A coherence-controlled holographic microscope (CCHM) enables quantitative phase imaging with coherent as well as incoherent illumination. The low spatially coherent light induces a coherence gating effect, which makes observation of samples possible also through scattering media. The paper describes theoretically and simulates numerically imaging of a two-dimensional object through a static scattering layer by means of CCHM, with the main focus on the quantitative phase imaging quality. The authors have investigated both strongly and weakly scattering media characterized by different amounts of ballistic and diffuse light. It is demonstrated that the phase information can be revealed also for the case of the static, strongly scattering layer. The dependence of the quality of imaging process on the spatial light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data gained with a model phase object, as well as living carcinoma cells treated in an optically turbid emulsion.
- MeSH
- Algorithms MeSH
- Holography methods MeSH
- Humans MeSH
- Microscopy methods MeSH
- Cell Line, Tumor MeSH
- Computer Simulation MeSH
- Image Processing, Computer-Assisted methods MeSH
- Scattering, Radiation MeSH
- Light MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Conformational changes associated with ribosome function have been identified by X-ray crystallography and cryo-electron microscopy. These methods, however, inform poorly on timescales. Neutron scattering is well adapted for direct measurements of thermal molecular dynamics, the 'lubricant' for the conformational fluctuations required for biological activity. The method was applied to compare water dynamics and conformational fluctuations in the 30 S and 50 S ribosomal subunits from Haloarcula marismortui, under high salt, stable conditions. Similar free and hydration water diffusion parameters are found for both subunits. With respect to the 50 S subunit, the 30 S is characterized by a softer force constant and larger mean square displacements (MSD), which would facilitate conformational adjustments required for messenger and transfer RNA binding. It has been shown previously that systems from mesophiles and extremophiles are adapted to have similar MSD under their respective physiological conditions. This suggests that the results presented are not specific to halophiles in high salt but a general property of ribosome dynamics under corresponding, active conditions. The current study opens new perspectives for neutron scattering characterization of component functional molecular dynamics within the ribosome.
- MeSH
- RNA, Archaeal chemistry MeSH
- Haloarcula marismortui chemistry MeSH
- Ribosome Subunits, Small, Archaeal chemistry MeSH
- RNA, Messenger chemistry MeSH
- Neutron Diffraction MeSH
- Molecular Dynamics Simulation * MeSH
- Ribosome Subunits, Large, Archaeal chemistry MeSH
- Publication type
- Journal Article MeSH
Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.
We have developed a novel simple method for effective preparing gold nanoparticles (AuNPs) intended for utilization in biomedicine. The method is based on gold sputtering into liquid poly(ethylene glycol) (PEG). The PEG was used as a basic biocompatible stabilizer of the AuNP colloid. In addition, two naturally occurring polysaccharides - Chitosan (Ch) and Methylcellulose (MC) - were separately diluted into the PEG base with the aims to enhance the yield of the sputtering without changing the sputtering parameters, and to further improve the stability and the biocompatibility of the colloid. The colloids were sterilized by steam, and their stability was measured before and after the sterilization process by dynamic light scattering and UV-Vis spectrophotometry. The results indicated a higher sputtering yield in the colloids containing the polysaccharides. The colloids were also characterized by atomic absorption spectroscopy (AAS) to reveal the composition of the prepared nanoparticles by transmission electron microscopy (TEM) to visualize the nanoparticles and to evaluate their size and clustering, and by rheometry to estimate the viscosity of the colloids. The zeta-potential of the AuNPs was also determined as an important parameter indicating the stability and the biocompatibility of the colloid. In addition, in vitro tests of antimicrobial activity and cytotoxicity were carried out to estimate the biological activity and the biocompatibility of the colloids. Antimicrobial tests were performed by a drip test on two bacterial strains - Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli. AuNP with chitosan proved to possess the highest antibacterial activity, especially towards the Gram-positive S. epidermidis. In vitro tests on eukaryotic cells, i.e. human osteoblastic cell line SAOS-2 and primary normal human dermal fibroblasts (NHDF), were performed after a 7-day cultivation to determine the effect and the toxic dose of the colloids on human cells. The studied colloid concentrations were in the range from 0.6 μg/ml to 6 μg/ml. Toxicity of the colloids started to reappear at a concentration of 4.5 μg/ml, especially with chitosan in the colloid, where the colloid with a concentration of 6 μg/ml proved to be the most toxic, especially towards the SAOS-2 cell line. However, the PEG and PEG-MC containing colloids proved to be relatively non-toxic, even at the highest concentration, but with a slowly decreasing tendency of the cell metabolic activity.
