Engineering a therapeutic IgG molecule to address cysteinylation,aggregation and enhance thermal stability and expression

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T_m, 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.     

DISC1 and the aggresome

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

Stimulation of autophagy is neuroprotective in a mouse model of human tauopathy

The most common neurodegenerative diseases are characterized by the accumulation of misfolded proteins. Tauopathies, which include Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, Pick disease and cases of frontotemporal dementia and parkinsonism linked to chromosome 17, are characterized by the accumulation of hyperphosphorylated and filamentous MAPT/tau protein. The pathological mechanisms involved in MAPT protein accumulation are not well understood, but a possible impairment of protein degradation pathways has been suggested. We investigated the effects of autophagy stimulation on MAPT pathology in a model tauopathy, the human mutant P301S MAPT transgenic mouse line. In the brain of the trehalose-treated mutant mice, autophagy is activated and a reduced number of neurons containing MAPT inclusions, as well as a decreased amount of insoluble MAPT, are observed. The improvement of MAPT pathology is associated with increased nerve cell survival. Moreover, MAPT inclusions colocalize with SQSTM1/p62- and LC3-positive puncta, suggesting the colocalization of MAPT aggregates with autophagic vacuoles. Autophagy is not activated in the spinal cord of the human P301S MAPT transgenic mice and neuronal survival, as well as MAPT pathology, is unaffected. This study supports a role for autophagy stimulation in the degradation of MAPT aggregates and opens new perspectives for the investigation of autophagy as a pathological mechanism involved in neurodegenerative diseases.     

Timing alters how a heat shock affects a host-parasitoid interaction

Abiotic factors' effects on species are now well-studied, yet they are still often difficult to predict, especially for strongly interacting species. If these altered abiotic factors and species interactions occur as discrete events in time, such complications may occur because of the events’ relative timing. One such discrete abiotic factor is the short-duration, large magnitude increase in temperature called a heat shock. This study investigates how the timing of heat shocks affects the successful attack and reproduction of a parasitoid wasp (Aphidius ervi) attacking its host, the pea aphid (Acyrthosiphon pisum). We tested three relative timings: 1) heat shock before the wasp attacks hosts, 2) heat shock while the wasp is foraging, and 3) heat shock after the wasp has attacked hosts. In each scenario we compared wasp mummy production (pupal stage) with and without a heat shock. Our results showed that a heat shock had the largest effect when it occurred while wasps actively foraged, with fewer mummies produced when exposed to a heat shock compared to the no heat shock control. Follow-up behavioral tests suggest this was caused by wasps becoming inactive during heat shocks. In contrast, when heat shocks were applied three days before or after foraging, we found no difference in mummy production between the heat shock treatment and no heat shock control. These results show the potential importance of timing when considering the ramifications of an altered abiotic factor, especially with relatively discrete abiotic events and interactions.     

Investigating food limitations in wild fisheries: the attendance and growth responses of fish at an anthropogenic feeding station within a temperate estuary

To investigate whether wild fish populations are food limited, this study explored whether the provision of supplementary food had a positive effect on the abundance, condition and growth characteristics of estuarine fish assemblages in New Zealand. Feed (7690 kg) was delivered from an anthropogenic feeding station over a 60-week period to a naturally occurring assemblage of wild fish. Yellow-eyed mullet (Aldrichetta forsteri) of juvenile, sub-adult and newly matured size classes were the dominant species actively foraging at the feeding station throughout its operation, whereas larger piscivorous species visited and foraged from the feed station over the summer period only. Other species were present in the wider area of Nelson Haven, but not at the feed station. No obvious changes in the condition factor of yellow-eyed mullet were observed across the period of monitoring, and no changes in their catchability were detected – although marked seasonal variation in catch rates was observed. Results from tag-recapture data identify that the length-based growth rates of yellow-eyed mullet recaptured near the feed station were higher than those of tagged individuals recaptured at a nearby comparison site, and were higher than the growth rates observed in natural populations of yellow-eyed mullet in the wider region. Shifts in the median size of fish, as observed by acoustic observations, agreed with the tag-recapture data. Although some of the results identified were not amenable to statistical analyses, the attendance of yellow-eyed mullet at the feeding station, in combination with the improved estimates of growth by most of the techniques employed, indicates that yellow-eyed mullet are food limited in their natural environment and that the growth performance of these fish can be positively affected by the increased availability of food.     

Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

Life history shifts and alterations in the early development of parasitic wasps

Summary

In insects, the regulation of embryonic development has been intensively studied in model species like Drosophila melanogaster. Previous comparative studies have suggested that the developmental processes documented in Drosophila well describe embryogenesis of holometabolous insects generally. However, there have been few attempts to take into account how life history has influenced insect embryogenesis or to characterize early development of species with life histories fundamentally different from flies. Our studies of advanced insects in the order Hymenoptera suggest that punctuated shifts in life history can profoundly influence these events. In particular, alterations associated with the evolution of endoparasitism argue that departures from the fly paradigm may occur commonly among insects that develop under conditions different from typical terrestrial species.     

Aerosol generation using nanometer liposome suspensions for pulmonary drug delivery applications

Abstract

Pulmonary lung targeting finds applications in drug delivery to the lung itself and to other body organs, via blood circulation following transfer across alveolar membranes. Understanding pulmonary drug delivery systems towards improving their efficacy needs identification of particle sizes of relevance and elucidation of links between suspension properties, techniques of atomisation and properties of the generated aerosols. This review article is focussed on understanding the elements of pulmonary drug delivery, specifically related to suspensions of small liposomes. Specific objectives of this review include (a) understanding aerosol particle deposition and absorption on pulmonary surface, (b) links between properties of aerosol generation and colloidal drug carriers used for drug encapsulation, and (c) investigation on the controlled properties of liposome aerosols generated using different atomisation techniques for efficacious aerosol therapy.     

Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot–Marie–Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.     

Fibrillation of human serum albumin shows nonspecific coordination on stoichiometric increment of Copper(II)

Protein aggregation is related to a series of pathological disorders the main cause of which are the fibrillar species generated during the process. Human serum albumin (HSA) undergoes rapid fibrillation in the presence of Cu(II) at pH 7.4 in 60% ethanol after 6-h incubation (～65?°C) followed by room temperature incubation. Here, we have investigated the effect of a stoichiometric variation of Cu(II) on the self-assembly of HSA using Congo red and thioflavin T dye-binding studies, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, fluorescence microscopy and transmission electron microscopy. The simulation of EPR spectra suggests that with the increment in Cu(II) ion concentration, there is a change in ligand field coordination. Kinetic parameters indicate reduced cooperativity that may be related to the nonspecific coordination on increment of Cu(II) concentration. Cu(II) is also able to direct the accumulation of a large number of fibers along with a formation of dense fibrillar network which is evident from microscopic images.