Enhanced Adhesion of Electrospun Polycaprolactone Nanofibers to Plasma-Modified Polypropylene Fabric.

Excellent adhesion of electrospun nanofiber (NF) to textile support is crucial for a broad range of their bioapplications, e.g., wound dressing development. We compared the effect of several low- and atmospheric pressure plasma modifications on the adhesion between two parts of composite-polycaprolactone (PCL) nanofibrous mat (functional part) and polypropylene (PP) spunbond fabric (support). The support fabrics were modified before electrospinning by low-pressure plasma oxygen treatment or amine plasma polymer thin film or treated by atmospheric pressure plasma slit jet (PSJ) in argon or argon/nitrogen. The adhesion was evaluated by tensile test and loop test adapted for thin NF mat measurement and the trends obtained by both tests largely agreed. Although all modifications improved the adhesion significantly (at least twice for PSJ treatments), low-pressure oxygen treatment showed to be the most effective as it strengthened adhesion by a factor of six. The adhesion improvement was ascribed to the synergic effect of high treatment homogeneity with the right ratio of surface functional groups and sufficient wettability. The low-pressure modified fabric also stayed long-term hydrophilic (ten months), even though surfaces usually return to a non-wettable state (hydrophobic recovery). In contrast to XPS, highly surface-sensitive water contact angle measurement proved suitable for monitoring subtle surface changes.

The subjective relevance of perceived sound aspects in remote singing education.

One of the consequences of the pandemic is a transition to remote education and the use of network audiovisual communication tools for education in musical disciplines. The circumstances of such education can differ and might influence the perceived sound or the education. The research observes the ratings of perceived aspects in singing lessons taught in three settings (common, reference, and direct). A variance of several aspects that relate to the perceived sound (temporal qualities and qualities of the sound and room) is observed in the remote forms, suggesting that these can be impaired in some settings and significant in the experience. The findings are discussed in relation to the perceived conditions and present practice.

Preoperative Chemoradiotherapy for Gastroesophageal Junction Adenocarcinoma Modified by PET/CT: Results of Virtual Planning Study.

Background and Objectives: The treatment of gastroesophageal junction (GEJ) adenocarcinoma consists of either perioperative chemotherapy or preoperative chemoradiotherapy. Radiotherapy (RT) in the neoadjuvant setting is associated with a higher probability of resections with negative margins (R0) and better tumor regression rate, which might be enhanced by incrementing RT dose with potential impact on treatment results. This virtual planning study demonstrates the feasibility of increasing the dose to GEJ tumor and involved nodes using PET/CT imaging. Materials and Methods: 16 patients from the chemoradiotherapy arm of the phase II GastroPET study were treated by a prescribed dose of 45.0 Gray (Gy) in 25 fractions. PET/CT was performed before treatment. The prescribed dose was virtually boosted on PET/CT-positive areas to 54.0 Gy by 9 Gy in 5 fractions. Dose-volume histograms (DVH) were compared, and normal tissue complication (NTCP) modeling was performed for both dose schedules. Results: DVHs were exceeded in mean heart dose in one case for 45.0 Gy and two cases for 54.0 Gy, peritoneal space volume criterion V45Gy < 195 ccm in three cases for 54.0 Gy and V15Gy < 825 ccm in one case for both dose schedules. The left lung volume of 25 Gy isodose exceeded 10% in most cases for both schedules. The NTCP values for the heart, spine, liver, kidneys and intestines were zero for both schemes. An increase in NTCP value was for lungs (median 3.15% vs. 4.05% for 25 × 1.8 Gy and 25 + 5 × 1.8 Gy, respectively, p = 0.013) and peritoneal space (median values for 25 × 1.8 Gy and 25 + 5 × 1.8 Gy were 3.3% and 14.25%, respectively, p < 0.001). Conclusion: Boosting PET/CT-positive areas in RT of GEJ tumors is feasible, but prospective trials are needed.

Predictive and prognostic significance of tumour subtype, SSTR1-5 and e-cadherin expression in a well-defined cohort of patients with acromegaly.

