Conditions for Handling and Optimal Storage of Mycolactone.

The successful isolation of mycolactone in a laboratory or from a clinical sample relies on proper handling and storage of the toxin. Mycolactone is a light-sensitive and an amphiphilic toxin produced by Mycobacterium ulcerans. The biochemistry of the toxin makes it unstable in aqueous matrices such as blood, which causes it to self-aggregate or present in complex with carrier molecules.

Interaction of amphiphilic lipoarabinomannan with host carrier lipoproteins in tuberculosis patients: Implications for blood-based diagnostics.

Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion.

Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone.

Mycolactone, the amphiphilic macrolide toxin secreted by , plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development.

Membrane perturbing properties of toxin mycolactone from Mycobacterium ulcerans.

Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic.

Detection of Lipomannan in Cattle Infected with Bovine Tuberculosis.

Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM.

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding.

Detection of stealthy small amphiphilic biomarkers.

Pathogen-specific biomarkers are secreted in the host during infection. Many important biomarkers are not proteins but rather small molecules that cannot be directly detected by conventional methods. However, these small molecule biomarkers, such as phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and Mycobactin T (MbT) of Mycobacterium tuberculosis, are critical to the pathophysiology of infection, and may be important in the development of diagnostics, vaccines, and novel therapeutic strategies.

Association of lipoarabinomannan with high density lipoprotein in blood: implications for diagnostics.

Understanding the pathophysiology of tuberculosis, and the bio-distribution of pathogen-associated molecules in the host is essential for the development of efficient methods of intervention. One of the key virulence factors in the pathology of tuberculosis infection is Lipoarabinomannan (LAM). Previously, we have demonstrated the reliable detection of LAM in urine from tuberculosis patients in a sandwich immunoassay format.

Taste of sugar at the membrane: thermodynamics and kinetics of the interaction of a disaccharide with lipid bilayers.

Sugar recognition at the membrane is critical in various physiological processes. Many aspects of sugar-membrane interaction are still unknown. We take an integrated approach by combining conventional molecular-dynamics simulations with enhanced sampling methods and analytical models to understand the thermodynamics and kinetics of a di-mannose molecule in a phospholipid bilayer system.

Rapid detection of Mycobacterium tuberculosis biomarkers in a sandwich immunoassay format using a waveguide-based optical biosensor.

Early diagnosis of active tuberculosis (TB) remains an elusive challenge, especially in individuals with disseminated TB and HIV co-infection. Recent studies have shown a promise for the direct detection of pathogen-specific biomarkers such as lipoarabinomannan (LAM) for the diagnosis of TB in HIV-positive individuals. Currently, traditional immunoassay platforms that suffer from poor sensitivity and high non-specific interactions are used for the detection of such biomarkers.

Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures.

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers.

Quantitative multiplex detection of pathogen biomarkers on multichannel waveguides.

No single biomarker can accurately predict disease. An ideal biodetection technology should be capable of the quantitative, reproducible, and sensitive detection of a limited suite of such molecules. To this end, we have developed a multiplex biomarker assay for protective antigen and lethal factor of the Bacillus anthracis lethal toxin using semiconductor quantum dots as the fluorescence reporters on our waveguide-based biosensor platform.

An intrinsically fluorescent recognition ligand scaffold based on chaperonin protein and semiconductor quantum-dot conjugates.

Genetic engineering of a novel protein-nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18-subunit double ring, which self-assembles in the presence of Mg ions and ATP.

Optimizing a waveguide-based sandwich immunoassay for tumor biomarkers: evaluating fluorescent labels and functional surfaces.

The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the detection of biomarkers associated with disease. We have previously demonstrated the application of this technology to the sensitive detection of carcinoembryonic antigen in serum and nipple aspirate fluid from breast cancer patients. In this publication, we report improvements to this technology that will facilitate transition to a point-of-care diagnostic system and/or robust research tool.

Waveguide-based biosensors for pathogen detection.

Optical phenomena such as fluorescence, phosphorescence, polarization, interference and non-linearity have been extensively used for biosensing applications. Optical waveguides (both planar and fiber-optic) are comprised of a material with high permittivity/high refractive index surrounded on all sides by materials with lower refractive indices, such as a substrate and the media to be sensed. This arrangement allows coupled light to propagate through the high refractive index waveguide by total internal reflection and generates an electromagnetic wave-the evanescent field-whose amplitude decreases exponentially as the distance from the surface increases.

Detection of intact influenza viruses using biotinylated biantennary S-sialosides.

We report the modular synthesis of robust, biotinylated biantennary sialylglycoconjugates and their ability to differentiate between two type A influenza strains. This is the first demonstration of glycoconjugate-based discriminatory capture and detection of two strains of intact influenza virus, in the presence of the innate enzymatic activity of viral neuraminidases. We also demonstrate a "carboassay" using glycoconjugates as capture and reporter elements, which therefore, does not require antibodies.

Functional PEG-modified thin films for biological detection.

We report a general procedure to prepare functional organic thin films for biological assays on oxide surfaces. Silica surfaces were functionalized by self-assembly of an amine-terminated silane film using both vapor- and solution-phase deposition of 3'-aminopropylmethyldiethoxysilane (APMDES). We found that vapor-phase deposition of APMDES under reduced pressure produced the highest quality monolayer films with uniform surface coverage, as determined by atomic force microscopy (AFM), ellipsometry, and contact angle measurements.

Integrated optical biosensor for detection of multivalent proteins.

We have developed a simple, highly sensitive and specific optical waveguide sensor for the detection of multivalent proteins. The optical biosensor is based on optically tagged glycolipid receptors embedded within a fluid phospholipid bilayer membrane formed upon the surface of a planar optical waveguide. Binding of multivalent cholera toxin triggers a fluorescence resonance energy transfer that results in a two-color optical change that is monitored by measurement of emitted luminescence above the waveguide surface.

Neutron reflectivity study of lipid membranes assembled on ordered nanocomposite and nanoporous silica thin films.

Single bilayer membranes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were formed on ordered nanocomposite and nanoporous silica thin films by fusion of small unilamellar vesicles. The structure of these membranes was investigated using neutron reflectivity. The underlying thin films were formed by evaporation induced self-assembly to obtain periodic arrangements of silica and surfactant molecules in the nanocomposite thin films, followed by photocalcination to oxidatively remove the organics and render the films nanoporous.

Femtosecond vibrational dynamics of self-trapping in a quasi-one-dimensional system.

We have directly time resolved the lattice motions associated with the formation of the self-trapped exciton in the quasi-one-dimensional system [Pt(en)(2)] [Pt(en)2Br2];(PF6)(4) ( en = ethylene-diamine, C2H8N2), using femtosecond impulsive excitation techniques. A strongly damped, low-frequency wave packet modulation at approximately 110 cm(-1) accompanies the formation of the self-trapped exciton on a approximately 200 fs time scale following excitation of the intervalence charge-transfer transition. Coherent oscillations at the ground state vibrational frequency and its harmonics are also detected.

Direct, ultrasensitive, and selective optical detection of protein toxins using multivalent interactions.

Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1.

Effect of direct-fed fibrolytic enzymes on the lactational performance of dairy cows.

In trial 1, 30 midlactation (213 d in milk) Holstein cows were randomly assigned to a control or enzyme treatment in a two-period crossover design and were fed a total mixed ration based on alfalfa hay and silage. Cows on the enzyme treatment received an enzyme solution containing cellulases and xylanases, which was sprayed on the forage component of the ration at a rate of 1.65 ml/kg of forage dry matter (DM) between 8 and 24 h prior to feeding.