Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.     

The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry

It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wt-hGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the beta-sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.     

Motor training programs of arm and hand in patients with MS according to different levels of the ICF: a systematic review

Background

Breast cancer survivors, particularly those treated with chemotherapy, are at significantly increased risk for long-term cognitive and neurobiologic impairments. These deficits tend to involve skills that are subserved by distributed brain networks. Additionally, neuroimaging studies have shown a diffuse pattern of brain structure changes in chemotherapy-treated breast cancer survivors that might impact large-scale brain networks.

Methods

We therefore applied graph theoretical analysis to compare the gray matter structural networks of female breast cancer survivors with a history of chemotherapy treatment and healthy age and education matched female controls.

Results

Results revealed reduced clustering coefficient and small-world index in the brain network of the breast cancer patients across a range of network densities. In addition, the network of the breast cancer group had less highly interactive nodes and reduced degree/centrality in the frontotemporal regions compared to controls, which may help explain the common impairments of memory and executive functioning among these patients.

Conclusions

These results suggest that breast cancer and chemotherapy may decrease regional connectivity as well as global network organization and integration, reducing efficiency of the network. To our knowledge, this is the first report of altered large-scale brain networks associated with breast cancer and chemotherapy.     

Differential effects of pre and post-payment on neurologists' response rates to a postal survey

Background  

Monetary incentives are an effective way of increasing response rates to surveys, though they are generally less effective in physicians, and are more effective when the incentive is paid up-front rather than when made conditional on completion.     

Use of cervicovaginal fluid for the identification of biomarkers for pathologies of the female genital tract

Cervicovaginal fluid has an important function in the homeostasis and immunity of the lower female genital tract. Analysis of the cervicovaginal fluid proteome may therefore yield important information about the pathogenesis of numerous gynecological pathologies. Additionally, cervicovaginal fluid has great potential as a source of biomarkers for these conditions.     

Angiotensin II and angiotensin-(1-7) decrease sFlt1 release in normal but not preeclamptic chorionic villi: an <Emphasis Type="Italic">in vitro</Emphasis> study

Background  

During preeclampsia, placental angiogenesis is impaired. Factors released from the placenta including vascular endothelial growth factor (VEGF), placental growth factor (PLGF), soluble VEGF receptor 1 (sFlt1), and soluble endoglin (sEng) are regulatory molecules of placental development and function. While the renin angiotensin system has been shown to regulate angiogenic factors in other research fields, these mechanisms have not been extensively studied during pregnancy.     

Investigation of the effect of Interleukin-1 receptor antagonist (IL-1ra) on markers of inflammation in non-ST elevation acute coronary syndromes (The MRC-ILA-HEART Study)

Background

Point of care testing (PoCT) may be a useful adjunct in the management of chronic conditions in general practice (GP). The provision of pathology test results at the time of the consultation could lead to enhanced clinical management, better health outcomes, greater convenience and satisfaction for patients and general practitioners (GPs), and savings in costs and time. It could also result in inappropriate testing, increased consultations and poor health outcomes resulting from inaccurate results. Currently there are very few randomised controlled trials (RCTs) in GP that have investigated these aspects of PoCT.

Design/Methods

The Point of Care Testing in General Practice Trial (PoCT Trial) was an Australian Government funded multi-centre, cluster randomised controlled trial to determine the safety, clinical effectiveness, cost effectiveness and satisfaction of PoCT in a GP setting. The PoCT Trial covered an 18 month period with the intervention consisting of the use of PoCT for seven tests used in the management of patients with diabetes, hyperlipidaemia and patients on anticoagulant therapy. The primary outcome measure was the proportion of patients within target range, a measure of therapeutic control. In addition, the PoCT Trial investigated the safety of PoCT, impact of PoCT on patient compliance to medication, stakeholder satisfaction, cost effectiveness of PoCT versus laboratory testing, and influence of geographic location.

Discussion

The paper provides an overview of the Trial Design, the rationale for the research methodology chosen and how the Trial was implemented in a GP environment. The evaluation protocol and data collection processes took into account the large number of patients, the broad range of practice types distributed over a large geographic area, and the inclusion of pathology test results from multiple pathology laboratories. The evaluation protocol developed reflects the complexity of the Trial setting, the Trial Design and the approach taken within the funding provided. The PoCT Trial is regarded as a pragmatic RCT, evaluating the effectiveness of implementing PoCT in GP and every effort was made to ensure that, in these circumstances, internal and external validity was maintained.

Trial Registration

12612605000272695     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

The first semi-synthetic serine protease made by native chemical ligation

Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure-function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory practice. Here, we describe a new strategy and provide a proof of concept for engineering semi-synthetic proteins. We chose a serine protease Streptomyces griseus trypsin (SGT) for this study to show that it is possible to efficiently couple a synthetic peptide containing a catalytically critical residue to a recombinant fragment containing the other active site residues. The 223-residue hybrid SGT molecule was prepared by fusing a chemically synthesized N-terminal peptide to a large C-terminal fragment of recombinant origin using native chemical ligation. This C-terminal polypeptide was produced from full-length SGT by cyanogen bromide cleavage at a genetically engineered Met57 position. This semi-synthetic hybrid trypsin is fully active, showing kinetics identical to the wild-type enzyme. Thus, we believe that it is an ideal model enzyme for studying the catalytic mechanisms of serine proteases by providing a straightforward approach to incorporate non-natural amino acids in the N-terminal region of the protein. In particular, this strategy will allow for replacement of the catalytic His57 residue and the buried N-terminus, which is thought to help align the active site, with synthetic analogs. Our approach relies on readily available recombinant proteins and small synthetic peptides, thus having general applications in chemical engineering of large proteins where the N-terminal region is the focal interest.