Nanoparticle-Modified Apoferritin Nanotransfer for Targeted Cytostatic Transport.

BACKGROUND: Ferritin is a globular intracellular protein that acts as the main reservoir for iron. Malignancies are associated with increased plasma ferritin concentrations. A number of studies show that tumor cells express high levels of transferrin receptors (TfR). Increased TfR expression was observed in prostate carcinoma. Apoferritin (APO) can be used as a protein nanotransporter into which a suitable medicinal substance can be encapsulated. Nanoparticles increase the permeability of tumor cells to nanotransporters and have a photothermal effect. The aim of this study was to encapsulate doxorubicin (DOX) into APO and to modify the resulting APO/DOX with gold (AuNPs) and silver nanoparticles prepared by green synthesis (AgNPsGS). METHODS: APO was characterized using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) - 120 V, 60 min, 24 mM Tris, 0.2 M glycine, 3 mM SDS. DOX fluorescence (Ex 480 nm; Em 650 nm) was observed, with a typical absorption maximum at 560 nm. Electrochemical measurement was performed in Brdicka solution (three-electrode setup). AgNPsGS were prepared by green synthesis using clover (Trifolium pratense L.). RESULTS: An electrophoretic study of APO and APO/DOX (5-100 μg/mL) was performed and the behavior of APO and APO/DOX (10 μM) as a function of pH was monitored. In an acidic environment, APO forms subunits of about 20 kDa; in an alkaline medium, it forms a globular protein of about 450 kDa. A change in APO/DOX mobility (about by 10%) was observed. A film of gold nanoparticles was applied to the APO/DOX surface. APO/DOX-AuNPs were washed with ultra-pure water. pH-dependent release of DOX a was monitored. The amount of DOX analyzed was increased by up to 50%. Furthermore, an AgNPsGS-DOX complex (1 mg AgNPsGS/100 μM DOX) was generated and prepared. Subsequently, the AgNPsGS-DOX complex was encapsulated into APO. To further improve therapeutic efficacy, the APO/AgNPsGS-DOX complex was coated with an Au layer. APO/AgNPsGS-DOX/AuNPs were stable and DOX was released from the complex after physical parameters had changed. CONCLUSION: APO nanocomplexes were prepared and modified to increase therapeutic efficacy against tumors. Tumor cell targeting was achieved by binding to TfR and via increased tumor cell permeability and retention. Release of the drug was made possible due to a pH change and photothermal activation that will now be tested. This work was supported by COST European Cholangiocarcinoma Network CA18122 and International Collaboration Project of The European Technology Platform for Nanomedicine. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 3. 2019 Accepted: 14. 5. 2019.

Resonances and Dissociative Electron Attachment in HNCO.

In a combined experimental and theoretical study, we probe the dissociative electron attachment in isocyanic acid HNCO. The experimental absolute cross section for the NCO^{-} fragment shows a sharp onset and fine structures near the threshold. The autoionizing state responsible for the dissociative attachment is found in both the R-matrix calculation and using analytic continuation in the coupling constant. The involved A^{'} resonance has a mixed π^{*}/σ^{*} character along the dissociating bond and thus combines the effects of nonzero electron angular momentum and dipole-supported states. This leads to unusual behavior of its width at various geometries. Because the potential energy gradient of the autoionizing state points essentially in the direction of the N─H bond, nuclear dynamics can be described by a one-dimensional nonlocal model. The results agree with the experiment both quantitatively and qualitatively. The present system may be a prototype for interpretation of the dissociative electron attachment process in a number of other polyatomic systems.

Nonclassical Light from Large Ensembles of Trapped Ions.

The vast majority of physical objects we are dealing with are almost exclusively made of atoms. Because of their discrete level structure, single atoms have proved to be emitters of light, which is incompatible with the classical description of electromagnetic waves. We demonstrate this incompatibility for atomic fluorescence when scaling up the size of the source ensemble, which consists of trapped atomic ions, by several orders of magnitude. The presented measurements of nonclassical statistics on light unconditionally emitted from ensembles containing up to more than a thousand ions promise further scalability to much larger emitter numbers. The methodology can be applied to a broad range of experimental platforms focusing on the bare nonclassical character of single isolated emitters.

Interaction of O(-) and H2 at low temperatures.

