Development and Characterization of Stable Glycoenzyme Conjugates

Optical glucose biosensors are being developed for long-term monitoring in diabetic individuals. These sensors rely upon the enzyme glucose oxidase, and loss of enzymatic activity leads to a need for frequent recalibration and eventually sensor replacement. Current enzyme stabilization strategies are effective, but generally result in a large increase in size and exclusion from the solution-phase. This sacrifice of native properties precludes the stabilized enzyme from incorporation into the aforementioned sensing platform, which requires that the enzyme be homogenously distributed and entrapped within a hydrogel. It is this incompatibility which provides the motivation for the development of new non-traditional enzyme stabilization strategies. Toward that end, this work focuses on the development and characterization of three enzyme modification strategies, all of which are intended to stabilize enzyme activity while permitting incorporation into an optical biosensing hydrogel. The first approach involves glycosylation site-targeted covalent attachment of poly(ethylene glycol) to glucose oxidase, which improves storage stability by 60%. The second approach builds upon the first, but subsequent modification of the poly(ethylene glycol)-modified glucose oxidase is performed to further stabilize the enzyme. This approach improves long-term storage stability by an order of magnitude. The final approach involves encasement of the glycoenzyme within a shell of albumin, wherein the inert protein is attached at the glycosylation sites in an orthogonal manner. This technique result in highly thermostable enzyme, retaining greater than 25 times more activity than native glucose oxidase following exposure to buffer at 60 °C. In summary, enzyme deactivation is expected to be a major barrier in the realization of long-term glucose sensing with fully implantable optical glucose biosensors, and this work represents a step towards overcoming that hurdle. Each enzyme modification strategy yields a stabilized enzyme under certain conditions, whether it be long-term storage, elevated temperature, or exposure to various solvents/additives. This work enables the stabilized enzymes to be incorporated into hydrogels for evaluation under simulated in vivo conditions, followed by in vivo evaluation. Finally, it is expected that these enzyme stabilization approaches will be advantageous in other applications as well, including in vitro diagnostics, tissue engineering, and therapeutic biologicals

Development and Characterization of Stable Glycoenzyme Conjugates

Optical glucose biosensors are being developed for long-term monitoring in diabetic individuals. These sensors rely upon the enzyme glucose oxidase, and loss of enzymatic activity leads to a need for frequent recalibration and eventually sensor replacement. Current enzyme stabilization strategies are effective, but generally result in a large increase in size and exclusion from the solution-phase. This sacrifice of native properties precludes the stabilized enzyme from incorporation into the aforementioned sensing platform, which requires that the enzyme be homogenously distributed and entrapped within a hydrogel. It is this incompatibility which provides the motivation for the development of new non-traditional enzyme stabilization strategies. Toward that end, this work focuses on the development and characterization of three enzyme modification strategies, all of which are intended to stabilize enzyme activity while permitting incorporation into an optical biosensing hydrogel. The first approach involves glycosylation site-targeted covalent attachment of poly(ethylene glycol) to glucose oxidase, which improves storage stability by 60%. The second approach builds upon the first, but subsequent modification of the poly(ethylene glycol)-modified glucose oxidase is performed to further stabilize the enzyme. This approach improves long-term storage stability by an order of magnitude. The final approach involves encasement of the glycoenzyme within a shell of albumin, wherein the inert protein is attached at the glycosylation sites in an orthogonal manner. This technique result in highly thermostable enzyme, retaining greater than 25 times more activity than native glucose oxidase following exposure to buffer at 60 °C. In summary, enzyme deactivation is expected to be a major barrier in the realization of long-term glucose sensing with fully implantable optical glucose biosensors, and this work represents a step towards overcoming that hurdle. Each enzyme modification strategy yields a stabilized enzyme under certain conditions, whether it be long-term storage, elevated temperature, or exposure to various solvents/additives. This work enables the stabilized enzymes to be incorporated into hydrogels for evaluation under simulated in vivo conditions, followed by in vivo evaluation. Finally, it is expected that these enzyme stabilization approaches will be advantageous in other applications as well, including in vitro diagnostics, tissue engineering, and therapeutic biologicals

Evaluation of Esophageal Motility Utilizing the Functional Lumen Imaging Probe

© 2016 by the American College of Gastroenterology. Objectives:Esophagogastric junction (EGJ) distensibility and distension-mediated peristalsis can be assessed with the functional lumen imaging probe (FLIP) during a sedated upper endoscopy. We aimed to describe esophageal motility assessment using FLIP topography in patients presenting with dysphagia.Methods:In all, 145 patients (aged 18-85 years, 54% female) with dysphagia that completed up per endoscopy with a 16-cm FLIP assembly and high-resolution manometry (HRM) were included. HRM was analyzed according to the Chicago Classification of esophageal motility disorders; major esophageal motility disorders were considered "abnormal". FLIP studies were analyzed using a customized program to calculate the EGJ-distensibility index (DI) and generate FLIP topography plots to identify esophageal contractility patterns. FLIP topography was considered "abnormal" if EGJ-DI was < 2.8 mm 2 /mm Hg or contractility pattern demonstrated absent contractility or repetitive, retrograde contractions.Results:HRM was abnormal in 111 (77%) patients: 70 achalasia (19 type I, 39 type II, and 12 type III), 38 EGJ outflow obstruction, and three jackhammer esophagus. FLIP topography was abnormal in 106 (95%) of these patients, including all 70 achalasia patients. HRM was "normal" in 34 (23%) patients: five ineffective esophageal motility and 29 normal motility. In all, 17 (50%) had abnormal FLIP topography including 13 (37%) with abnormal EGJ-DI.Conclusions:FLIP topography provides a well-tolerated method for esophageal motility assessment (especially to identify achalasia) at the time of upper endoscopy. FLIP topography findings that are discordant with HRM may indicate otherwise undetected abnormalities of esophageal function, thus FLIP provides an alternative and complementary method to HRM for evaluation of non-obstructive dysphagia.Link_to_subscribed_fulltex

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells

Actin depletion initiates events leading to granule secretion at the immunological synapse.

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.Funding was provided by the Wellcome Trust through Principal Research Fellowships (075880 and 103930) to G.M.G. and a Strategic Award (100140) to the Cambridge Institute for Medical Research (CIMR). A.T.R. is an NIH-OxCam scholar supported by funding to J.L.-S. from the Eunice Shriver National Institute of Child Health and Human Development.This is the final version. It was first published by Elsevier at http://www.cell.com/immunity/abstract/S1074-7613%2815%2900173-9

Mitigation of Quantum Dot Cytotoxicity by Microencapsulation

When CdSe/ZnS-polyethyleneimine (PEI) quantum dots (QDs) are microencapsulated in polymeric microcapsules, human fibroblasts are protected from acute cytotoxic effects. Differences in cellular morphology, uptake, and viability were assessed after treatment with either microencapsulated or unencapsulated dots. Specifically, QDs contained in microcapsules terminated with polyethylene glycol (PEG) mitigate contact with and uptake by cells, thus providing a tool to retain particle luminescence for applications such as extracellular sensing and imaging. The microcapsule serves as the “first line of defense” for containing the QDs. This enables the individual QD coating to be designed primarily to enhance the function of the biosensor

Macrocephaly and developmental delay caused by missense variants in RAB5C

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway