The purpose of this work is to explore the preparation of nanofibrous orally dispersible films (ODFs) by needleless electrospinning from the active pharmaceutical ingredient (API) Tadalafil using particles suspended in a solution of polymers and other excipients. The prepared films were characterized by a combination of scanning electron microscopy, mechanical tests, measurements of the disintegration time and dissolution characteristic, X-ray diffraction, and differential scanning calorimetry. Furthermore, we investigated the impact of lamination pressures in the range of 0 to 5 bars combined with films at various relative humidity values on the mechanical properties of the ODF. An increase in lamination pressure resulted in higher Young's modulus values, with the maximum value observed for a sample laminated at a pressure of 5 bar and the maximum stress and strain of the prepared ODF at a lamination pressure of 1.2 bar. Moreover, there was a significant increase in the disintegration time with increase in lamination pressure. The disintegration time ranged from 0.35 s for non-laminated samples to 12 s for samples laminated at a pressure of 5 bar. On the contrary, the lamination pressure did not reveal to have any impact on the dissolution kinetics. These results confirmed that the lamination pressure can improve the processability of ODFs without affecting the API dissolution kinetics.
A comprehensive approach to biological monitoring of 44 workers occupationally exposed to styrene in a hand lamination plant was performed by using several end-points: styrene in workplace air, styrene in exhaled air, styrene in blood, DNA strand breaks (SBs) and oxidised bases in mononuclear leukocytes, chromosomal aberrations in lymphocytes, immune parameters and genotyping of polymorphic genes of some xenobiotic-metabolizing enzymes (CYP 1A1, EPHX, GSTM1 and GSTP1). We found a significantly higher number of DNA SBs, measured by a modified comet assay, in mononuclear leukocytes of the styrene-exposed workers compared with results from 19 unexposed controls (P<0.001). A fairly strong correlation was observed between SBs and years of exposure (P<0.001, r=0.545). The styrene-exposed workers also showed a significantly increased frequency of chromosomal aberrations (P<0.0001 for highly exposed group, P<0.004 for medium-exposed group, and P=0.0001 for low-exposed group). The proliferative response of T-lymphocytes stimulated with concanavalin A was significantly suppressed in people exposed to styrene (P<0.05). We recorded a significant increase of the percentage of monocytes in differential white blood cell counts in the exposed group (P<0.05). Using flow cytometry, we found an increased expression of adhesion molecules CD62L, CD18, CD11a, CD11b, CD49d and CD54 in the exposed workers as compared with the control group (P<0.05).
- MeSH
- Lymphocyte Activation drug effects MeSH
- Biomarkers MeSH
- Chromosome Aberrations MeSH
- Adult MeSH
- Enzymes genetics metabolism MeSH
- Genotype MeSH
- Air Pollutants, Occupational toxicity MeSH
- Humans MeSH
- Cell Adhesion Molecules MeSH
- Environmental Monitoring * methods MeSH
- Plastics MeSH
- DNA Damage MeSH
- Occupational Exposure * MeSH
- Case-Control Studies MeSH
- Styrene * toxicity MeSH
- T-Lymphocytes immunology drug effects MeSH
- In Vitro Techniques MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.
- MeSH
- DNA Adducts analysis MeSH
- Biomarkers MeSH
- Air Pollutants, Occupational MeSH
- Humans MeSH
- Plastics MeSH
- Occupational Exposure adverse effects MeSH
- Styrenes analysis adverse effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Currently, due to the relatively high number of active users with amputation of the lower limbs, it is important to increase user comfort, innovate and optimize the connection between the residual limb and the prosthesis, i.e. the prosthetic socket. The purpose of this work is to combine the potential and advantages of both conventional and innovative (modern) production processes for the design and fabrication of personalized hybrid sockets to optimize production, comfort, and patient safety. The socket was designed and constructed for a highly active user (Functional Classification Level 4) to ensure comfort and safety during high-stress sports activities (skiing). Unlike traditional plastering, a 3D scanner was used to take measurement data. The 3D model of the stump was edited in a software environment instead of laborious and lengthy processing of the plaster positive. Subsequently, a matrix of the prosthetic socket was made from PETG material using FFF 3D printing, which was laminated to increase strength. 3D printed samples of PETG material were tested for tension and pressure according to the relevant standard (EN ISO 527-2: 1996). The last phase was static and dynamic testing of the hybrid socket. No deviations were recorded in the monitored parameters, both at a slow (1.0 km/h) and at a standard (2.5 km/h) walking speed. Once the socket integrity has been assessed, a greater dynamic load was initiated in the form of activities with higher dynamic levels (lateral leaning on the knee and jumps). According to the test results, there has been no change in the shape or integrity of the socket, and the subjective point of view of the volunteer rated the hybrid prosthetic socket as comfortable.