- MeSH
- Anti-Bacterial Agents chemistry pharmacology MeSH
- Cell Line MeSH
- Chitosan chemistry MeSH
- Dynamic Light Scattering MeSH
- Escherichia coli drug effects MeSH
- Colloids chemistry MeSH
- Metal Nanoparticles chemistry MeSH
- Humans MeSH
- Methylcellulose chemistry MeSH
- Polysaccharides chemistry MeSH
- Drug Stability MeSH
- Staphylococcus epidermidis drug effects MeSH
- Sterilization MeSH
- Particle Size MeSH
- Gold chemistry pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Vibrational optical activity (VOA) such as vibrational circular dichroism and Raman optical activity comprises developing spectroscopic techniques providing additional information on molecules. The information depends on theoretical modeling of the spectra. The review deals with the physics of the phenomena, their advantages and problems in experiments and spectral interpretation, applications and simulation methods. Application potentials in large, hydrated and flexible systems are documented on insuline, lactamide and a recently discovered natural molecule, "SynoxA". VOA is able to afford unique structural information; however, better instruments as well as more accurate simulations are still desirable.
- MeSH
- Amides MeSH
- Circular Dichroism * MeSH
- Cell Physiological Phenomena MeSH
- Insulin physiology MeSH
- Quantum Theory MeSH
- Molecular Structure MeSH
- Oxazolidinones MeSH
- Computer Simulation * MeSH
- Spectrum Analysis, Raman * MeSH
- Fluorescence Resonance Energy Transfer MeSH
- Molecular Dynamics Simulation MeSH
- Spectrum Analysis * MeSH
- Vibration MeSH
- Diffusion Tensor Imaging MeSH
Background The effect of infarct pattern on functional outcome in acute ischemic stroke is incompletely understood. Purpose To investigate the association of qualitative and quantitative infarct variables at 24-hour follow-up noncontrast CT and diffusion-weighted MRI with 90-day clinical outcome. Materials and Methods The Safety and Efficacy of Nerinetide in Subjects Undergoing Endovascular Thrombectomy for Stroke, or ESCAPE-NA1, randomized controlled trial enrolled patients with large-vessel-occlusion stroke undergoing mechanical thrombectomy from March 1, 2017, to August 12, 2019. In this post hoc analysis of the trial, qualitative infarct variables (predominantly gray [vs gray and white] matter involvement, corticospinal tract involvement, infarct structure [scattered vs territorial]) and total infarct volume were assessed at 24-hour follow-up noncontrast CT or diffusion-weighted MRI. White and gray matter infarct volumes were assessed in patients by using follow-up diffusion-weighted MRI. Infarct variables were compared between patients with and those without good outcome, defined as a modified Rankin Scale score of 0-2 at 90 days. The association of infarct variables with good outcome was determined with use of multivariable logistic regression. Separate regression models were used to report effect size estimates with adjustment for total infarct volume. Results Qualitative infarct variables were assessed in 1026 patients (mean age ± standard deviation, 69 years ± 13; 522 men) and quantitative infarct variables were assessed in a subgroup of 358 of 1026 patients (mean age, 67 years ± 13; 190 women). Patients with gray and white matter involvement (odds ratio [OR] after multivariable adjustment, 0.19; 95% CI: 0.14, 0.25; P < .001), corticospinal tract involvement (OR after multivariable adjustment, 0.06; 95% CI: 0.04, 0.10; P < .001), and territorial infarcts (OR after multivariable adjustment, 0.22; 95% CI: 0.14, 0.32; P < .001) were less likely to achieve good outcome, independent of total infarct volume. Conclusion Infarct confinement to the gray matter, corticospinal tract sparing, and scattered infarct structure at 24-hour noncontrast CT and diffusion-weighted MRI were highly predictive of good 90-day clinical outcome, independent of total infarct volume. Clinical trial registration no. NCT02930018 © RSNA, 2021 Online supplemental material is available for this article. See also the editorial by Mossa-Basha in this issue.
- MeSH
- Arterial Occlusive Diseases diagnostic imaging pathology therapy MeSH
- Diflucortolone MeSH
- Diffusion Magnetic Resonance Imaging * MeSH
- Double-Blind Method MeSH
- Drug Combinations MeSH
- Ischemic Stroke diagnostic imaging pathology therapy MeSH
- Humans MeSH
- Lidocaine MeSH
- Neuroprotective Agents therapeutic use MeSH
- Tomography, X-Ray Computed * MeSH
- Prognosis MeSH
- Aged MeSH
- Thrombectomy MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Randomized Controlled Trial MeSH
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.
