Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

Factors affecting the length of the breeding cycle and the frequency of nest attendance by Grey Herons Ardea cinerea

An investigation of the length of the breeding cycle and of the frequency of nest visits by Grey Herons at 3 heronries with different degrees of breeding success showed that the duration of reproduction, particularly the chickrearing period, decreased and the frequency of daily nest visits increased with increasing average breeding success of a colony. Successful pairs also seemed to spend less time displaying and incubating eggs. Herons arrived at their nests most frequently during twilight, especially in the morning. The mean time absent from the nest was greatest during incubation and smallest when feeding small chicks. The total reproductive period tended to be longer for those pairs whose periods of absence from the nest were longer.     

The potato tuber,maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.     

Pollen morphology of Diosma and Coleonema (Rutaceae: Diosminae) and its taxonomic implications

The pollen morphology of Diosma and Coleonema of the taxonomically problematic subtribe Diosminae (Rutaceae) was investigated to determine its taxonomic relevance. Pollen of 26 of the 28 species of Diosma and of each of the eight species of Coleonema was investigated by LM, SEM and TEM. In pollen of Diosma the exine sculpturing is uniform, and only one pollen type, reticulate, could be distinguished. In contrast, exine sculpturing shows interspecific variation in Coleonema and three distinct types, striate, reticulate and perforate, could be discerned. Species groupings indicated by the pollen types suggest possible relationships between species within Coleonema and with other genera that were not previously apparent. Pollen data support macromorphological evidence that suggests the generic re-classification of the Diosminae.     

Development,validation and pilot screening of an in vitro multi-cellular three-dimensional cancer spheroid assay for anti-cancer drug testing

It has been demonstrated that two-dimensional (2D) monolayer cancer cell proliferation assay for anti-cancer drug screening is a very artificial model and cannot represent the characteristics of three-dimensional (3D) solid tumors. The multi-cellular in vitro 3D tumor spheroid model is of intermediate complexity, and can provide a bridge to the gap between the complex in vivo tumors and simple in vitro monolayer cell cultures. In this study, a simple and cost-effective cancer 3D spheroid assay suitable for small molecule anti-cancer compound screening was developed, standardized and validated on H292 non-small lung cancer cell line. A pilot screening with this assay was performed utilizing a compound library consisting of 41 anti-cancer agents. The traditional 2D monolayer cell proliferation assay was also performed with the same cell line and compounds. A correlational study based on the IC₅₀ values from the 2D and 3D assays was conducted. There is low correlation with the two sets of biological data, suggesting the two screening methods provide different information regarding the potency of the tested drug candidates.     

Disentangling the effects of global climate and regional land-use change on the current and future distribution of mangroves in South Africa

The mangrove distribution in South Africa is fragmented and restricted to small forest patches occupying only 16 % of the estuaries within the current range. In this study we used species distribution models to test (1) whether the absence of mangrove forest and its species (Avicennia marina, Bruguiera gymnorrhiza and Rhizophora mucronata) within their current range is driven by climate or by climate combined with human or geomorphic perturbation and (2) how climate change may potentially affect the latitudinal limit of the mangrove forests and its species in South Africa. We used three modelling techniques (generalized linear models, generalized additive models and gradient boosting machines) and a set of three climate-based predictive variables (minimum air temperature of the coldest month, waterbalance and growing-degree days) combined separately with an index of human or geomorphic perturbation. Climate variables for the future projections were derived from two general circulation models driven by two socio-economic scenarios (A2a and B2a). Within the range of the mangrove forest, the fragmented distribution of the mangroves in South Africa was not explained by our set of climate variables alone. The index of human perturbations slightly improved the predictions but the index of geomorphic perturbation did not. Climate change will create climatically suitable sites for the mangrove forest and the two species A. marina and B. gymnorrhiza beyond their current limits, but model outcomes did not agree on the future potential distribution of R. mucronata. We were able to successfully predict range limits and to detect future climatically suitable sites beyond the current limits. Factors controlling mangrove distribution within its range are still to be identified although absences were partly explained by human perturbations.     

Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity

Background

Matrix metalloproteinase (MMP)-dependent extracellular matrix (ECM) remodeling is a key feature in cardiometabolic syndrome-associated adipogenesis and atherosclerosis. Activation of membrane-tethered (MT) 1-MMP depends on furin (PCSK3). However, the regulation and function of the natural furin-inhibitor serpinB8 and thus furin/MT1-MMP-activity in obesity-related tissue inflammation/remodeling is unknown. Here we aimed to determine the role of serpinB8/furin in obesity-associated chronic inflammation.

Methods and Results

Monocyte → macrophage transformation was characterized by decreases in serpinB8 and increases in furin/MT1-MMP. Rescue of serpinB8 by protein overexpression inhibited furin-dependent pro-MT1-MMP activation in macrophages, supporting its role as a furin-inhibitor. Obese white adipose tissue-facilitated macrophage migration was inhibited by furin- and MMP-inhibition, stressing the importance of the furin-MMP axis in fat tissue inflammation/remodeling. Monocytes from obese patients (body mass index (BMI) >30kg/m²) had higher furin, MT1-MMP, and resistin gene expression compared to normal weight individuals (BMI<25kg/m²) with significant correlations of BMI/furin and furin/MT1-MMP. In vitro, the adipocytokine resistin induced furin and MT1-MMP in mononuclear cells (MNCs), while MCP-1 had no effect.

Conclusions

Acquisition of the inflammatory macrophage phenotype is characterized by an imbalance in serpinB8/furin, leading to MT1-MMP activation, thereby enhancing migration. Increases in MT1-MMP and furin are present in MNCs from obese patients. Dissecting the regulation of furin and its inhibitor serpinB8 should facilitate targeting inflammation/remodeling in cardiometabolic diseases.     

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.     

Assessing in vivo dynamics of multiple quality attributes from a therapeutic IgG4 monoclonal antibody circulating in cynomolgus monkey

Characterization of biopharmaceutical proteins and assessment and understanding of the critical quality attributes (CQAs) is a significant part of biopharmaceutical product development and is routinely performed in vitro. In contrast, systematic analysis of the quality attributes in vivo is not as widespread, although metabolism and clearance of multiple variants of therapeutic proteins administered to non-human primates and human subjects may have a different impact on safety, efficacy and exposure. The major hurdles of such studies are usually sample availability and technical capability. In this study, we used affinity purification coupled with liquid chromatography and mass spectrometric analysis of the digested protein for consistent and simultaneous detection of the full amino acid sequence of a therapeutic IgG4 monoclonal antibody, MAB1. This methodology allowed us to assess in vivo changes of all sequence-related modifications and quality attributes of MAB1 over the duration of a preclinical pharmacokinetic study in cynomolgus monkeys.