation inhibition of hdc decreased the number of hcrt neurons in a Hrh1-dependent manner. The reduction was rescued by overexpression of hdc mRNA. hdc mRNA injection alone led to an up-regulation of hcrt neuron numbers. These results suggest that histamine is essential for the development of a functional and intact hcrt system and that histamine has a bidirectional effect on the development of the hcrt neurons. In summary, our findings provide evidence that these two systems are linked both functionally and developmentally, which may have important implications in sleep disorders and narcolepsy. development via histamine receptor H1 in zebrafish.     

Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein

Objective

Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL.

Methods and Results

Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR^−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans.

Conclusion

Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.     

Quantitative genetics of temperature performance curves of Neurospora crassa

Earth's temperature is increasing due to anthropogenic CO emissions; and organisms need either to adapt to higher temperatures, migrate into colder areas, or face extinction. Temperature affects nearly all aspects of an organism's physiology via its influence on metabolic rate and protein structure, therefore genetic adaptation to increased temperature may be much harder to achieve compared to other abiotic stresses. There is still much to be learned about the evolutionary potential for adaptation to higher temperatures, therefore we studied the quantitative genetics of growth rates in different temperatures that make up the thermal performance curve of the fungal model system Neurospora crassa. We studied the amount of genetic variation for thermal performance curves and examined possible genetic constraints by estimating the G -matrix. We observed a substantial amount of genetic variation for growth in different temperatures, and most genetic variation was for performance curve elevation. Contrary to common theoretical assumptions, we did not find strong evidence for genetic trade-offs for growth between hotter and colder temperatures. We also simulated short-term evolution of thermal performance curves of N. crassa, and suggest that they can have versatile responses to selection.     

Biologically Fe<Superscript>2+</Superscript> oxidizing fluidized bed reactor performance and controlling of Fe<Superscript>3+</Superscript> recycle during heap bioleaching: an artificial neural network-based model

The performance of a biological Fe²⁺ oxidizing fluidized bed reactor (FBR) was modeled by a popular neural network-back-propagation algorithm over a period of 220 days at 37 °C under different operational conditions. A method is proposed for modeling Fe³⁺ production in FBR and thereby managing the regeneration of Fe³⁺ for heap leaching application, based on an artificial neural network-back-propagation algorithm. Depending on output value, relevant control strategies and actions are activated, and Fe³⁺ production in FBR was considered as a critical output parameter. The modeling of effluent Fe³⁺ concentration was very successful, and an excellent match was obtained between the measured and the predicted concentrations.     

Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display

The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.