Reactive collisions between O(-) and H2 have been studied experimentally at temperatures ranging from 10 K to 300 K using a cryogenic radiofrequency 22-pole ion trap. The rate coefficients for associative detachment, leading to H2O + e(-), increase with decreasing temperature and reach a flat maximum of 1.8 × 10(-9) cm(3) s(-1) at temperatures between 20 K and 80 K. There, the overall reaction probability is in good agreement with a capture model indicating efficient non-adiabatic couplings between the entrance potential energy surfaces. Classical trajectory calculations on newly calculated potential energy surfaces as well as the topology of the conical intersection seam leading to the neutral surface corroborate this. The formation of OH(-) + H via hydrogen transfer, although occurring with a probability of a few percent only (about 5 × 10(-11) cm(3) s(-1) at temperatures 10-300 K), indicates that there are reaction paths, where electron detachment is avoided.

IT delivery of ChABC modulates NG2 and promotes GAP-43 axonal regrowth after spinal cord injury.

Chondroitin sulphate proteoglycans (CSPGs) with the major component NG2 have an inhibitory effect on regeneration of damaged axons after spinal cord injury. In this study, we investigate whether the digestion of CSPGs by chondroitinase ABC (ChABC) may decrease the NG2 expression and promote axon regrowth through the lesion site. Rats underwent spinal cord compression injury and were treated with ChABC or vehicle through an intrathecal catheter delivery at 2, 3, and 4 days after injury. In addition, animals were behaviorally scored using BBB test in weekly intervals after SCI. Based on immunocytochemical analyses, we have quantified distribution of NG2 glycoprotein and GAP-43 in spinal cord tissue in both experimental groups. Multiple injections of ChABC caused decrease of NG2 expression at lesion site at 5 and 7 days, but not at 14 and 28 days in comparison with vehicle-treated rats and significantly enhanced GAP-43 expression during the entire survival. The densitometry analysis showed significantly higher GAP-43 immunoreactivity (1.8-2.2-fold) in the regrowing axons and cell bodies within the central lesion cavity when compared with vehicle group. Longitudinally oriented and disorganized GAP-43-labeled axons were able to infiltrate and penetrate damaged tissue. The outgrowth of GAP-43 axons after CHABC delivery was significantly longer (≤0.457 mm) when compared with the length of axons in vehicle-treated rats (≤0.046 mm). Present findings suggest that degradation of NG2 with acute IT ChABC treatment may promote ongoing (long-lasting) axonal regenerative processes at late survival (14 and 28 days), but with no significant impact on the improvement of motor function.

[Electronic system for correct head position control after some vitreoretinal surgeries]. / Elektronický systém pro kontrolu správné polohy hlavy po nekterých vitreoretinálních operacích.

Team of authors consisting of vitreoretinal surgeons and biomedical engineers developed and tested an electronic system helping the patients after some vitreoretinal surgeries to keep the recommended head position. The authors describe the principle of this system and its use in clinical practice.

Experimental results for H2 formation from H- and H and implications for first star formation.

During the epoch of first star formation, molecular hydrogen (H2) generated via associative detachment (AD) of H- and H is believed to have been the main coolant of primordial gas for temperatures below 10(4) kelvin. The uncertainty in the cross section for this reaction has limited our understanding of protogalaxy formation during this epoch and of the characteristic masses and cooling times for the first stars. We report precise energy-resolved measurements of the AD reaction, made with the use of a specially constructed merged-beams apparatus. Our results agreed well with the most recent theoretically calculated cross section, which we then used in cosmological simulations to demonstrate how the reduced AD uncertainty improves constraints of the predicted masses for Population III stars.

Enrichment of rat oligodendrocyte progenitor cells by magnetic cell sorting.

The embryonic, neonatal, as well as adult rat spinal cords harbor a pool of neural stem cells (NSCs), which may be easily isolated and used to replace neuronal cell loss or remyelinate damaged axons following various neurodegenerative disorders. In the present study we have used magnetic cell sorting (MACs) technology to generate enriched oligodendroglial cell populations from the embryonic (E16) rat spinal cord. Target cells were separated by positive selection, using specific A2B5 antibody-labeled MicroBeads achieving optimal recovery and high purity of pro-oligodendroglial cells. Based on immunocytochemical analyses for oligodendroglial developmental markers (A2B5, NG2, RIP and MBP) we were able to characterize and quantify oligodendroglial progenitors (OPCs) and mature oligodendroglial cells in: (i) unseparated heterogeneous population of NSCs, or in (ii) antigen-antibody separated NSCs. Our results showed that MACs technology enable us to gain enriched OPCs from heterogeneous population of spinal NSCs, resulting in a 58-61% of mature oligodendrocytes content (MBP+, RIP+) in comparison to 6-12% of oligodendroglial cells acquired from unseparated population. In addition, the enriched OPCs could be cultured in vitro for several >8 passages, giving rise to a high number of newly formed spheres, as well as high expansion potential. These experiments indicate that MACs technology provide a feasible approach for experimental cell enrichment of desired oligodendroglial progeny, which may be used in future trials for cell-based therapies to treat spinal cord injury.

