Co‐expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor‐β1 into a biologically active protein

Transforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.     

Combined experimental and theoretical study on the removal of pollutant compounds by peroxidases: affinity and reactivity toward a bioremediation catalyst

Water pollution is a significant and growing problem throughout the world, especially in developing countries. In order to minimize environmental problems, catalysts have increasingly been designed to remove pollutants from the water. In an attempt to innovate by the creation of new low-cost alternatives to efficiently remove pollutants, the enzymatic treatment has been intensely studied for this purpose. Reactions catalyzed by enzymes are able to perform specific treatments, commonly with high rates of the final products. With this, the enzyme, peroxidase, is a promising candidate as a bioremediation catalyst. The efficiency of oxidoreductive enzymes, such as horseradish peroxidase (HRP) and soybean peroxidase (SP) have been studied, given that their performance depends on the substrate. In this investigation, experimental techniques and theoretical calculations have been employed in order to investigate the oxidative process for the ferulic acid and bromophenol blue dyes, performed by HRP and SP. Both enzymes showed a comparable behavior with respect to ferulic acid substrate. On the other hand, by utilizing bromophenol blue dye as a substrate, the behavior of the employed catalysts was significantly different. Experimental data have shown that HRP was more active toward bromophenol blue when compared to ferulic acid, being more rapidly degraded by the HRP enzyme. This tendency was confirmed by our theoretical docking, PM6 semi-empirical method, and DFT calculation results, in which the interaction, binding energies, and transition states were determined.     

Terrestrial Mammal Occupancy in the Context of Widespread Forest Loss and a Proposed Interoceanic Canal in Nicaragua's Decreasingly Remote South Caribbean Region

Central America is experiencing rapid forest loss and habitat degradation both inside and outside of protected areas. Despite increasing deforestation, the Caribbean region of Nicaragua plays an important role in the survival or extinction of large mammal populations in Central America given that it still retains core areas of habitat for large mammal species. The proposed interoceanic canal project that would bisect the southern half of this Caribbean region represents a new threat that, combined with an advancing agricultural frontier, could affect populations of large mammal species such as jaguars, white-lipped peccaries, and Baird’s tapirs. We used occupancy models to examine the relative occupancy probabilities for an assemblage of terrestrial mammals in the south Caribbean region of Nicaragua to identify current core areas for our study species and conduct a preliminary evaluation of the potential impacts of the proposed interoceanic canal. We modeled a community level distribution of eight species with varying levels of sensitivity to human encroachment and a range of habitat associations. Our model results reveal three priority areas for terrestrial mammal conservation in our study area. The mapped predictions show that the only remaining area of suitable habitat for large mammals in the path of the proposed interoceanic canal is a relatively thin strip of forest that runs along the Caribbean Coast. In light of these findings, we propose five recommendations that will help ensure the conservation of this area of the proposed canal route as suitable habitat for our study species.     

The Effect of Social Parasitism by Polyergus breviceps on the Nestmate Recognition System of Its Host,Formica altipetens

Highly social ants, bees and wasps employ sophisticated recognition systems to identify colony members and deny foreign individuals access to their nest. For ants, cuticular hydrocarbons serve as the labels used to ascertain nest membership. Social parasites, however, are capable of breaking the recognition code so that they can thrive unopposed within the colonies of their hosts. Here we examine the influence of the socially parasitic slave-making ant, Polyergus breviceps on the nestmate recognition system of its slaves, Formica altipetens. We compared the chemical, genetic, and behavioral characteristics of colonies of enslaved and free-living F. altipetens. We found that enslaved Formica colonies were more genetically and chemically diverse than their free-living counterparts. These differences are likely caused by the hallmark of slave-making ant ecology: seasonal raids in which pupa are stolen from several adjacent host colonies. The different social environments of enslaved and free-living Formica appear to affect their recognition behaviors: enslaved Formica workers were less aggressive towards non-nestmates than were free-living Formica. Our findings indicate that parasitism by P. breviceps dramatically alters both the chemical and genetic context in which their kidnapped hosts develop, leading to changes in how they recognize nestmates.     

A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation

Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.