1,027 research outputs found

    Structural and biophysical analysis of important biomedical enzymes and nano-architectures

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    Dopa decarboxylase (DDC) is an important enzyme in the catecholamine biosynthesis pathways. Catecholamines, e.g., dopamine, serotonin, etc. often are the major neuromodulators or neurotransmitters. Hence, DDC plays a key role in regulation of neurodegenerative diseases like Parkinson’s disease (PD). In order to achieve a medicine for PD, a successful inhibitor for DDC, that could reduce the activity of DDC in the blood while making it more effective in brain, is required. An effective design of an inhibitor requires a detailed structural study of human DDC. It was aimed to solve the DDC structure by X-ray crystallography. In order to have enough protein the DDC encoding gene has been cloned in the pET21d vector which was later termed as pET-DDC-His. However, it required numerous trials and errors until a suitable condition for soluble DDC expression was found. Addition of additives like PLP, ethanol, a complex of sorbitol and betaine in the growth medium of the bacteria did not help bring the protein in the soluble part as it formed inclusion bodies. Several soluble protein fusions with DDC, like Thioredoxin and Glutathione-S-transferase were also not quite helpful towards achieving soluble expression of DDC. Finally, a coexpression of DDC along with bacterial chaperone proteins, e.g., GroEL and GroES (after cotransforming both the DDC and Chaperone protein encoding plasmid in the same E.coli cell, used for expression) lead to solubilization of recombinant human DDC. This enzyme was then purified to homogeneity by successively passing the crude bacterial proteins through Ni-chelate-affinity chromatography and Size Exclusion Chromatography. The purified protein (>90 % purity) did not produce a good yield (4mg/ 8L culture), but this was enough to start the initial crystallization trial. Using a scale up to a 50 L culture, quite a good amount of protein was achieved. The homogeneity of DDC was further confirmed by using Multi-Angle Light Scattering and Blue Native PAGE. The dimeric enzyme preparation was then utilized for crystallization using the Hanging Drop Vapor Diffusion method. In a particular condition of the crystal screens trigonal bipyramidal crystals formed. However, these crystals did not show good diffraction when bombarded with X-ray beams. Later, this particular crystallization condition remained irreproducible. The peptide nanoparticle, designed and produced in our lab, could possibly be a very valuable tool in biomedical applications, e.g., in designing vaccines, delivering drugs, bioimaging, serodiagnosis, etc. The design of the peptide nanoparticles is based on the application of the symmetry elements of virus icosahedral capsid on a specially designed building block peptide. The designed peptide building block contains two oligomerization motifs, i.e., a trimeric coiled coil and a pentameric coiled coil joined by a linker region. Sixty such peptide units, upon self-assembly, would produce peptide nanoparticle mimicking a small icosahedral virus particle. The peptide chains in the building block provide flexibility in the design so that an additional peptide could be attached to it at the C-terminus in order to functionalize the peptide nanoparticle for various biomedical applications. First of all, the functional peptide at the C-terminus could be an epitope for the antibody of a life threatening disease like HIV. These peptide nanoparticles can then function as the potent vaccine candidate for that particular disease. In this thesis work, I have attached the two epitopes against the two broadly neutralizing classes of antibody for HIV infection, 2F5 and 4E10, to the peptide nanoparticle. Secondly, another sequence of peptide, which proved to have the capacity of seeding gold on its surface, was attached to the building block peptide unit. The nanoparticle, functionalized with such a peptide, can decorate a gold layer surrounding it. Gold coating on the peptide nanoparticle scaffold can provide a nanostructure, called ‘nanoshells’, which could be very important in the field of therapeutics because of its ability in easy detection and quick treatment of cancer cells. Lastly, I added three peptides; those are recognized in the culture filtrates of M.tuberculosis isolated from TB patients, separately, to the basic peptide construct to form three different nanoparticles. Also, I tried to make a single nanoparticle that displays all the three peptides on its surface. Such a nanoparticle could be a very useful tool in the serodiagnosis or the antibody-based rapid detection of the deadly disease- Tuberculosis. The nanoparticle formation in each of the above-mentioned cases was more or less successful. One of the constructs could successfully even produce gold shells on the peptide nanoparticle