Experimental and theoretical evidence for long-lived molecular hydrogen anions H2- and D2-.

The existence of (metastable) molecular hydrogen anions H2(-), D2(-), and H3(-) is demonstrated. These anion species were produced by sputtering of TiH2 and TiD2 targets with Cs+ ions and were identified by accelerator mass spectrometry. From the respective flight times through the spectrometer, lifetimes for H2(-) and D2(-) of at least 3 micros and 4 micros, respectively, can be inferred. Theoretical calculations within the nonlocal resonance model predict the existence of highly rotationally excited anions with lifetimes in the micros range. It is proposed that in sputtering molecular hydrogen species with high rotational and vibrational excitation are formed that are stable on the time scale of the experiment.

Experimental cauda equina compression induces HSP70 synthesis in dog.

The heat shock protein 70 (HSP70) is a key component of the stress response induced by various noxious conditions such as heat, oxygen stress, trauma and infection. In present study we have assessed the consequences of the compression of lower lumbar and sacral nerve roots caused by a multiple cauda equina constrictions (MCEC) on HSP70 immunoreactivity (HSP70-IR) in the dog. Our data indicate that constriction of central processes evokes HSP70 up-regulation in the spinal cord (L7, S1-Co3) as well as in the corresponding dorsal root ganglion cells (DRGs) (L7-S1) two days following injury. A limited number of bipolar or triangular HSP-IR neurons were found in the lateral collateral pathway (LCP) as well as in the pericentral region (lamina X) of the spinal cord. In contrast, a high number of HSP70 exhibiting motoneurons with fine processes appeared in the ventral horn (laminae VIII-IX) of lumbosacral segments. Concomitantly, close to them a few lightly HSP70-positive neuronal somata or cell bodies lacking the HSP70-IR occurred. In the DRGs, HSP70 expression was mildly up-regulated in small and medium-sized neurons and in satellite cells. On the contrary, DRGs from intact or sham-operated dogs did not reveal HSP70 specific neuronal staining. In conclusion, we have demonstrated that the MCEC in dogs mimicking the cauda equina syndrome in clinical settings evokes expression of HSP70 synthesis in specific neurons of the lumbo-sacro-coccygeal spinal cord segments and in small and medium sized neurons of corresponding DRGs. This suggests that HSP70 may play an active role in neuroprotective processes partly by maintaining intracellular protein integrity and preventing the neuronal degeneration in this experimental paradigm.

Effects of interchannel coupling in associative detachment: electron spectra for H+Cl- and H+Br- collisions.

We present experimental and theoretical energy spectra of the electrons detached in collisions of slow Cl- and Br- ions with atomic hydrogen. Nonlocal resonance theory predicts two kinds of features in the spectra: steplike structures associated with rovibrational onsets and steep rises associated with interchannel coupling, the latter being absent in a calculation using the simpler local-complex potential theory. Our experimental spectra confirm the presence of both types of structures and thus the necessity of including interchannel coupling to properly describe the product-state distribution.

The effect of lactobacilli inoculation on organic acid levels in the mucosal film and the small intestine contents in gnotobiotic pigs.

The effect of the inoculation of three Lactobacillus plantarum strains upon lactic, acetic, acetoacetic and propionic acid levels in the mucosal film (F) and the jejunal and ileal contents (O) has been investigated in gnotobiotic pigs. In the jejunum of the inoculated animals, the mucosal film revealed significantly increased levels of lactic, propionic and acetoacetic acids when compared to the contents (25.3 vs. 10.8 mmol.l-1, 18.5 vs. 5 mmol.l-1 and 29.7 vs. 11.2 mmol.l-1, respectively) as well as insignificantly increased acetic acid levels (11.0 vs. 5.8 mmol.l-1). In the ileum of gnotobiotic pigs, propionic acid levels of the mucosal film were significantly higher than those of the contents (21.2 vs. 9.5 mmol.l-1, p < 0.05). In comparison to the contents, the increased lactic, acetic and acetoacetic acid levels in the film proved to be insignificant. The above results suggest that the significantly increased levels of the Lactobacilli-produced organic acids may present an efficient barrier inhibiting the adherence of digestive tract pathogens to the intestinal mucosa.

Interactions of Lactobacillus spp. and enteropathogenic Escherichia coli under in vitro and in vivo conditions.

