Discovery of new PPARγ agonists based on arylopeptoids

In this study we present the design, synthesis and biological evaluation of a small, first-generation library of small molecule aromatic amides based on the arylopeptoid skeleton. The compounds were efficiently synthesized using a highly convenient submonomer solid-phase methodology which potentially allows for access to great product diversity. The synthesized compounds were tested for their ability to activate peroxisome proliferator-activated receptors (PPARs) and they all acted as PPARγ agonists in the μM range spanning from 2.5- to 14.7-fold activation of the receptor. This is the first discovery of bioactive molecules based on the arylopeptoid architecture.     

Anti-Oxidative Effects of Melatonin Receptor Agonist and Omega-3 Polyunsaturated Fatty Acids in Neuronal SH-SY5Y Cells: Deciphering Synergic Effects on Anti-Depressant Mechanisms

Omega-3 polyunsaturated fatty acids (n-3 or omega-3 PUFAs) and melatonin receptor agonist ramelteon (RMT) both display antidepressant effects, while their cellular effects on anti-oxidative and neuroprotective mechanisms might be different. In this study, we aimed to decipher the individual and synergistic actions of n-3 PUFAs and RMT, as compared with the conventional antidepressant fluoxetine (FLX), in a cellular model of oxidative stress, which might play an important role in the pathophysiology of depression and associated disorders. We investigated the rescue and prevention effects of FLX, RMT, and n-3 PUFAs, e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), by using cell viability in SH-SY5Y cells under oxidative stress along with measurements of key cellular markers of oxidative stress, inflammatory, and neuroprotection. The results revealed that the RMT and EPA combination significantly increased the cell viability in a dose-dependent manner. RMT showed preventive effects, FLX and DHA possessed rescue effects, while EPA showed both rescue and preventive effects. We observed the dose-dependent activation and translocation of nuclear factor-κB to the nucleus augmented by the expressions of peroxisome proliferator activator receptor-gamma, tyrosine hydroxylase, c-Fos expression, and reactive oxygen species, implying that RMT and EPA combination reversed oxidative and neuroinflammatory pathophysiology and protected the neuronal cells from further damage. The results demonstrated that RMT and EPA synergistically provide effective neuroprotective, anti-oxidative/inflammatory effect against oxidative stress. Our study provides pre-clinical evidence to conduct future clinical trials of using n-3 PUFAs/RMT combination in depressive disorders.     

Immune Evasion,Stress Resistance,and Efficient Nutrient Acquisition Are Crucial for Intracellular Survival of Candida glabrata within Macrophages

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes'' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.     

Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD′

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD′. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent − 1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD′₂C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD′, has been identified. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.     

A Statistical Model for Estimation of Fish Density Including Correlation in Size,Space, Time and between Species from Research Survey Data

Trawl survey data with high spatial and seasonal coverage were analysed using a variant of the Log Gaussian Cox Process (LGCP) statistical model to estimate unbiased relative fish densities. The model estimates correlations between observations according to time, space, and fish size and includes zero observations and over-dispersion. The model utilises the fact the correlation between numbers of fish caught increases when the distance in space and time between the fish decreases, and the correlation between size groups in a haul increases when the difference in size decreases. Here the model is extended in two ways. Instead of assuming a natural scale size correlation, the model is further developed to allow for a transformed length scale. Furthermore, in the present application, the spatial- and size-dependent correlation between species was included. For cod (Gadus morhua) and whiting (Merlangius merlangus), a common structured size correlation was fitted, and a separable structure between the time and space-size correlation was found for each species, whereas more complex structures were required to describe the correlation between species (and space-size). The within-species time correlation is strong, whereas the correlations between the species are weaker over time but strong within the year.     

Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Background  

Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS) is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference.     

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract.  Objective : In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20-mm sciatic nerve defect in rats. Materials and methods : We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. Results : Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S-100 and neurofilament indicated existence of Schwann cells and axonal re-growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell-free constructs based on vein and spider silk was highly effective in nerve regeneration. Conclusion : We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re-growth in a long-distance model for peripheral nerve regeneration.