Monitoring the dynamic vulnerability of an Arctic subsistence food system to climate change: The case of Ulukhaktok, NT.

Vulnerability to climate change is highly dynamic, varying between and within communities over different timescales. This paper draws upon complex adaptive systems thinking to develop an approach for capturing, understanding, and monitoring climate vulnerability in a case study from northern Canada, focusing on Inuit food systems. In the community of Ulukhaktok, Northwest Territories, we followed 10 hunters over a 2-year period, asking them to document their harvesting activities and discuss their lived experience of harvesting under changing environmental and societal conditions. GPS monitoring and participatory mapping sessions were used to document 23,996km of trails (n = 409), with conversational bi-weekly semi-structured interviews and secondary instrumental weather data used to contextualise climate change within a nexus of other socioeconomic, cultural, and political stressors that also affect harvesting. Our results demonstrate that climate change has considerable potential to affect harvesting activities, particularly when its impacts manifest as anomalous/extreme events. However, climate change impacts are not necessarily the most salient issues affecting harvesting on a day-to-day basis. Instead, factors relating to economics (particularly financial capital and the wage-based economy), social networks, and institutions are found to have a greater influence, either as standalone factors with cascading effects or when acting synchronously to augment the impacts of environmental change.

Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes.

Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (-4 to -8°C) and high concentrations of ethanol 8-40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80-90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.

Validation of concurrent preimplantation genetic testing for polygenic and monogenic disorders, structural rearrangements, and whole and segmental chromosome aneuploidy with a single universal platform.

Preimplantation genetic testing (PGT) has been successfully applied to reduce the risk of miscarriage, improve IVF success rates, and prevent inheritance of monogenic disease and unbalanced translocations. The present study provides the first method capable of simultaneous testing of aneuploidy (PGT-A), structural rearrangements (PGT-SR), and monogenic (PGT-M) disorders using a single platform. Using positive controls to establish performance characteristics, accuracies of 97 to >99% for each type of testing were observed. In addition, this study expands PGT to include predicting the risk of polygenic disorders (PGT-P) for the first time. Performance was established for two common diseases, hypothyroidism and type 1 diabetes, based upon availability of positive control samples from commercially available repositories. Data from the UK Biobank, eMERGE, and T1DBASE were used to establish and validate SNP-based predictors of each disease (7,311 SNPs for hypothyroidism and 82 for type 1 diabetes). Area under the curve of disease status prediction from genotypes alone were 0.71 for hypothyroidism and 0.68 for type 1 diabetes. The availability of expanded PGT to evaluate the risk of polygenic disorders in the preimplantation embryo has the potential to lower the prevalence of common genetic disease in humans.

Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats.

For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

Purification of pegylated proteins.

Separation of PEGylated proteins is challenging because PEG itself is a relatively inert, neutral, hydrophilic polymer and the starting point for PEGylation is a pure protein. Thus, other than molecular weight and size, differences in the physicochemical properties typically used to fractionate proteins may be slight between different PEGylated forms of a protein. The usual properties of electrostatic charge and molecular weight (size) form the basis of the most commonly used separation techniques, particularly IEC, SEC, and ultrafiltration. The main effect of PEGylation on ion-exchange separations is to shield the electrostatic charges on the protein surface and to reduce the strength of interactions with higher PEG chain molecular weight or higher PEGylation extent. Thus, ion exchange can be used very effectively to separate on the basis of PEGylation extent for low extents, but as N increases, the effectiveness of separation rapidly diminishes. Separation of positional isomers is possible by RPC or ion exchange at analytical scale, but it is problematic at the preparative scale due to the small size of the differences in electrostatic interactions between isomers. PEGylation imparts significant changes in molecular weight with each chain added to a protein and there are corresponding increases in molecular size, so SEC and ultrafiltration (and dialysis) are effective methods for separating native and PEGylated proteins. However, the relative size difference between variants differing in PEGylation extent by one adduct reduces with N, so that efficient SEC separation between PEGylated species differing by one PEG chain is not likely to be economic at the preparative scale for N > 3. This holds true even for PEG proteins produced with large PEG polymers (Mr > or =20 kDa). For small PEG polymers (Mr = 2 kDa), only native and PEGylated species can be separated effectively. At the analytical scale, with proper calibration, SEC can provide valuable information on PEGylation extent. Membranes can be used to reduce the concentration of smaller molecular weight species by dialysis but cannot fully remove them, and an operational trade-off between purity and yield is required. Gel electrophoresis can confirm PEGylation reactions have proceeded and indicate the relative purity of products, but its use to confirm PEGylation extent requires further research. The main drawback of separations based solely upon molecular size is that they cannot differentiate between positional isomers. Capillary electrophoresis is an exception, quantitatively combining any or all of size, shape, conformational freedom, and small differences in protein surface properties to separate by both PEGylation extent and positional isomerism. Relative hydrophobicity is a useful property for analytical separations using RPC, but HIC, which is used routinely for production-scale purification of proteins, does not appear to be particularly useful for separation of PEGylated species.

