Clare Lefave

Bio: Clare Lefave is an academic researcher from Hoffmann-La Roche. The author has contributed to research in topics: Epigenomics & Rod. The author has an hindex of 5, co-authored 7 publications receiving 169 citations. Previous affiliations of Clare Lefave include Columbia University Medical Center & University of Notre Dame.

Nano-biosensor development for bacterial detection during human kidney infection: use of glycoconjugate-specific antibody-bound gold NanoWire arrays (GNWA)

Abstract: Infectious disease, commonly caused by bacterial pathogens, is now the world's leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations.

Reversible suppression of T cell function in the bone marrow microenvironment of acute myeloid leukemia.

Abstract: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.

Integrative epigenomic and genomic filtering for methylation markers in hepatocellular carcinomas

Abstract: Epigenome-wide studies in hepatocellular carcinoma (HCC) have identified numerous genes with aberrant DNA methylation. However, methods for triaging functional candidate genes as useful biomarkers for epidemiological study have not yet been developed. We conducted targeted next-generation bisulfite sequencing (bis-seq) to investigate associations of DNA methylation and mRNA expression in HCC. Integrative analyses of epigenetic profiles with DNA copy number analysis were used to pinpoint functional genes regulated mainly by altered DNA methylation. Significant differences between HCC tumor and adjacent non-tumor tissue were observed for 28 bis-seq amplicons, with methylation differences varying from 12% to 43%. Available mRNA expression data in Oncomine were evaluated. Two candidate genes (GRASP and TSPYL5) were significantly under-expressed in HCC tumors in comparison with precursor and normal liver tissues. The expression levels in tumor tissues were, respectively, 1.828 and − 0.148, significantly lower than those in both precursor and normal liver tissue. Validations in an additional 42 paired tissues showed consistent under-expression in tumor tissue for GRASP (−7.49) and TSPYL5 (−9.71). A highly consistent DNA hypermethylation and mRNA repression pattern was obtained for both GRASP (69%) and TSPYL5 (73%), suggesting that their biological function is regulated by DNA methylation. Another two genes (RGS17 and NR2E1) at Chr6q showed significantly decreased DNA methylation in tumors with loss of DNA copy number compared to those without, suggesting alternative roles of DNA copy number losses and hypermethylation in the regulation of RGS17 and NR2E1. These results suggest that integrative analyses of epigenomic and genomic data provide an efficient way to filter functional biomarkers for future epidemiological studies in human cancers.

Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling

Abstract: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg−1 dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 106 mm2 s−1), vascular perfusion/permeability (Ktrans, min−1) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour Ktrans (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis. Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

A Review on the Electrochemical Sensors and Biosensors Composed of Nanowires as Sensing Material.

Abstract: The development and application of nanowires for electrochemical sensors and biosensors are reviewed in this article. Next generation sensor platforms will require significant improvements in sensitivity, specificity and parallelism in order to meet the future needs in variety of fields. Sensors made of nanowires exploit some fundamental nanoscopic effect in order to meet these requirements. Nanowires are new materials, which have the characteristic of low weight with extraordinary mechanical, electrical, thermal and multifunctional properties. The advantages such as size scale, aspect ratio and other properties of nanowires are especially apparent in the use of electrical sensors such as electrochemical sensors and in the use of field-effect transistors. The preparation methods of nanowires and their properties are discussed along with their advantages towards electrochemical sensors and biosensors. Some key results from each article are summarized, relating the concept and mechanism behind each sensor, with experimental conditions as well as their behavior at different conditions.

Nanotechnology for Early Cancer Detection

Abstract: Vast numbers of studies and developments in the nanotechnology area have been conducted and many nanomaterials have been utilized to detect cancers at early stages. Nanomaterials have unique physical, optical and electrical properties that have proven to be very useful in sensing. Quantum dots, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, gold nanowires and many other materials have been developed over the years, alongside the discovery of a wide range of biomarkers to lower the detection limit of cancer biomarkers. Proteins, antibody fragments, DNA fragments, and RNA fragments are the base of cancer biomarkers and have been used as targets in cancer detection and monitoring. It is highly anticipated that in the near future, we might be able to detect cancer at a very early stage, providing a much higher chance of treatment.

Self‐Immolative Spacers: Kinetic Aspects, Structure–Property Relationships, and Applications

Abstract: Self-immolative spacers are covalent assemblies tailored to correlate the cleavage of two chemical bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller molecules. Originally introduced to overcome limitations for drug delivery, self-immolative spacers have gained wide interest in medicinal chemistry, analytical chemistry, and material science. For most applications, the kinetics of the disassembly of the activated self-immolative spacer governs functional properties. This Review addresses kinetic aspects of self-immolation. It provides information for selecting a particular self-immolative motif for a specific demand. Moreover, it should help researchers design kinetic experiments and fully exploit the rich perspectives of self-immolative spacers.

Use of Electrochemical DNA Biosensors for Rapid Molecular Identification of Uropathogens in Clinical Urine Specimens

Abstract: We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.