In somatotroph pituitary tumours, somatostatin analogue (SSA) therapy outcomes vary throughout the studies. We performed an analysis of cohort of patients with acromegaly from the Czech registry to identify new prognostic and predictive factors. Clinical data of patients were collected, and complex immunohistochemical assessment of tumour samples was performed (SSTR1-5, dopamine D2 receptor, E-cadherin, AIP). The study included 110 patients. In 31, SSA treatment outcome was evaluated. Sparsely granulated tumours (SGST) differed from the other subtypes in expression of SSTR2A, SSTR3, SSTR5 and E-cadherin and occurred more often in young. No other clinical differences were observed. Trouillas grading system showed association with age, tumour size and SSTR2A expression. Factors significantly associated with SSA treatment outcome included age, IGF1 levels, tumour size and expression of E-cadherin and SSTR2A. In the group of SGST, poor SSA response was observed in younger patients with larger tumours, lower levels of SSTR2A and higher Ki67. We observed no relationship with expression of other proteins including AIP. No predictive value of E-cadherin was observed when tumour subtype was considered. Multiple additional factors apart from SSTR2A expression can predict treatment outcome in patients with acromegaly.

Well-Blended PCL/PEO Electrospun Nanofibers with Functional Properties Enhanced by Plasma Processing.

Biodegradable composite nanofibers were electrospun from poly(ε-caprolactone) (PCL) and poly(ethylene oxide) (PEO) mixtures dissolved in acetic and formic acids. The variation of PCL:PEO concentration in the polymer blend, from 5:95 to 75:25, revealed the tunability of the hydrolytic stability and mechanical properties of the nanofibrous mats. The degradation rate of PCL/PEO nanofibers can be increased compared to pure PCL, and the mechanical properties can be improved compared to pure PEO. Although PCL and PEO have been previously reported as immiscible, the electrospinning into nanofibers having restricted dimensions (250-450 nm) led to a microscopically mixed PCL/PEO blend. However, the hydrolytic stability and tensile tests revealed the segregation of PCL into few-nanometers-thin fibrils in the PEO matrix of each nanofiber. A synergy phenomenon of increased stiffness appeared for the high concentration of PCL in PCL/PEO nanofibrous mats. The pure PCL and PEO mats had a Young's modulus of about 12 MPa, but the mats made of high concentration PCL in PCL/PEO solution exhibited 2.5-fold higher values. The increase in the PEO content led to faster degradation of mats in water and up to a 20-fold decrease in the nanofibers' ductility. The surface of the PCL/PEO nanofibers was functionalized by an amine plasma polymer thin film that is known to increase the hydrophilicity and attach proteins efficiently to the surface. The combination of different PCL/PEO blends and amine plasma polymer coating enabled us to tune the surface functionality, the hydrolytic stability, and the mechanical properties of biodegradable nanofibrous mats.

Plasma-Coated Polycaprolactone Nanofibers with Covalently Bonded Platelet-Rich Plasma Enhance Adhesion and Growth of Human Fibroblasts.

Biodegradable nanofibers are extensively employed in different areas of biology and medicine, particularly in tissue engineering. The electrospun polycaprolactone (PCL) nanofibers are attracting growing interest due to their good mechanical properties and a low-cost structure similar to the extracellular matrix. However, the unmodified PCL nanofibers exhibit an inert surface, hindering cell adhesion and negatively affecting their further fate. The employment of PCL nanofibrous scaffolds for wound healing requires a certain modification of the PCL surface. In this work, the morphology of PCL nanofibers is optimized by the careful tuning of electrospinning parameters. It is shown that the modification of the PCL nanofibers with the COOH plasma polymers and the subsequent binding of NH2 groups of protein molecules is a rather simple and technologically accessible procedure allowing the adhesion, early spreading, and growth of human fibroblasts to be boosted. The behavior of fibroblasts on the modified PCL surface was found to be very different when compared to the previously studied cultivation of mesenchymal stem cells on the PCL nanofibrous meshes. It is demonstrated by X-ray photoelectron spectroscopy (XPS) that the freeze-thawed platelet-rich plasma (PRP) immobilization can be performed via covalent and non-covalent bonding and that it does not affect biological activity. The covalently bound components of PRP considerably reduce the fibroblast apoptosis and increase the cell proliferation in comparison to the unmodified PCL nanofibers or the PCL nanofibers with non-covalent bonding of PRP. The reported research findings reveal the potential of PCL matrices for application in tissue engineering, while the plasma modification with COOH groups and their subsequent covalent binding with proteins expand this potential even further. The use of such matrices with covalently immobilized PRP for wound healing leads to prolonged biological activity of the immobilized molecules and protects these biomolecules from the aggressive media of the wound.

Safety of Anti-TNF-Alpha Therapy During Pregnancy on Long-term Outcome of Exposed Children: A Controlled, Multicenter Observation.