New drug formulations are sought for poorly water-soluble substances because there is a risk of compromised bioavailability if such substances are administered orally. Such active pharmaceutical ingredients can be reformulated as solid dispersions with suitable water-soluble polymers. In this contribution, formulation of a novel and physically stable dispersion of Simvastatin in poly(2-hydroxypropyl) methacrylamide (pHPMA) is demonstrated. Due to the limited water sorption of pHPMA and a high Tg, the prepared dispersion is more suited for oral administration and storage compared with neat amorphous Simvastatin. Surprisingly, the rate of global reorientation and the internal motion of Simvastatin molecules were enhanced and exhibited dynamical heterogeneities when incorporated into the pHPMA matrix. As revealed by solid-state nuclear magnetic resonance combined with Raman spectroscopy exploiting the fluorescence phenomenon the mobility of the ester and lactone components increased considerably, whereas the naphthalene ring remained rigid. Furthermore, the solid dispersion was found to be nano-heterogeneous with nanometer-sized Simvastatin domains. The presence of these clusters had no impact on the dynamics of the rigid pHPMA chains. Thus, the diffusion of Simvastatin molecules through the glassy pHPMA walls and the subsequent transformation of the clusters into larger crystallites were prevented. No crystallization was detected for more than two years.
- MeSH
- Adsorption MeSH
- Calorimetry, Differential Scanning MeSH
- Polymethacrylic Acids chemistry MeSH
- Magnetic Resonance Spectroscopy MeSH
- Molecular Structure MeSH
- Spectrum Analysis, Raman MeSH
- Simvastatin chemistry MeSH
- Drug Stability MeSH
- Water chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Telomeres are protective structures at chromosome ends which shorten gradually with increasing age. In chronic lymphocytic leukemia (CLL), short telomeres have been associated with unfavorable disease outcome, but the link between clonal evolution and telomere shortening remains unresolved. METHODS: We investigated relative telomere length (RTL) in a well-characterized cohort of 198 CLL patients by qPCR and focused in detail on a subgroup 26 patients who underwent clonal evolution of TP53 mutations (evolTP53). In the evolTP53 subgroup we explored factors influencing clonal evolution and corresponding changes in telomere length through measurements of telomerase expression, lymphocyte doubling time, and BCR signaling activity. RESULTS: At baseline, RTL of the evolTP53 patients was scattered across the entire RTL spectrum observed in our CLL cohort. RTL changed in the follow-up samples of 16/26 (62%) evolTP53 cases, inclining to reach intermediate RTL values, i.e., longer telomeres shortened compared to baseline while shorter ones prolonged. For the first time we show that TP53 clonal shifts are linked to RTL change, including unexpected RTL prolongation. We further investigated parameters associated with RTL changes. Unstable telomeres were significantly more frequent among younger patients (P = 0.032). Shorter telomeres were associated with decreased activity of the B-cell receptor signaling components p-ERK1/2, p-ZAP-70/SYK, and p-NFκB (P = 0.04, P = 0.01, and P = 0.02, respectively). CONCLUSIONS: Our study revealed that changes of telomere length reflect evolution in leukemic subclone proportion, and are associated with specific clinico-biological features of the explored cohort.
- MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell genetics MeSH
- Clonal Evolution genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Mutation MeSH
- Tumor Suppressor Protein p53 genetics MeSH
- Proto-Oncogene Proteins c-bcr metabolism MeSH
- Signal Transduction MeSH
- Telomerase genetics MeSH
- Telomere ultrastructure MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
The article describes the development of calibration procedures for quantitative evaluation of powder blends utilizing non-contact probe of Raman spectrometer. Two model blends containing ascorbic acid and lactose were chosen. They differed in the used sieve fractions. A new procedure combining convective-diffusive and shear mixing was used for the development of PLS (partial least squares regression) calibration methods. This procedure significantly improves the homogeneity of the mixtures containing fine particles of the size below 100 ??????m. The newly developed methods were further utilized to study blending and the impact of vibrations on the powder blend. The results imply that the particles size has a statistically significant impact on the change of homogeneity in the course of vibration action. The results demonstrated the potential of Raman spectroscopy to be used as a PAT (process analytical technology) method in pharmaceutical analysis.
