Application of phage display and plasmid display to broaden the specificity of human Fbs1 for capture of N-glycosylated peptides

The objective of this study was to develop a method for selective and comprehensive enrichment of N-linked glycopeptides to facilitate biomarker discovery. The natural function of human Fbs1 is to bind misfolded N-linked glycoproteins by recognition of the common pentasaccharide core motif (Man3GlcNAc2) of the N-glycan. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules, however, wild-type Fbs1 preferentially binds high mannose containing glycans. To reduce the bias during N-glycomolecule enrichment experiments, we isolated Fbs1 variants with altered specificity through mutagenesis and plasmid display selection. Five cycles of E. coli propagation and in vitro panning against immobilized fetuin resulted in a pool of variants with improved binding for complex N-glycopeptides. The most valuable Fbs1 variant enabled substantially unbiased N-glycopeptide enrichment from a level of 3.5% to 66% when applied to IgG-depleted serum. Importantly, plasmid display is a rapid method for altering substrate binding specificity that is an attractive alternative to phage display. M13 phage display of Fbs1 was also accomplished by non-standard methodology. Since Fbs1 folding is impaired by disulfide bond formation in the E.coli periplasm, a mutant E.coli host was critical for proper display on the surface of M13 phage. Furthermore, display of functional Fbs1 could only be achieved by expressing the pIII-Fbs1 fusion protein with an SRP-dependent signal peptide. Significance: The Fbs1 study revealed that plasmid display is a powerful alternative to phage display. In particular, plasmid display is appropriate for non-secretory proteins. The plasmid display selection process is very rapid as 5 cycles may be performed in 5 days. Our efforts to display Fbs1 by M13 phage display were met by complication. In response, we developed a method capable of functional display of non-secretory protein on the surface of M13 phage. Reference: Chen M and Samuelson JC “A DsbA-deficient Periplasm Enables Functional Display of a Protein with Redox-Sensitive Folding on M13 Phage” Biochemistry (2016) 55(23):3175-9

E. coli strain engineering to minimize host cell protein contamination of recombinant target protein

Microbial hosts are preferentially employed for recombinant protein production, as clone generation is easier relative to eukaryotic host systems. Also, methods for manipulation of microbial genomes are generally more straightforward. Using E.coli as a model, we demonstrate a rapid genome engineering method named FAST-GE and compare this method to several other common techniques. Examples of protein expression host genome modification are highlighted where the final objective is isolation of highly pure target protein. Host cell protein (HCP) analysis is a highly sensitive quality assurance test and is a standard during the purification of therapeutic proteins. In some cases, host genome modification may be necessary to eliminate specific contaminating proteins since contaminant removal by conventional chromatography methods reduces target protein yield too severely. The NiCo21(DE3) strain of E.coli is a BL21(DE3) derivative engineered to aid in the isolation of poly-histidine tagged recombination protein. The advantages of employing NiCo21(DE3) for recombinant protein expression will be described

Contraceptive discontinuation and switching behavior among family planning clinic clients in Dalhatu Araf Specialist Hospital, Lafia

Background: Contraceptives are effective ways with which couples can limit or space the number of children they have. Several methods of contraception exist, both modern and traditional methods. Couples have a myriad of these from which to choose from. However, contraceptive discontinuation and switching are a reality. The dynamics of contraceptive use, discontinuation and switching are important markers of how well the programs are meeting the family planning needs of women and couples. The aim of the study was to ascertain the magnitude of women who wanted to discontinue or switch their present contraceptive methods and establish the reasons why. Methods: Our study was a cross sectional descriptive study of women attending the family planning clinic of Dalhatu Araf Specialist Hospital, Lafia over a 12 month period. A self-administered structured questionnaire was administered to the family planning clinic clients after obtaining a written informed consent. Results: Contraceptive discontinuation rate was 36.5%, and the switching rate was 5.2%. The commonest reasons for discontinuing contraception were; desirous of pregnancy (43%), side effects of method (28.2%), husband’s disapproval (16.7%), marital dissolution (4.2%), inconvenience of use (3.1%), failure of method (1.6%) and menopause (0.4%). The reasons for switching were also similar and include; side effects of the method (51.4%), inconvenience of use (16.2%), husband’s disapproval (8.1%), personal choice (5.4%) and marital dissolution (2.7%). Conclusions: We concluded that the contraceptive discontinuation rate was moderately high, while the switching rate was low. We recommend adequate counseling of clients before contraceptive uptake to forestall this

A mathematical model of the Gini coefficient and evaluation of the redistribution function of the tax system in the Czech Republic

The paper deals with two related topics – a mathematical model of the Gini coefficient, and its application in the taxation theory. However, the mathematical part of the paper should not only be understood as a traditional methodological starting point, but it has also didactic character and correctly describes the properties of the Gini coefficient, pointing to misinterpretation deficiencies found in a number of contemporary works. In the application part of the paper, we study the fulfillment of redistribution function of the tax system in the Czech Republic by using the Musgrave-Thin index coefficient M. Our research implies that the global progressivity of the tax allowance system in the Czech Republic was moderately progressive throughout the analyzed period 2006–2016. The considerable change in the personal income tax structure made in 2008 did not essentially reflect the global tax progressivity.Web of Science66675073

Proteinase K goes thermo-labile

Proteinase K, originally from the fungus Tritirachium album, is a highly active serine protease with broad cleavage specificity. This enzyme is widely used to remove proteins/enzymes in nucleic acid samples. However, use of wildtype proteinase K (WTPK) in multi-step enzymatic workflows such as next generation sequencing (NGS) is limited due to its extreme thermostability and ineffective removal by heat treatment. The purpose of this study was to engineer a thermolabile Proteinase K (TLPK) as active as WTPK, which may be fully inactivated at 65°C or below to minimize DNA/RNA damage. Using molecular engineering approaches, we have successfully obtained TLPK. As shown in Figure 1, TLPK is almost as active as WTPK at 37°C using native bovine serum albumin (BSA) as substrate. Importantly, TLPK can be efficiently inactivated within the temperature range of 55°C to 65°C, which is demonstrated by loss of protease activity on bovine serum albumin (BSA) substrate (Figure 2a) and a colorimetric peptide substrate (Figure 2b) after heat treatment. Compared to WTPK, TLPK shows over 20°C more labile to heat inactivation. The melting temperature (Tm) of TLPK is also around 25°C lower than that of WTPK, decreasing from 75.9°C to 50.9°C. TLPK greatly outperforms a broad specificity protease isolated from an arctic marine microbial source, both by specific enzyme activity and thermolability. One of the TLPK applications is it can inactivate heat resistant restriction enzymes such as PvuII and PstI without affecting downstream reactions. The mainstream applications may be its incorporation into multi-step enzymatic workflows such as NGS sample preparation. Unlike WTPK, TLPK can be used to eliminate an enzyme function without contaminating the next enzymatic step in the same reaction vessel. New England Biolabs has tested TLPK and found it to simplify and improve NGS workflows. Please click Additional Files below to see the full abstract

Measurement in Economics and Social Science

The paper discusses measurement, primarily in economics, from both analytical and historical perspectives. The historical section traces the commitment to ordinalism on the part of economic theorists from the doctrinal disputes between classical economics and marginalism, through the struggle of orthodox economics against socialism down to the cold-war alliance between mathematical social science and anti-communist ideology. In economics the commitment to ordinalism led to the separation of theory from the quantitative measures that are computed in practice: price and quantity indexes, consumer surplus and real national product. The commitment to ordinality entered political science, via Arrow’s ‘impossibility theorem’, effectively merging it with economics, and ensuring its sterility. How can a field that has as its central result the impossibility of democracy contribute to the design of democratic institutions? The analytical part of the paper deals with the quantitative measures mentioned above. I begin with the conceptual clarification that what these measures try to achieve is a restoration of the money metric that is lost when prices are variable. I conclude that there is only one measure that can be embedded in a satisfactory economic theory, free from unreasonable restrictions. It is the Törnqvist index as an approximation to its theoretical counterpart the Divisia index. The statistical agencies have at various times produced different measures for real national product and its components, as well as related concepts. I argue that all of these are flawed and that a single deflator should be used for the aggregate and the components. Ideally this should be a chained Törnqvist price index defined on aggregate consumption. The social sciences are split. The economic approach is abstract, focused on the assumption of rational and informed behavior, and tends to the political right. The sociological approach is empirical, stresses the non-rational aspects of human behavior and tends to the political left. I argue that the split is due to the fact that the empirical and theoretical traditions were never joined in the social sciences as they were in the natural sciences. I also argue that measurement can potentially help in healing this split

Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5′-GCGGCCGC-3′) has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5′-NCGGCCGN-3′ sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5′-GCTGCCGC-3′. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5′-GCTGCCGC-3′ with a specific activity of 8.2 × 10(4) U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

AbstractThe effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 μg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 μg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host’s immunomodulatory responses