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

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

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

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

Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI

BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0), respectively. We have purified and partially characterized these two enzymes, and analyzed the genes that encode them. BsrDI and BtsI are unusual in two respects: each cleaves DNA as a heterodimer of one large subunit (B subunit) and one small subunit (A subunit); and, in the absence of their small subunits, the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions: GCAATG (−/0) and GCAGTG (−/0). We refer to the single subunit, the bottom-strand nicking forms as ‘hemidimers’. Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites: a canonical PD-Xn-EXK and a second non-canonical PD-Xn-E-X12-QR. Interestingly, the other family members, which include BsrI (ACTGG 1/−1) and BsmI/Mva1269I (GAATGC 1/−1) are single polypeptide chains, i.e. monomers, rather than heterodimers. In BsrDI and BtsI, the two catalytic sites are found in two separate subunits. Site-directed mutagenesis confirmed that the canonical catalytic site located at the N-terminus of the large subunit is responsible for the bottom-strand cleavage, whereas the non-canonical catalytic site located in the small subunit is responsible for hydrolysis of the top strand. Top-strand specific nicking variants, Nt.BsrDI and Nt.BtsI, were successfully engineered by combining the catalytic-deficient B subunit with wild-type A subunit

Centerscope

Centerscope, formerly Scope, was published by the Boston University Medical Center "to communicate the concern of the Medical Center for the development and maintenance of improved health care in contemporary society.

Overexpression of sICAM-1 in the Alveolar Epithelial Space Results in an Exaggerated Inflammatory Response and Early Death in Gram Negative Pneumonia

Abstract Background A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1) plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs), we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM) function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1) is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection. Methods Using the surfactant protein C (SPC) promoter, we developed a transgenic mouse (SPC-sICAM-1) that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae. Results SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups. Conclusions We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.http://deepblue.lib.umich.edu/bitstream/2027.42/112728/1/12931_2010_Article_1038.pd

Improving the Study of Protein Glycosylation with New Tools for Glycopeptide Enrichment

High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides

Postoperative Complications in the Ahmed Baerveldt Comparison Study During Five Years of Follow-up

To compare the late complications in the Ahmed Baerveldt Comparison Study during 5 years of follow-up