In the present study, the effect of Lactobacillus spp. against enteropathogenic Escherichia coli 08:K88+Ent+ under in vitro and in vivo conditions has been compared and the effect of inoculation of Lactobacillus spp. upon the colonization of both the jejunum and ileum by enteropathogenic E. coli 08:K88+Ent+ in 9 gnotobiotic pigs has been observed. Under in vitro conditions, the strain Lactobacillus spp. showed the inhibition of 2.1 +/- 0.1 mm against enteropathogenic E. coli 08:K88+Ent+. Two days after the inoculation, the enteropathogenic E. coli 08:K88+Ent+, inoculated to the control group of gnotobiotic pigs (E), colonized the mucosa of both jejunum and ileum of gnotobiotic pigs at counts of 6.41 and 6.08 log 10/cm2, respectively. In experimental group (L-E), the counts of adhered enteropathogenic E. coli in the identical sections of the small intestine, following the inoculation by Lactobacillus spp., amounted to 6.35 and 6.43 log/cm2, respectively. In both groups, numbers of E. coli in the intestinal content of both jejunum and ileum were nearly the same (group E 9.03 and 9.31 log 10/ml; group L-E 8.97 and 9.11 log 10/ml). Two to five days after E. coli inoculation, Lactobacillus spp. counts adhered to the jejunal wall ranged from 5.4 to 6.49 log 10 cm2; in the ileum they ranged from 6.05 to 6.77 log 10 cm2. In the jejunal content, the lactobacilli counts ranged from 6.81 to 8.86 log 10/ml and in the ileum from 8.5 to 898 log 10/ml. Two days after the E. coli inoculation, the concentration of lactic acid in the content of jejunum in gnotobiotic pigs of the groups E and L-E was 16.3 mmol/l and 23.6 mmol/l, respectively. The concentration of acetic acid in the jejunum of the pigs of E and L-E groups was 15.9 mmol/l and 19.6 mmol/l, respectively. Similarly, the higher concentrations of both acids were found also in the ileum of the L-E pigs. The results obtained indicate that the used strain of Lactobacillus spp. which has been preventively inoculated to gnotobiotic pigs, did not prevent the adhesion of enteropathogenic E. coli 08:K88+Ent+ to the mucosa of both jejunum and ileum also despite of the demonstrated inhibitory effect against enteropathogenic E. coli under in vitro conditions and despite good adherent ability in vivo. In both groups of animals, the diseases with pronounced clinical signs as well as losses have occurred.

[Colonization of the digestive tract by defined lactoflora in gnotobiotic and conventional lambs]. / Kolonizácia tráviaceho traktu gnotobiotických a konvencných jahniat definovanou laktoflórou.

The effect of Lactobacillus casei 249/89 on the colonization of the intestinal tract and selected indicators of the metabolic profile of gnotobiotic and conventional lambs from the viewpoint of its possible utilization in the prevention of diarrhoeal syndrome of bacterial etiology in young animals has been studied. Five gnotobiotic and three conventional lambs were used in these studies. The lambs were slaughtered at 3, 6, 10, 15 and 21 days of age. The population of L. casei colonizing the intestinal epithelium was at the age dynamics on average higher in gnotobiotic lambs compared with lactobacilli in conventional lambs (Fig. 1). The significant difference (p < 0.001) was noted at the age of 3 days (gnotobiotic lambs = 3.40 log 10/cm2 and conventional lambs = 1.08 log 10/cm2). Also the counts of lactobacilli colonizing individual sections of the intestine (Fig. 2) was on average higher in gnotobiots with significant differences in jejunum and colon (p < 0.05). In both groups, the highest number of lactobacilli was observed on the intestinal epithelium in lower sections of the digestive tract. In gnotobiots, the population of L. casei colonizing the duodenum was highest at the age of 3 days (3.49 log 10/cm2), in jejunum (3.74 log 10/cm2) and in ileum 4.37 log 10/cm2) at the age of 6 days and in the colon (4.7 log 10/cm2) at the age of 15 days (Fig. 3). In conventional lambs, the population of lactobacilli colonizing individual sections of the intestinum was increasing with age (Fig. 4). The number of L. casei in the intestinal content of gnotobiotic lambs was on average higher than that in conventional animals but the differences were not significant. In both groups, the highest number of lactobacilli was noted at the age of 10 days (gnotobiotic lambs = 5.9 log 10/ml; the conventional lambs = 4.6 log 10/ml). From the viewpoint of individual sections of the intestine, the population of L. casei in the intestinal content of gnotobiotic lambs was higher than the lactobacilli count in conventional animals with significant difference in jejunum (p < 0.01). In gnotobiots, the highest lactobacilli count was in the colon (6.17 log 10/ml); in conventional lambs in the ileum (4.71 log 10/ml).(ABSTRACT TRUNCATED AT 400 WORDS)