Optical trapping with holographically structured light for single-cell studies.

A groundbreaking work in 1970 by Arthur Ashkin paved the way for developing various optical trapping techniques. Optical tweezers have become an established method for the manipulation of biological objects, due to their noninvasiveness and precise controllability. Recent innovations are accelerating and now enable single-cell manipulation through holographic light structuring. In this review, we provide an overview of recent advances in optical tweezer techniques for studies at the individual cell level. Our review focuses on holographic optical tweezers that utilize active spatial light modulators to noninvasively manipulate live cells. The versatility of the technology has led to valuable integrations with microscopy, microfluidics, and biotechnological techniques for various single-cell studies. We aim to recapitulate the basic principles of holographic optical tweezers, highlight trends in their biophysical applications, and discuss challenges and future prospects.

Color-Tuning Mechanism of the Lit Form of Orange Carotenoid Protein.

Orange carotenoid protein (OCP) of photosynthetic cyanobacteria binds to ketocarotenoids noncovalently and absorbs excess light to protect the host organism from light-induced oxidative damage. Herein, we found that mutating valine 40 in the α3 helix of Gloeocapsa sp. PCC 7513 (GlOCP1) resulted in blue- or red-shifts of 6-20 nm in the absorption maxima of the lit forms. We analyzed the origins of absorption maxima shifts by integrating X-ray crystallography, homology modeling, molecular dynamics simulations, and hybrid quantum mechanics/molecular mechanics calculations. Our analysis suggested that the single residue mutations alter the polar environment surrounding the bound canthaxanthin, thereby modulating the degree of charge transfer in the photoexcited state of the chromophore. Our integrated investigations reveal the mechanism of color adaptation specific to OCPs and suggest a design principle for color-specific photoswitches.

The effect of rotation moment on the stability of immediately loaded orthodontic miniscrews: a pilot study.

The aim of this study was to evaluate the effect of the direction and magnitude of the rotation moment to loaded orthodontic miniscrews on stability. In six adult male beagle dogs (12 months old), 36 orthodontic miniscrews were inserted into the mandibular buccal alveolar bone without drilling. Immediately after insertion, 24 miniscrews were loaded with 1- and 2-Ncm nickel titanium (NiTi) coil springs with either a clockwise or counterclockwise rotation moment while 12 miniscrews were left unloaded. Following an observation period of 3 or 12 weeks, the animals were killed and the miniscrews with the surrounding bone were prepared for histomorphometric evaluation. Bone-to-implant contact (BIC) and the ratio of bone volume/total volume (BV/TV) were measured. BIC and BV/TV were statistically compared using the Kruskal-Wallis H and Mann-Whitney U tests. Three of the miniscrews loaded with 2 Ncm counterclockwise rotation moment were lost within 3 weeks. At 12 weeks after insertion, the counterclockwise group showed a statistically significantly lower BIC in comparison with the clockwise group. The results suggest that a rotation moment to loaded orthodontic miniscrews, as well as the direction and magnitude of the rotation moment, can influence miniscrew stability. Counterclockwise rotational moments can be a risk factor impairing miniscrew stability.

1-Arylsulfonyl-2-(pyridylmethylsulfinyl) benzimidazoles as new proton pump inhibitor prodrugs.

New arylsulfonyl proton pump inhibitor (PPI) prodrug forms were synthesized. These prodrugs provided longer residence time of an effective PPI plasma concentration, resulting in better gastric acid inhibition.

Autophosphorylation of DNA-dependent protein kinase regulates DNA end processing and may also alter double-strand break repair pathway choice.