Multipurpose peptide tags for protein isolation.

A multifunctional peptide tag (HYDHYD) consisting of histidine, tyrosine and aspartate residues was fused to the N-terminal ends of green fluorescent protein (GFP), lactate dehydrogenase (LDH) and human hemoglobin (Hb), proteins which were subjected to ion-exchange chromatography (IEC), aqueous two-phase system partition, immobilized metal-ion affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC). Tagged GFP was retained significantly longer (>1 column volume) in both HIC and IEC. It exhibited 3x greater partition in favor of the hydrophobic phase in a two-phase system and 96% could be bound to an IMAC column which did not bind native GFP.

Novel in situ polymerized coatings for hydrophobic interaction chromatography media.

Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(R) polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, alpha-chymotrypsinogen A, myoglobin and alpha-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70mgprotein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (YPYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media.

Ion exchange chromatography of antibody fragments.

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.

Modeling of protein interactions with surface-grafted charged polymers. Correlations between statistical molecular modeling and a mean field approach.

Ion exchange media involving charge groups attached to flexible polymers are widely used for protein purification. Such media often provide enhanced target protein purity and yield. Yet, little is understood about protein interaction with such media at the molecular level, or how different media architectures might affect separation performance. To gain a better understanding of such adsorptive systems, statistical mechanical perturbation calculations, utilizing a Debye-Hückel potential, were performed on surface-grafted charged polymers and their interaction with model proteins. The studied systems were weakly charged, and the polymers were linear and relatively short (degree of polymerization is 30). Segment distributions from the surface were also determined. The interaction of spherical model protein particles of 12-30 A radius were investigated with respect to polymer grafting density, distance from matrix surface, protein charge, and ionic strength. The partitioning coefficient of the model proteins was determined for different distances from the surface. An empirical mean field theory that scales the entropy of the protein with the square of the protein radius correlates well to Monte Carlo statistical modeling results. Upon adsorption to the polymer layers, the model proteins exhibit a critical surface charge density that is proportional to the ionic strength, independent of the grafting density, and appears to be a fundamental determinant of protein adsorption. Partitioning of protein-like nanoparticles to the charged polymer surface is only favored above the particle critical charge density.

Prediction of the viscosity radius and the size exclusion chromatography behavior of PEGylated proteins.

Size exclusion chromatography (SEC) was used to determine the viscosity radii of equivalent spheres for proteins covalently grafted with poly(ethylene glycol) (PEG). The viscosity radius of such PEGylated proteins was found to depend on the molecular weight of the native protein and the total weight of grafted PEG but not on PEG molecular weight, or PEG-to-protein molar grafting ratio. Results suggest grafted PEG's form a dynamic layer over the surface of proteins. The geometry of this layer results in a surface area-to-volume ratio approximately equal to that of a randomly coiled PEG molecule of equivalent total molecular weight. Two simple methods are given to predict the viscosity radius of PEGylated proteins. Both methods accurately predicted (3% absolute error) the viscosity radii of various PEG-proteins produced using three native proteins, alpha-lactalbumin (14.2 kDa MW), beta-lactoglobulin dimer (37.4 kDa MW), and bovine serum albumin (66.7 kDa MW), three PEG reagents (2400, 5600, and 22500 MW), and molar grafting ratios of 0 to 8. Accurate viscosity radius prediction allows calculation of the distribution coefficient, K(av), for PEG-proteins in SEC. The suitability of a given SEC step for the analytical or preparative fractionation of different PEGylated protein mixtures may therefore be assessed mathematically. The methods and results offer insight to several factors related to the production, purification, and uses of PEGylated proteins.

N-terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition.

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.

Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins.

Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated. Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.