BACKGROUND: Evidence of the impact of in utero exposure to anti-tumor necrosis factor (TNF)-alpha on long-term childhood development is limited. The aim was to assess the impact of in utero exposure to anti-TNF-alpha due to mothers' inflammatory bowel disease (IBD) on long-term postnatal development of exposed children. METHODS: We included consecutive children (≥12 months of age) born to mothers with IBD (2007-2016) treated with anti-TNF-alpha during pregnancy in 3 centers in the Czech Republic. A control group was comprised of unexposed children of non-IBD mothers undergoing mandatory check-ups at general pediatricians' offices. Data on perinatal period, psychomotor development, vaccination, infections, antibiotics, and allergy were collected by treating pediatricians using a predefined questionnaire. RESULTS: Seventy-two exposed and 69 unexposed children were included (median age, 35 and 50 months, respectively). Exposed children had growth and psychomotor development similar to controls. There was no significant difference in infectious complications within the first year of life (23.9% vs 17.4%; P = 0.36) or during the whole follow-up between exposed infants and controls (P = 0.32). Concomitant immunosuppressants during pregnancy and anti-TNF-alpha levels in cord blood were not associated with elevated infection rate within the first year of life (P > 0.05). Over 95% of exposed children had adequate serologic response to vaccination, except for haemophilus and mumps vaccines. Clinically manifested allergy was similar between the groups (P = 0.98). CONCLUSIONS: Anti-TNF-alpha exposure in utero does not seem to have a negative impact on postnatal development of children with regard to infectious complications, allergy, growth, or psychomotor development when compared with unexposed children of non-IBD women.

Importance of Cadherins Methylation in Ovarian Cancer: a Next Generation Sequencing Approach.

Epigenetic aberrations are well known to play an important role in carcinogenesis, and also have a great potential to serve as biomarkers in many types of cancers, including ovarian cancer in which sensitive and specific biomarkers and detection methods are critically needed. The aim of this study was to investigate methylation of cadherin genes CDH10, CDH13 and CDH18 in ovarian cancer tissue by comparison with control tissue. The study group consisted of 38 patients with ovarian cancer and 25 control patients. For detection of epigenetic events we used next generation sequencing, the most important data were confirmed using high-resolution melting analysis and real-time PCR. We observed significantly higher methylation in CDH13, sporadic methylation in CDH10 and loss of methylation in CDH18 in the ovarian cancer group compared with the control group. These observations suggest that changes in methylation of cadherin genes may be one of the major mechanisms associated with ovarian cancer progression. In addition, because of the high frequency of methylation of the CDH13 gene in the early stages of ovarian cancer, the analyzed CpG sites might be good targets for next study of potential ovarian cancer screening biomarkers.

Immobilization of Platelet-Rich Plasma onto COOH Plasma-Coated PCL Nanofibers Boost Viability and Proliferation of Human Mesenchymal Stem Cells.

The scaffolds made of polycaprolactone (PCL) are actively employed in different areas of biology and medicine, especially in tissue engineering. However, the usage of unmodified PCL is significantly restricted by the hydrophobicity of its surface, due to the fact that its inert surface hinders the adhesion of cells and the cell interactions on PCL surface. In this work, the surface of PCL nanofibers is modified by Ar/CO2/C2H4 plasma depositing active COOH groups in the amount of 0.57 at % that were later used for the immobilization of platelet-rich plasma (PRP). The modification of PCL nanofibers significantly enhances the viability and proliferation (by hundred times) of human mesenchymal stem cells, and decreases apoptotic cell death to a normal level. According to X-ray photoelectron spectroscopy (XPS), after immobilization of PRP, up to 10.7 at % of nitrogen was incorporated into the nanofibers surface confirming the grafting of proteins. Active proliferation and sustaining the cell viability on nanofibers with immobilized PRP led to an average number of cells of 258 ± 12.9 and 364 ± 34.5 for nanofibers with ionic and covalent bonding of PRP, respectively. Hence, our new method for the modification of PCL nanofibers with PRP opens new possibilities for its application in tissue engineering.

Importance of promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium.

