Tyrosine bioconjugation - an emergent alternative

Protein bioconjugation is an increasingly important field of research, with wide-ranging applications in areas such as therapeutics and biomaterials. Traditional cysteine and lysine bioconjugation strategies are widely used and have been extensively researched, but in some cases they are not appropriate and alternatives are needed or they are not compatible with one another to enable the formation of dually (and distinctly) modified dual-conjugates (an increasingly desired class of bioconjugates). Here we review the heretofore less explored approach of tyrosine bioconjugation, which is rapidly becoming a constructive alternative/complement to the more well-established strategies. Herein we present an overview of the field, and then focus on promising recent methods that can achieve high conversion and chemoselectivity. This suggests that not only can tyrosine bioconjugation be used in conjunction with cysteine and lysine modification to obtain proteins with multiple different modifications, it is also becoming a stand-alone alternative to these more traditional methods

A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability

Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide–thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C–S and C–N linked thio-bioconjugates demonstrated C–S functionalized linkers to be cleavable and C–N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments

Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation

Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-target modification with blood thiols (e.g., glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker

Having a lot of a good thing: multiple important group memberships as a source of self-esteem.

Copyright: © 2015 Jetten et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedMembership in important social groups can promote a positive identity. We propose and test an identity resource model in which personal self-esteem is boosted by membership in additional important social groups. Belonging to multiple important group memberships predicts personal self-esteem in children (Study 1a), older adults (Study 1b), and former residents of a homeless shelter (Study 1c). Study 2 shows that the effects of multiple important group memberships on personal self-esteem are not reducible to number of interpersonal ties. Studies 3a and 3b provide longitudinal evidence that multiple important group memberships predict personal self-esteem over time. Studies 4 and 5 show that collective self-esteem mediates this effect, suggesting that membership in multiple important groups boosts personal self-esteem because people take pride in, and derive meaning from, important group memberships. Discussion focuses on when and why important group memberships act as a social resource that fuels personal self-esteem.This study was supported by 1. Australian Research Council Future Fellowship (FT110100238) awarded to Jolanda Jetten (see http://www.arc.gov.au) 2. Australian Research Council Linkage Grant (LP110200437) to Jolanda Jetten and Genevieve Dingle (see http://www.arc.gov.au) 3. support from the Canadian Institute for Advanced Research Social Interactions, Identity and Well-Being Program to Nyla Branscombe, S. Alexander Haslam, and Catherine Haslam (see http://www.cifar.ca)

Factors affecting phage D29 infection: a tool to investigate different growth states of mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay

Incorporating conceptual site models into national-scale environmental risk assessments for legacy waste in the coastal zone

Solid wastes deposited in the coastal zone that date from an era of lax environmental regulations continue to pose significant challenges for regulators and coastal managers worldwide. The increasing risk of contaminant release from these legacy disposal sites, due to a range of factors including rising sea levels, associated saline intrusion, and greater hydrological extremes, have been highlighted by many researchers. Given this widespread challenge, and the often-limited remedial funds available, there is a pressing need for the development of new advanced site prioritization protocols to limit potential pollution risks to sensitive ecological or human receptors. This paper presents a multi-criteria decision analysis that integrates the principles of Conceptual Site Models (Source-Pathway-Receptor) at a national scale in England and Wales to identify legacy waste sites where occurrence of pollutant linkages are most likely. A suite of spatial data has been integrated in order to score potential risks associated with waste type (Source), likelihood of pollutant release relating to current and future flood and erosion climate projections, alongside current management infrastructure (Pathway), and proximity to sensitive ecological features or proxies of human use in coastal areas (Receptors). Of the 30,281 legacy waste deposits identified in England and Wales, 3,219 were located within the coastal zone, with coastal areas containing a density of legacy wastes (by area) 10.5 times higher than inland areas. Of these, 669 were identified as priority sites in locations without existing coastal defences or flood management infrastructure, with 2550 sites identified in protected areas where contaminant transfer risks could still be apparent. The majority (63 %) of the priority sites have either undefined source terms, or are classified as mixed wastes. Mining and industrial wastes were also notable waste categories, and displayed strong regional distributions in the former mining areas of north-east and south-west of England, south Wales, and post-industrial estuaries. The large-scale screening process presented here could be used by environmental managers as a foundation to direct more high-resolution site assessment and remedial work at priority sites, and can be used as a tool by governments for directing funding to problematic sites

Asthmatics Exhibit Altered Oxylipin Profiles Compared to Healthy Individuals after Subway Air Exposure

Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications.This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air.Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information.Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E(2) (PGE(2)). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change.Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

Background: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phageresistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD 50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophag

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion