Validation of Humanized Mouse Antibodies

Antibody therapy is being developed and tested as one of the most promising agents for treatment of various human diseases. As of March 2016, 350 antibody candidates are in clinical trials. Many of these antibodies have been taken from animals and “humanized” by genetic modification. Our experiment tests monoclonal antibodies that have been harvested from mouse hybridoma (spleen-derived) cells and cloned until the heavy and light chains of the antibody can be recognized by human cells. Because of this “humanization” procedure, basic antibody assays are needed to demonstrate that the binding, specificity and functional parameters of the antibodies are not lost during cloning. The purpose of this research is to perform this validation through assays. The antibodies are harvested from cell supernatants and purified using affinity chromatography. Then, the antibody fractions are tested for reactivity with human target protein PTP-Beta, via western blot and ELISA procedures. Cross-reactivity of the antibody is tested against human eta and cynomolgus beta proteins. The work presented in this poster describes results from one particular mouse antibody, R15, which has been humanized to functionally enhance endothelial survival. The goal is to generate a therapeutic antibody candidate that improves endothelium survival and stability

Protein-mediated dethreading of a biotin-functionalised pseudorotaxane

In this article, we describe the synthesis of new biotin-functionalised naphthalene derivatives 3 and 4 and their complexation behaviour with avidin and neutravidin using a range of analytical techniques. We have shown using 2-(4prime or minute-hydroxyazobenzene)benzoic acid displacement and ITC experiments{,} that compounds 3 and 4 have the propensity to form reasonably high-affinity bioconjugates with avidin and neutravidin. We have also demonstrated using 1H NMR{,} UV-vis and fluorescence spectroscopy that the naphthalene moiety of 3 and 4 facilitates the formation of pseudorotaxane-like structures with 1 in water. We have then investigated the ability of avidin and neutravidin to modulate the complexation between 1 and 3 or 4. UV-vis and fluorescence spectroscopy has shown that in both cases the addition of the protein disrupts complexation between the naphthalene moieties of 3 and 4 with 1

Are Cell Death Proteins/Antigens Found on Interdigital Cells Dying During Limb Development Expressed in a Simple Organism Such As Tetrahymena?

Numerous studies have been published that describe the genes and proteins that control cell death in various biological systems including normal embryonic development and in disease such as cancer. We describe attempts to look at a possible conserved cell death antigen in the simple organism Tetrahymena, using a unique monoclonal antibody that recognizes only dying cells in the chick limb. The main impetus for the research is to answer the question; does the cell death process have key proteins that exist in the dying process that can be modulated prior to the completion of the cell death process? Using various stimuli to induce cell death in tetrahymena thermophila including staurosporine, hypoxia and other know cell death modulators, we describe the preliminary methods used to verify that cells across two species may express conserved cell death proteins at certain times during the death process. The goal is to demonstrate that normal interdigit cell death is an ideal system for isolating programmed cell death antigens and provides a way to identify common mediators/markers in other model systems such as tetrahymena thermophila

Biomacromolecular stereostructure mediates mode hybridization in chiral plasmonic nanostructures

The refractive index sensitivity of plasmonic fields has been exploited for over 20 years in analytical technologies. While this sensitivity can be used to achieve attomole detection levels, they are in essence binary measurements that sense the presence/absence of a predetermined analyte. Using plasmonic fields, not to sense effective refractive indices but to provide more “granular” information about the structural characteristics of a medium, provides a more information rich output, which affords opportunities to create new powerful and flexible sensing technologies not limited by the need to synthesize chemical recognition elements. Here we report a new plasmonic phenomenon that is sensitive to the biomacromolecular structure without relying on measuring effective refractive indices. Chiral biomaterials mediate the hybridization of electric and magnetic modes of a chiral solid-inverse plasmonic structure, resulting in a measurable change in both reflectivity and chiroptical properties. The phenomenon originates from the electric-dipole–magnetic-dipole response of the biomaterial and is hence sensitive to biomacromolecular secondary structure providing unique fingerprints of α-helical, β-sheet, and disordered motifs. The phenomenon can be observed for subchiral plasmonic fields (i.e., fields with a lower chiral asymmetry than circularly polarized light) hence lifting constraints to engineer structures that produce fields with enhanced chirality, thus providing greater flexibility in nanostructure design. To demonstrate the efficacy of the phenomenon, we have detected and characterized picogram quantities of simple model helical biopolymers and more complex real proteins

Triple-Negative Breast Cancer: A Review of Conventional and Advanced Therapeutic Strategies

Triple-negative breast cancer (TNBC) cells are deficient in estrogen, progesterone and ERBB2 receptor expression, presenting a particularly challenging therapeutic target due to their highly invasive nature and relatively low response to therapeutics. There is an absence of specific treatment strategies for this tumor subgroup, and hence TNBC is managed with conventional therapeutics, often leading to systemic relapse. In terms of histology and transcription profile these cancers have similarities to BRCA-1-linked breast cancers, and it is hypothesized that BRCA1 pathway is non-functional in this type of breast cancer. In this review article, we discuss the different receptors expressed by TNBC as well as the diversity of different signaling pathways targeted by TNBC therapeutics, for example, Notch, Hedgehog, Wnt/b-Catenin as well as TGF-beta signaling pathways. Additionally, many epidermal growth factor receptor (EGFR), poly (ADP-ribose) polymerase (PARP) and mammalian target of rapamycin (mTOR) inhibitors effectively inhibit the TNBCs, but they face challenges of either resistance to drugs or relapse. The resistance of TNBC to conventional therapeutic agents has helped in the advancement of advanced TNBC therapeutic approaches including hyperthermia, photodynamic therapy, as well as nanomedicine-based targeted therapeutics of drugs, miRNA, siRNA, and aptamers, which will also be discussed. Artificial intelligence is another tool that is presented to enhance the diagnosis of TNBC

Protein Analysis of Human Lacrimal Fluid in Varying Age Groups

Purpose: The objective of this research project was to identify proteins secreted from human lacrimal fluids onto the extra-ocular surface of the eye that could be later used to predict eye health, disease, and age-related changes. The identification of specific lacrimal proteins in relative quantities and patterns in younger versus older patients may reflect both ocular and extra-ocular disease states. Methods: This observational study collected samples of lacrimal fluid from 20 subjects between the ages of 18 and 25 years and 20 subjects over the age of 50 years with the use of Schirmer strips. The protein composition of these lacrimal fluid samples was then analyzed to determine specific proteins that evidenced unique patterns among the subject populations. Results: The protein concentrations between the two age groups (n = 40) was significantly higher in the younger patient group (1408.3 ug/mL versus 1152.5 ug/mL, p = 0.03). No consistent qualitative differences in the protein bands were observed between the two different patient age groups. However, excising and analyzing the outlying protein bands revealed unique proteins within the older patient group (aldehyde dehydrogenase and serotransferrin precursor). Preliminary attempts were made to determine the presence of proteins in lacrimal fluid that may originate from cells lining the ducts and blood vessels associated with the ocular environment. Conclusion: These preliminary results in age related differences in eye lacrimal fluid will contribute to future research endeavors in order to determine which specific proteins were increased or decreased quantitatively in the younger population, if any, and what role they might have in eye health, disease, and age-related changes

Accepting higher morbidity in exchange for sacrificing fewer animals in studies developing novel infection-control strategies.

Preventing bacterial infections from becoming the leading cause of death by the year 2050 requires the development of novel, infection-control strategies, building heavily on biomaterials science, including nanotechnology. Pre-clinical (animal) studies are indispensable for this development. Often, animal infection outcomes bear little relation to human clinical outcome. Here, we review conclusions from pathogen-inoculum dose-finding pilot studies for evaluation of novel infection-control strategies in murine models. Pathogen-inoculum doses are generally preferred that produce the largest differences in quantitative infection outcome parameters between a control and an experimental group, without death or termination of animals due to having reached an inhumane end-point during the study. However, animal death may represent a better end-point for evaluation than large differences in outcome parameters or number of days over which infection persists. The clinical relevance of lower pre-clinical outcomes, such as bioluminescence, colony forming units (CFUs) retrieved or more rapid clearance of infection is unknown, as most animals cure infection without intervention, depending on pathogen-species and pathogen-inoculum dose administered. In human clinical practice, patients suffering from infection present to hospital emergency wards, frequently in life-threatening conditions. Animal infection-models should therefore use prevention of death and recurrence of infection as primary efficacy targets to be addressed by novel strategies. To compensate for increased animal morbidity and mortality, animal experiments should solely be conducted for pre-clinical proof of principle and safety. With the advent of sophisticated in vitro models, we advocate limiting use of animal models when exploring pathogenesis or infection mechanisms

A Pharmacogenomic and Protein Analysis of Human Lacrimal Fluid in Varying Age Groups

Proteins are large biological molecules located within all cells. They are considered the basic functional components of cells that allow them to operate appropriately. Genes consist of both DNA and RNA, and are the cellular components that code for the proteins. A biomarker is any cellular component that is an indication of a biological state. Therefore, genetic and protein biomarkers are specific genes and proteins, respectively, present in cells that indicate a specific biological state of a cell. Identification of proteins and genetic biomarkers in relative quantities has been found to reflect various disease states and age groups in humans. Comparisons of possible techniques for collecting lacrimal fluids from human subjects which could potentially be utilized in the design of the study

3D Printed PLA Scaffolds to Promote Healing of Large Bone Defects

One challenge modern medicine faces is the ability to repair large bone defects and stimulate healing. Small defects typically heal naturally, but large bone defects do not and current solutions are to replace the missing tissue with biologically inert materials such as titanium. This limits the amount of bone healing as the defect is not repaired but rather replaced. The focus of our research is to develop a method of using 3D printing to create biodegradable scaffolds which promote bone in-growth and replacement. To accomplish this we used poly lactic acid (PLA) filament and a desktop 3D printer. To promote bone healing and provide mechanical support our team investigated different design methodologies to provide a scaffold of customizable stiffness while allowing cell attachment and in-growth. Our team used CAD modeling to create unique architecture design systems which we analyzed for stiffness using Finite Element Analysis (FEA). We developed a unit cell method of scaffold construction that allowed for customized stiffness of irregular shapes. We 3D printed our designs using a desktop 3D printer and verified our stiffness through mechanical tension and compression testing. We investigated cell viability of the scaffolds by immersing test specimens in culturing media and fibroblast cells. Fibroblast cells are from the same lineage as osteoblast cells but are much faster growing, allowing for more efficient testing. Specimens were left in the media for one week then a total cell count was performed. Scaffold designs were then evaluated based on stiffness and cell viability. We have produced several different viable models with appropriate stiffness for human trabecular bone and good cellular adhesion