    Molecular SPECT Imaging: An Overview

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    Molecular imaging has witnessed a tremendous change over the last decade. Growing interest and emphasis are placed on this specialized technology represented by developing new scanners, pharmaceutical drugs, diagnostic agents, new therapeutic regimens, and ultimately, significant improvement of patient health care. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) have their signature on paving the way to molecular diagnostics and personalized medicine. The former will be the topic of the current paper where the authors address the current position of the molecular SPECT imaging among other imaging techniques, describing strengths and weaknesses, differences between SPECT and PET, and focusing on different SPECT designs and detection systems. Radiopharmaceutical compounds of clinical as well-preclinical interest have also been reviewed. Moreover, the last section covers several application, of μSPECT imaging in many areas of disease detection and diagnosis

    High-potency ligands for DREADD imaging and activation in rodents and monkeys

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    Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping

    Molecular fMRI

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    Comprehensive analysis of brain function depends on understanding the dynamics of diverse neural signaling processes over large tissue volumes in intact animals and humans. Most existing approaches to measuring brain signaling suffer from limited tissue penetration, poor resolution, or lack of specificity for well-defined neural events. Here we discuss a new brain activity mapping method that overcomes some of these problems by combining MRI with contrast agents sensitive to neural signaling. The goal of this “molecular fMRI” approach is to permit noninvasive whole-brain neuroimaging with specificity and resolution approaching current optical neuroimaging methods. In this article, we describe the context and need for molecular fMRI as well as the state of the technology today. We explain how major types of MRI probes work and how they can be sensitized to neurobiological processes, such as neurotransmitter release, calcium signaling, and gene expression changes. We comment both on past work in the field and on challenges and promising avenues for future development.National Institutes of Health (U.S.) (Grants R21-MH102470 and U01-NS09045)Massachusetts Institute of Technology. Simons Center for the Social Brain (Seed Grant

    Optimal set of EEG features for emotional state classification and trajectory visualization in Parkinson's disease

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    In addition to classic motor signs and symptoms, individuals with Parkinson's disease (PD) are characterized by emotional deficits. Ongoing brain activity can be recorded by electroencephalograph (EEG) to discover the links between emotional states and brain activity. This study utilized machine-learning algorithms to categorize emotional states in PD patients compared with healthy controls (HC) using EEG. Twenty non-demented PD patients and 20 healthy age-, gender-, and education level-matched controls viewed happiness, sadness, fear, anger, surprise, and disgust emotional stimuli while fourteen-channel EEG was being recorded. Multimodal stimulus (combination of audio and visual) was used to evoke the emotions. To classify the EEG-based emotional states and visualize the changes of emotional states over time, this paper compares four kinds of EEG features for emotional state classification and proposes an approach to track the trajectory of emotion changes with manifold learning. From the experimental results using our EEG data set, we found that (a) bispectrum feature is superior to other three kinds of features, namely power spectrum, wavelet packet and nonlinear dynamical analysis; (b) higher frequency bands (alpha, beta and gamma) play a more important role in emotion activities than lower frequency bands (delta and theta) in both groups and; (c) the trajectory of emotion changes can be visualized by reducing subject-independent features with manifold learning. This provides a promising way of implementing visualization of patient's emotional state in real time and leads to a practical system for noninvasive assessment of the emotional impairments associated with neurological disorders

    The role of molecular imaging in modern drug development

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    Drug development represents a highly complex, inefficient and costly process. Over the past decade, the widespread use of nuclear imaging, owing to its functional and molecular nature, has proven to be a determinant in improving the efficiency in selecting the candidate drugs that should either be abandoned or moved forward into clinical trials. This helps not only with the development of safer and effective drugs but also with the shortening of time-to-market. The modern concept and future trends concerning molecular imaging will assumedly be hybrid or multimodality imaging, including combinations between high sensitivity and functional (molecular) modalities with high spatial resolution and morphological techniques

    Revolutionary impact of PET and PET-CT on the day-to-day practice of medicine and its great potential for improving future health care

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    In this communication, we present an overview of the impact and advantages of PET and PET-CT fusion imaging in the practice of medicine. We also discuss the evolution of this promising molecular imaging technique since its inception and the future prospects of the combined structure-function approach. Superior contrast resolution, accurate quantification and above all optimal image quality aid in improved diagnosis of many serious disorders including cancer. We speculate that this powerful imaging approach will almost completely replace most other conventional methods in the future. Currently, 18[F]-fluorode- -oxyglucose (FDG) is the main radiopharmaceutical employed for PET studies around the globe. With the availability of high quality PET images on a routine basis in most centres around the world and the likelihood that several other useful PET tracers will be approved in the near future for routine clinical applications, this technique will likely become essential in almost any medical disorder
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