Biochemical and computational studies towards selective inhibition of the immunoproteasome

The proteasome pathway degrades >90% of cytosolic proteins deemed redundant, misfolded or toxic, thereby influencing key regulatory pathways including: cell cycle control, DNA repair and apoptosis. As such, proteasome inhibitors (PI) have exhibited broad therapeutic applications, particularly for multiple myeloma and mantle cell lymphoma with 3 inhibitors gaining FDA approval. However, covalent binding and lack of targeted action cause severe toxicity. Upon stimulation by inflammatory cytokines, constitutive proteasome (CP) active sites β1c, β2c and β5c are replaced with corresponding β1i, β2i and β5i subunits; forming the immunoproteasome (IP). The abundant CP is required for regular cell function, however due to upregulation in diseased states selective IP inhibition is associated with an increased therapeutic index. Recent identification of structural differences between CP and IP specificity pockets (S1-4) allows structure-based drug design. The cyclic peptides argyrin A and F exhibit potent, reversible CP inhibition with mechanisms distinct to existing therapeutics. In this project, argyrin B inhibition and binding interactions between the CP and IP are investigated, using purified enzyme assays alongside computational molecular modelling. Kinetic assays revealed argyrin B IC_50 values of 146.5 μM and 8.76 μM at β1c and β1i, respectively; a 16-fold difference with statistical significance. Whilst argyrin B also showed slight preference towards β5i over β5c, with low micromolar IC_50 values. The same trends were supported by Ki values and molecular docking estimated binding energies. AutoDock and FRED simulations suggest increased β1i S1 pocket hydrophobicity, T21S and G97H substitutions from β1c to β1i as key towards favourable β1i binding. At β5c, small, hydrophobic characteristics of S2 become polar in β5i that enhances argyrin B interactions. These findings facilitate design of further IP selective inhibitors, whilst the identification of the first known β1i selective and non-covalent PI shows great therapeutic potential with reduced toxicity proposed in comparison to existing therapeutics

A continuum approach to lifestyle entrepreneurship.

Recent years have witnessed an increased interest in small independently operated businesses with researchers expressing an interest in the manner in which the independent owner-operator is motivated to enter the market and their subsequent approach to business. Research into these small firms has shown that the owner/operators may create their businesses for a variety of reasons. However an emerging perspective is that not all individuals will actively pursue traditional objectives such as growth and profit maximisation, rather they are increasingly choosing their occupation to suit their style of life. Lifestyle motivations have thus been recognised in the literature as important stimuli to small business formation. Various research has been undertaken into the lifestyle construct and the impact on the motivations and behaviours of the individual towards the creation and development of smallscale enterprises. This study aims to build on recent work in this area to provide an enhanced understanding of lifestyle theory. This study adopts an interpretivist approach to understand the fundamental meanings attached to lifestyle entrepreneurship in the context of the small business. Bed and Breakfast (B&B) accommodation operations are used as a frame through which to understand the motivations of the individual towards venture creation. This study develops the ideal typifications of Business Orientated Lifestylers, Lifestyle Focused Business Performers and Self Expressive Homemakers to help explain the complex and dynamic range of lifestyle motivations and objectives present, and proposes the model of a continuum to portray these lifestyle business owners as being between low intensity and high intensity lifestyle goals and objectives. It is suggested that the typologies and subsequent lifestyle continuum presented in this study can be used by researchers, policy makers and practitioners to better understand the lifestyle entrepreneur and the environment within which they operate, and further, to support these lifestyle entrepreneurs in the operation of their business

Cellulose and hemicellulose digestion by herbivorous terrestrial crustaceans

Cellulose, the main component of plant cell walls, is insoluble and difficult to digest enzymatically. This research discovered that herbivorous land crabs have an efficient gastric mill in the stomach which disrupts this insoluble material, and a range of highly specialised enzymes that can then break down the cellulose

Argyrin B a non-competitive inhibitor of the human immunoproteasome exhibiting preference for β1i

Inhibitors of the proteasome have found broad therapeutic applications however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity

“In there but not in there”:Sibling sexual abuse as a disruptor in the field of child sexual abuse

In this commentary paper, which draws on the authors’ own past research and practice experience in the field of child sexual abuse prevention as well as a thorough knowledge of the extant literature, we argue that sibling sexual abuse challenges conventional thinking about child sexual abuse, both in terms of how the general public conceptualises child sexual abuse and in terms of our practice responses to it. Traditional service responses are often inadequate and inappropriate in situations involving sexual abuse between siblings. The question is further raised as to whether traditional service responses are appropriate for other forms of child sexual abuse. We argue that instead of siloed, individualist therapy and criminal or youth justice responses, whole-family responses are required, which draw on the principles of family therapy and restorative justice

Purification and characterisation of endo-β-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor

Laminarinase and endo-&beta;-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a Vmax of 42.0 &micro;mol reducing sugars produced min&ndash;1 mg protein&ndash;1, a Km of 0.126% (w/v) and an optimum pH range of 5.5&ndash;7, and hydrolysed mainly &beta;-1,3-glycosidic bonds. In addition to the hydrolysis of &beta;-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of &beta;-1,4-glycosidic bonds. It had a Vmax of 19.6 &micro;mol reducing sugars produced min&ndash;1 mg protein&ndash;1, a Km of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-&beta;-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4&ndash;7. It mainly hydrolysed &beta;-1,4-glycosidic bonds, but was also capable of significant hydrolysis of &beta;-1,3-glycosidic bonds. Two endo-&beta;-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53&plusmn;3 and 52 kDa, were purified from C. destructor. Endo-&beta;-1,4-glucanase 1 was only capable of hydrolysing &beta;-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-&beta;-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose. <br /

Light in Medicine: The Interplay of Chemistry and Light

Photodynamic therapy (PDT) has had mixed reception in the clinic, with most success stories being based on the ablative capacity of PDT. In these applications, maximal combinations of light and an exogenous photosensitiser are used to generate high levels of reactive oxygen species (ROS) that induce cell death either directly via necrosis or indirectly via vascular damage. However, recent advances in understanding the complex role of ROS in cell signalling have revealed potential new applications for PDT. For example, the proliferative effects of low level ROS could be applied to wound healing or immunomodulation. These effects should also be considered in the ablative applications. With the decades of chemical advances for ablative PDT at hand – including targeting mechanisms to diseased cells and subcellular locations, optimisation of light absorption, and carrier mechanisms that modulate the therapeutic response – the application of PDT to other types of treatment could be relatively rapid. This review serves to summarise some of these developments and suggest future directions