15 research outputs found
Tracking Protein Motions using Serial Femtosecond Crystallography with X-Ray Free-Electron Laser
Get PDFMizohata E., Nango E., Tosha T., et al. Tracking Protein Motions using Serial Femtosecond Crystallography with X-Ray Free-Electron Laser. Current Protocols 5, e70212 (2025); https://doi.org/10.1002/cpz1.70212.Since the birth of biochemistry, researchers have investigated the structure–function relationship of a wide variety of proteins. However, until recently, when X-ray free-electron lasers (XFELs) became available, it was not possible to visualize the motion of proteins from moment to moment with excellent temporal and spatial resolution. Here, we introduce practical methods to visualize protein motions at room temperature using serial femtosecond crystallography (SFX) using XFELs. With the development of this technology, it will be possible to visualize the entire reaction mechanism of many proteins in the future. We first outline a streamlined microcrystallization workflow for hen egg-white lysozyme, enabling rapid detector calibration and data-collection optimization. Next, we present a rotational seeding approach refined on copper-containing nitrite reductase that yields homogeneous microcrystals suitable for high-resolution SFX and readily adaptable to other challenging targets. Finally, we describe a time-resolved strategy combining microcrystals of fungal nitric-oxide reductase with photolabile caged substrates and synchronized UV triggering, capturing catalytic intermediates on the millisecond timescale. Together, these procedures enable investigators to progress from preparing samples to capturing dynamic structural snapshots. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microcrystallization of lysozyme. Basic Protocol 2: Microcrystallization of copper-containing nitrite reductase. Basic Protocol 3: Time-resolved serial femtosecond crystallography
Development and Evaluation of Exenatide Loaded PLGA Nanoparticles for Intranasal Delivery in the Treatment of Obesity
No full textBackground: Obesity, considered a complex condition, is the fastest-growing public health concern worldwide. Its treatment is limited due to the side effects of pharmacological options available, outweighing their benefits.
Aim: The present study aims to formulate a novel biodegradable formulation of exenatide for direct brain delivery through the nasal route.
Methods: To formulate exenatide loaded poly (lactide-co-glycolide) (PLGA) nanoparticles, a double emulsion (w/o/w) solvent evaporation method was employed. A full factorial (33) design of the experiment was used to optimize the formulation.
Results: The entrapment efficiency and particle size of the optimized formulation were found to be 68% and 110 nm, respectively. The in-vitro drug release study indicated the sustained release of 48% drug in 5 days. The safety of drug-loaded PLGA nanoparticles for intranasal delivery was indicated by the sheep nasal toxicity study. The efficacy of the developed nanoparticles was demonstrated by an in-vivo pharmacodynamics study on Albino Wistar rats, showing a 6.2% weight reduction after 30 days of treatment.
Conclusion: Thus, exenatide is a novel peptide having significant weight loss benefits and no severe side effects. Long-term studies in at least two or more animal models followed by extensive clinical evaluation can safely result in a product for clinical use
Photolytic zirconium benzyl bond cleavage and subsequent aryl C−F activation in zirconium complexes of fluorinated aryl diamides
No full textPhotolysis of Zr(C6F5NCH2CH2OCH2)2(CH2Ph)2 at 435 nm results in formation of the metalated dimer {Zr[C6F4NCH2CH2OCH2CH2OCH2CH2NC6F5][CH2Ph]}2·(C7H8)2 (1), the bridging difluoride dimer {Zr[C6F5NCH2CH2OCH2CH2OCH2CH2NC6F5][F]}2[μ-F]2 (2), and bibenzyl. Complex 1 was characterized by NMR spectroscopy (1H, 19F, 13C) and X-ray crystallography. In the solid state 1 adopts a pentagonal bipyramidal geometry at each zirconium center with a η1-benzyl group occupying one of the axial positions. The bridging difluoride 2 was characterized by 1H and 19F NMR spectroscopy. For comparison purposes, Zr(C6F5NCH2CH2OCH2)2Me2 (3) was synthesized and characterized by NMR spectroscopy and X-ray crystallography. Photolysis of 3 at 435 nm does not result in any reaction.Peer reviewedFinal article publishe
An evaluation of the Atlanta Veterans Affairs Medical Center HIV program: provision of services to stabilize psychosocial functioning, 2005
Get PDFThis evaluation examines the effectiveness of the Atlanta Veterans Affairs Medical Center (VAMC) HIV Program in stabilizing the psychosocial functioning of its HIV/AIDS infected veteran patients. The Atlanta VAMC comprises an Infectious Disease Unit, under which the HIV program operates. This study evaluates the client services component of the program, which provides the psychosocial supports believed to sustain client functioning. The biopsychosocial model constitutes the conceptual framework for this study. The psychosocial challenges of the sample are articulated through this model with attention to physical health, psychological well being, and social functioning. The methodology for the evaluation includes an interview with the client services social worker regarding the program and a chart review. An evaluation tool is used to review 30 randomly selected client charts. Service data with attention to type and degree of psychosocial intervention is obtained from the chart. Client stabilization is assessed based on progress towards the intervention or fulfilling a psychosocial need. Data is analyzed to present descriptive findings and the outcome measure, psychosocial stabilization. The evaluator finds that primary psychosocial interventions including case management, substance abuse treatment and mental health are contributing to the stabilization of veterans functioning. Evidence of the biopsychosocial model is also indicated, as the interventions are addressing the relevant components of the model
Development and Validation of HPLC method for simultaneous estimation of Rifampicin and Ofloxacin using experimental design