BACKGROUND: Epigenetic changes are considered to be a frequent event during tumor development. Various methylation changes have been identified and show promise as potential cancer biomarkers. The aim of this study was to investigate promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium. METHODS: To search for promoter methylation of GATA4 and TP53 genes we used methylation-specific PCR (MSP) to compare the methylation status of 54 patients with endometrioid carcinoma of endometrium and 18 patients with normal endometrial tissue. RESULTS: In our study MSP revealed GATA4 promoter methylation in 44 of 54 in the carcinoma group (81.5%), and in none of the control group. No methylation was observed in TP53 gene. CONCLUSIONS: In conclusion, our study showed that there is significantly higher methylation in GATA4 gene in the endometrial cancer group compared with samples of non-neoplastic endometrium. The finding suggests the importance of hypermethylation of this gene in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies for endometrial cancer based on epigenetic changes.

Importance of promoter methylation of GATA4 gene in epithelial ovarian cancer.

AIMS: Ovarian cancer is the most lethal gynecological malignancy, with typically late diagnosis. Altered DNA methylation of tumor suppressor gene promoters probably plays a relevant role in ovarian carcinogenesis and frequently occurs as an early event in the development of different types of cancer including ovarian carcinoma. GATA4 methylation has been reported in a variety of human cancers. The aim of this study was to investigate promoter methylation of the GATA4 gene in ovarian cancer by comparison with that in normal ovarian tissue. METHODS: To search for promoter methylation of the GATA4 gene we used MSP (methylation-specific PCR) to compare the methylation status in 67 tissue samples of ovarian cancer with that in 40 control samples. RESULTS: In our study, methylation-specific PCR revealed GATA4 promoter methylation in 21 of 67 specimens with ovarian cancer (31.3%), and in none of the control ovarian tissue samples. CONCLUSION: These results confirm that methylation in the GATA4 promoter region could play an important role in ovarian carcinogenesis, and show new loci which are highly methylated only in ovarian cancer samples and which are associated predominantly with the endometrioid type of ovarian carcinoma.

Detection of the GPI-anchorless prion protein fragment PrP226* in human brain.

BACKGROUND: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2. METHODS: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot. RESULTS: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD). CONCLUSIONS: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.

Methylation analysis of tumor suppressor genes in endometroid carcinoma of endometrium using MS-MLPA.

BACKGROUND: Epigenetic changes are considered to be a frequent event during tumor development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumor suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in endometrial cancer by comparison with normal endometrial tissue. MATERIALS AND METHODS: We used MS-MLPA (Methylation-specific Multiplex ligation-dependent probe amplification) to compare the methylation status of 59 tissue samples of endometroid type of endometrial carcinoma with 20 control samples of non-neoplastic endometrium. RESULTS: Using 15% cut-off for methylation, we observed significantly higher methylation in the CDH13 gene in endometrial cancer group. We observed significantly higher methylation in both WT1 and GATA5 genes in IB stage of endometroid carcinoma. We also observed significantly higher methylation in GATA5 gene in the group of poorly differentiated endometroid carcinoma. CONCLUSION: The findings suggest the importance of hypermethylation of CDH13, WT1 and GATA5 genes in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies of endometrial cancer based on epigenetic changes.

Quinacrine reactivity with prion proteins and prion-derived peptides.

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to "covalently" modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV-vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.

Development of monoclonal antibodies specific for glycated prion protein.

Transmissive spongiform encephalopathies (TSE) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrP(TSE)) in brain. PrP(TSE) is at present the only specific biochemical marker of human and animal TSE. As deposits of PrP(TSE) remain in the body for long periods, there is substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrP(TSE) may have potential to serve as a diagnostic marker. Monoclonal antibodies specific for carboxymethyl lysine/arginine-modified prion protein were prepared. Recombinant human prion protein (rhPrP) was bacterially expressed and purified by affinity chromatography. rhPrP was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of 6 mice, and 960 hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of four promising clones. One of them (EM-31) strongly reacts with human and mouse recombinant PrP-CML, and three other clones react also with CML in vitro modified human and mouse brain PrP. Besides possible implication in TSE diagnostics, the antibodies may serve as tolls to advance our knowledge regarding the role of glycation in the prion pathophysiology.

Underestimation of the expression of cellular prion protein on human red blood cells.

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).

Focal sarcoid-like change of the thyroid gland. A possible consequence of aspiration cytology?

An unusual case of focal accumulation of sarcoid-like granulomas occurring within the thyroid gland of a 43-year-old female patient is reported. The granulomas were found solely at the site of previous fine needle aspiration biopsy. The follow-up did not show any symptoms of systemic granulomatous disease. The pathogenesis of this lesion is discussed.