- MeSH
- Printing, Three-Dimensional MeSH
- Amputation, Surgical MeSH
- Lower Extremity pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Prosthesis Design * MeSH
- Prostheses and Implants MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Case Reports MeSH
- MeSH
- Immunity, Cellular radiation effects MeSH
- Adult MeSH
- Air Pollutants, Occupational immunology blood adverse effects MeSH
- Leukocytes drug effects MeSH
- Middle Aged MeSH
- Humans MeSH
- Mitogens MeSH
- Occupational Exposure MeSH
- Styrenes immunology blood adverse effects MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Publication type
- Comparative Study MeSH
A- and C-type lamins are intermediate filament proteins responsible for the maintenance of nuclear shape and most likely nuclear architecture. Here, we propose that pronounced invaginations of A/C-type lamins into the nuclear interior represent channels for the transport of regulatory molecules to and from nuclear and nucleolar regions. Using fluorescent protein technology and immunofluorescence, we show that A-type lamin channels interact with several nuclear components, including fibrillarin- and UBF-positive regions of nucleoli, foci of heterochromatin protein 1 β, polycomb group bodies, and genomic regions associated with DNA repair. Similar associations were observed between A/C-type lamin channels and nuclear pores, lamin-associated protein LAP2α, and promyelocytic leukemia nuclear bodies. Interestingly, regions with high levels of A/C-type lamins had low levels of B-type lamins, and vice versa. These characteristics were observed in primary and immortalized mouse embryonic fibroblasts as well as human and mouse embryonic stem cell colonies exhibiting stem cell-specific lamin positivity. Our findings indicate that internal channels formed by nuclear lamins likely contribute to normal cellular processes through association with various nuclear and nucleolar structures.
- MeSH
- Cell Nucleus genetics MeSH
- Chromosomal Proteins, Non-Histone ultrastructure MeSH
- DNA-Binding Proteins metabolism ultrastructure MeSH
- Lamin Type A ultrastructure MeSH
- Lamin Type B ultrastructure MeSH
- Humans MeSH
- Membrane Proteins metabolism ultrastructure MeSH
- Mice MeSH
- DNA Repair genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.
- MeSH
- Cell Nucleus * metabolism MeSH
- Interphase MeSH
- Intermediate Filaments metabolism MeSH
- Lamin Type A * metabolism MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Recent studies have shown that histone code dictates the type and structure of chromatin. Bearing in mind the importance of A-type lamins for chromatin arrangement, we studied the effect of trichostatin A (TSA)-induced histone hyperacetylation in lamin A/C-deficient (LMNA-/-) fibroblasts. Lamin A/C deficiency caused condensation of chromosome territories and the nuclear reorganization of centromeric heterochromatin, which was accompanied by the appearance of a chain-like morphology of HP1beta foci. Conversely, histone deacetylase (HDAC) inhibition induced de-condensation of chromosome territories, which compensated the effect of lamin A/C deficiency on chromosome regions. The amount of heterochromatin in the area associated with the nuclear membrane was significantly reduced in LMNA-/- cells when compared with lamin A/C-positive (LMNA+/+) fibroblasts. TSA also decreased the amount of peripheral heterochromatin, similarly as lamin A/C deficiency. In both LMNA+/+ and LMNA-/- cells, physically larger chromosomes were positioned more peripherally as compared with the smaller ones, even after TSA treatment. Our observations indicate that lamin A/C deficiency causes not only reorganization of chromatin and some chromatin-associated domains, but also has an impact on the extent of chromosome condensation. As HDAC inhibition can compensate the lamin A/C-dependent chromatin changes, the interaction between lamins and specifically modified histones may play an important role in higher-order chromatin organization, which influences transcriptional activity.
- MeSH
- Acetylation drug effects MeSH
- Cell Nucleus metabolism MeSH
- Centromere metabolism MeSH
- Chromatin metabolism drug effects MeSH
- Fibroblasts metabolism MeSH
- Financing, Organized MeSH
- Heterochromatin metabolism MeSH
- Enzyme Inhibitors pharmacology MeSH
- Histone Deacetylase Inhibitors MeSH
- Nuclear Proteins metabolism MeSH
- Hydroxamic Acids pharmacology MeSH
- Lamin Type A genetics metabolism deficiency MeSH
- Lamin Type B metabolism MeSH
- Mice MeSH
- Chromatin Assembly and Disassembly drug effects MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal 'head-to-tail' interaction of lamin dimers (the so-called ACN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the ACN complex which features a 6.5 nm overlap of the rod ends.
- MeSH
- Mass Spectrometry MeSH
- Crystallography, X-Ray MeSH
- Lamin Type A chemistry MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