No full textA accurate, precise and robust isocratic HPLC method has been developed and validated for simultaneous determination of Rifampicin and Ofloxacin. The chromatographic separation was carried out on Kinetex C18, 100 A Phenomenex column with a mixture of 0.03M Potassium dihydrogen phosphate buffer pH 3.0: Acetonitrile (55:45) as mobile phase at 230 nm. The retention times were 2.91 and 4.87 min for Ofloxacin and Rifampicin, respectively. Calibration plots were linear over the concentration range 1–5 and 2–10 µg/ml for Rifampicin and Ofloxacin, respectively. The method was validated for linearity, sensitivity accuracy, precision, and robustness. Percent recoveries were found to be close to 100% with low variability. Fractional factorial design with four factors was chosen for robustness testing. The volume of acetonitrile and flow rate showed significant effect on retention factor of both the drugs and asymmetry factor of ofloxacin. The method may be adopted for routine analysis at industry
Masters Level Graduate Student Writing Groups: Exploring Academic Identity
Get PDFabstract: This action research project explores masters level graduate student writing and academic identity during one semester in an interdisciplinary masters program. Informing this study is a two part theoretical framework including the Academic Literacy Model (Lea and Street) and Wenger's concept of identity. The purpose of this exploration was to understand how first semester graduate students experienced academic writing and what characteristics of their academic identity emerged. A mixed-methods approach was used to collect both quantitative and qualitative data. Quantitative data included results from the Inventory of Processes in Graduate Writing (Lavelle and Bushrow, 2007) and the Graduate Student Identity Survey. Qualitative data was collected through researcher observations, student blog entries, writing group transcripts, and individual interviews. The following themes emerge from the data: a) graduate students attribute their successes in writing to previous experiences, b) graduate students experience struggles related primarily to academic quality and faculty expectations, c) graduate students negotiate ways of being in the academy through figuring out expectations of faculty and program, d) work done in the writing group meetings shows evidence of meaning-making for the graduate students, e) the focus of the MA program was critically important to graduate students in the graduate writing project, e) participants' role as graduate students felt most strongly in contexts that include academic activity, and f) students acknowledge change and increasingly identify themselves as writers. Ideas for future cycles of research are discussed.Dissertation/ThesisEd.D. Educational Leadership and Policy Studies 201
Mechanism of substrate inhibition in cytochrome-c dependent NO reductases from denitrifying bacteria (cNORs)
No full textSteady-state kinetics of cytochrome-c dependent denitrifying NO reductases (cNORs) show evidence of substrate inhibition at NO concentrations higher than 10 μM, but the mechanism of inhibition remains unclear. Here, we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to the oxidized cNORs. A differential signal at 1261 cm−1 that downshifts with 15NO and 15N18O is assigned to a ν(NO2) from a bridging diiron-nitrito complex at the heme-nonheme diron site. Theoretical calculations reproduces observed frequencies and isotope shifts. Our experimental results confirm a prior theoretical study by Blomberg and Siegbahn [Blomberg, M. R., and Siegbahn, P. E. M. Biochemistry 2012, 51, 5173–5186] that proposed substrate inhibition through a radical combination reaction between the diferric μ-oxo group and an NO molecule to form a heme Fe(III)-nitrito-FeB(II) inhibitory complex. Stopped-flow experiments suggest that substrate inhibition also occurs after a half-reduction cycle, i.e. when fully-reduced cNOR reduces two NO molecules at the heme-nonheme diferrous active site cluster to produce one N2O molecule and the diferric cluster. These results support catalytic mechanisms that proceed through isomerization of a diferric-hyponitrite transient complex to produce a bridging diferric μ-oxo group and N2O without protonation of the putative hyponitrite intermediate.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi
Development and optimization of dispersible tablet of Bacopa monnieri with improved functionality for memory enhancement
No full textEnhancement of Solubility of Artemisinin and Curcumin by Co-Solvency Approach for Application in Parenteral Drug Delivery System
No full textThe aim of present study was to enhance solubility of poorly soluble antimalarial drugs, Artemisinin and Curcumin by adopting Co-solvency approach and to develop parenteral aqueous injectable solution. Solubility enhancement of both drugs was achieved using co-solvency approach. The parenteral injection was prepared by using a ternary co-solvent system which comprised of benzyl alcohol, PEG 400 and tween 80 (as surfactant). Solubility of Artemisinin and Curcumin was found to be higher in benzyl alcohol and PEG 400. Co-solvent system comprising of benzyl alcohol, PEG 400 and tween 80 in volume fraction of 0.3, 0.9 and 0.2 respectively showed the minimum required solubility of Artemisinin (90 mg per ml) and Curcumin (180 mg per ml). The parenteral injectable formulation was characterized for pH, clarity, viscosity, osmolarity and sterility and the stated parameters were found in acceptable range. <em>In-vitro </em>erythrocyte toxicity study showed that intravenous administration of optimized formulation will be safe. <em>In-vitro </em>antimalarial assay indicated that efficacy of artemisinin and curcumin parenteral formulation was greater than quinine and combination of Artemether and Lumefantrine. Stability study of the optimized batch showed no change in physical and chemical characteristics. Based on study, one can conclude that Artemisinin and Curcumin can be successfully formulated as parenteral injectable formulation by co-solvency approach for the effective treatment of malarial infection</jats:p