Two highly conserved double-strand break (DSB) repair pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), function in all eukaryotes. How a cell chooses which pathway to utilize is an area of active research and debate. During NHEJ, the DNA-dependent protein kinase (DNA-PK) functions as a "gatekeeper" regulating DNA end access. Here, we provide evidence that DNA-PK regulates DNA end access via its own autophosphorylation. We demonstrated previously that autophosphorylation within a major cluster of sites likely mediates a conformational change that is critical for DNA end processing. Furthermore, blocking autophosphorylation at these sites inhibits a cell's ability to utilize the other major double-strand break repair pathway, HR. Here, we define a second major cluster of DNA-PK catalytic subunit autophosphorylation sites. Whereas blocking phosphorylation at the first cluster inhibits both end processing and HR, blocking phosphorylation at the second cluster enhances both. We conclude that separate DNA-PK autophosphorylation events may function reciprocally by not only regulating DNA end processing but also affecting DSB repair pathway choice.

Quaternary derivatives of granatanol diesters: potent, ultrashort acting non-depolarizing neuromuscular relaxants.

The purpose of this study was to explore the feasibility of utilizing the granatanol: N-methyl [9-azabicyclo (3.3.1) nonane] 3-alpha-ol as the terminal group in a series of new bisquaternary azabicycyclic diester-type neuromuscular blocking agents. Fifty two bisquaternary ammonium derivatives of several dicarboxylic acid esters of granatanol and three similar derivatives of pseudo granatanol have been investigated for neuromuscular blocking (NMB) potency (ED(50) s), onset and recovery of action and for cardiovascular side effects. All agents were studied first in anesthetized rats, and selected agents were subjected to further pharmacodynamic testing in rabbits, juvenile pigs, cats, dogs and monkeys. One agent was tested in continuous i.v. infusion mode in comparison with its corresponding tropine diester and the aminosteroid muscle relaxant, rocuronium. Several new and highly potent NMB granatanol derivatives are described, which are largely similar in NMB potency to the previously described tropine: N-methyl [8-azabicyclo (3.2.1)] 3-alpha-ol diester derivatives. The majority of the presently described granatanol derivatives displayed ultrashort onset and duration of actions. In that respect some of these agents proved to be the fastest and shortest acting non-depolarizing muscle relaxants described so far. On the negative side, many, but not all, granatanol derivatives produced cardiovascular side effects: e.g. changes in heart rate and blood pressure. Like with the similar tropinyl diester derivatives, cardiac vagal block was present with the majority of these agents as assessed in the rat, pig and cat. Few glutaryl, fumaryl and cyclobutane (trans) 1,2-dicarboxylyl granatanol diesters quaternized with disubstituted benzyl halides, bearing p-acyloxy radicals, showed excellent NMB profile. In these derivatives, however, the rapid decomposition of the p-acyloxy groups leads to formation of toxic quinone methene metabolites which precludes their further pharmaceutical development. The pseudo granatanol derivatives were less potent in the rat than the corresponding granatanols and were not further investigated. We conclude that the 9-azabicyclo (3.3.1) nonane (granatane) ring system can successfully replace the similar 8-azabicyclo (3.2.1) octane (tropane) ring system in building potent, utrashort acting NMB agents.

Neuromuscular pharmacology of TAAC3, a new nondepolarizing muscle relaxant with rapid onset and ultrashort duration of action.

UNLABELLED: We selected bis [N-(3,4-diacetoxybenzyl) tropanium-3alpha-yl] glutarate dibromide (TAAC3) from many new tropinyl diester derivatives to evaluate its neuromuscular blocking (NMB) and autonomic side effects on anesthetized rats, rabbits, guinea pigs, cats, pigs, dogs, and monkeys. NMB potency, onset, recovery index, and duration of action were determined. Comparisons of these pharmacologic variables were made between TAAC3 and rocuronium. In the cat, the degrees of train-of-four and tetanic fade, posttetanic potentiation, and pharmacologic antagonism were evaluated. For determination of the NMB maintenance dose, TAAC3 was also given to rabbits and pigs in the initial dose/maintenance infusion mode. Cardiac vagal block was evaluated in the rat, pig, cat, and guinea pig on the basis of the inhibition of the bradycardia to stimulation of the vagus nerve. Sympathetic ganglion block was studied on the superior cervical ganglion-nictitating membrane preparation of the cat. TAAC3 produced nondepolarizing NMB. Its NMB 90% effective doses ranged from 90 to 425 microg/kg, depending on the species. TAAC3 had a faster onset (0.8-1.0 min), shorter recovery index (0.6-1.1 min), and shorter duration of action (1.8-3.5 min) than rocuronium. It produced a slight cumulative effect on infusion, but not on repeated single-dose administration. Cardiac vagal block was present at doses exceeding the NMB 90% effective dose. In the cat and pig at equipotent NMB doses, the degree of cardiac vagal block was similar to that of rocuronium. There was no demonstrable sympathetic ganglion block in the cat. In view of its favorable NMB characteristics, TAAC3 is now undergoing detailed preclinical studies. IMPLICATIONS: We developed a new nondepolarizing muscle relaxant, TAAC3, and investigated it in several animal models. TAAC3 has shown a very rapid onset and an ultrashort duration of neuromuscular blocking action. A minor degree of cardiac vagal block was observed. TAAC3 is promising for further studies.

Single-step perfusion chromatography with a throughput potential for enhanced peptide detection by matrix-assisted laser desorption/ ionization-mass spectrometry.

Mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis can be routinely performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) which has become a standard tool. Since MALDI-MS detection relies heavily on the quality of the MALDI target, development of an efficient sample preparation technique for removal of sample contaminants is necessary. To date, among the several sample preparation techniques for MALDI targets available, multistep perfusion chromatography (MSPC) using Poros R2 and Oligo R3 has been most commonly used. However, MSPC requires at least four working steps and is not efficient for high-throughput analysis and recovery of low abundance proteins. During the course of proteomic analysis of a large set of rat liver tissues and the immortalized human sebaceous gland cells (SZ95 cells), we were interested in developing an alternative to MSPC. Here, we describe a single-step perfusion chromatography (SSPC) method for MALDI target preparation, which uses a tiny column packed with a mixture of Poros R2 and Oligo R3 resins. The SSPC method significantly improves not only detection of peptides but also efficiency of sample handling, thus enabling high-throughput sample preparation for analyzing large set of samples with high resolution and reproducibility.

Differential expression of the liver proteome in senescence accelerated mice.

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorders due to its inherited aging phenotype. To investigate proteins involved in the aging process in liver, we compared the young (4- or 20-week old) and the aged group (50-week-old) of SAMP8 (short life span) and SAMR1 (control) mice, and identified 85 differentially expressed distinct proteins comprising antioxidation, glucose/amino acid metabolism, signal transduction and cell cycle systems using proteomics tools. For the antioxidation system, the aged SAMP8 mice showed a large increase in glutathione peroxidase and decreases in glutathione-S-transferase and peroxiredoxin, ranging from 2.5- to 5-fold, suggesting lower detoxification potentials for oxidants in the aged SAMP8 liver. Similarly, levels of key glycolytic enzymes decreased greatly in the aged SAMP8 compared to SAMR1, indicating a disturbance in glucose homeostasis that may be closely related to the typical deficits in learning and memory of the aged SAMP8. Protein profiles of amino acid metabolic enzymes suggest that accumulation of glutamine and glutamate in tissues of the aged SAMP8 may be due to hyperexpression of ornithine aminotransferase and/or glutamate dehydrogenase. Decreases in levels of proteins involved in signal transduction/apoptosis (e.g., cathepsin B) in the aged SAMP8 may support the previously proposed negative relationship between apoptosis and aging. However, the changes described above were not markedly seen in the young group of both strains. For cell cycle systems, levels of selenium binding protein increased about four-fold with age in SAMP8. Yet, almost no change occurred in either the young or the aged SAMR1, which may explain problems associated with cell proliferation and tissue regeneration in the aged SAMP8. In conclusion, composite profiles of key proteins involved in age-related processes enable assessment of accelerated senescence and the appearance of senescence-related pathologies in the aged SAMP8.