Nanofibrous materials have great potential for use in tissue engineering due to their structure, which mimics the extracellular fibrous matrix. Increasing their biological activity is currently the ...main goal in the development of these scaffolds. From the standpoint of promoting healing and tissue regeneration, the use of human platelets containing hundreds of biologically active molecules is promising. The present work deals with the preparation of PVA-based nanofibrous material containing native platelet-derived proteins that are released in a sustained manner. The needleless electrospinning process of preparation of material that does not affect the activity of incorporated proteins has been optimized, and the resulting material can be produced on a large scale with a protein loading efficiency of 0.64%. The reasonable fiber diameter distribution (370±150 nm) with low defects ensures a homogeneous distribution of proteins. The use of high molecular weight PVA (125 000 g/mol) with a high degree of hydrolysis (98-98.8%) resulted in a 40% reduction in PVA solubility without the need for subsequent covalent crosslinking. This, in turn, results in a sustained release of proteins, where after an initial burstrelease of 90% of the proteins, 10% is gradually released over the next 7 days. Our results demonstrate the potential use of the platelet-lysate loaded PVA material in tissue engineering.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
A number of studies have reported aberrant glycosylation in connection with malignancy. Our investigation further expands on this topic through the examination of N-glycans, which could be associated ...with the resistance of advanced stage, high-grade non-mucinous ovarian cancer to platinum/taxane based chemotherapy. We used tissue samples of 83 ovarian cancer patients, randomly divided into two independent cohorts (basic and validation). Both groups involved either cases with/without postoperative tumor residue or the cases determined either resistant or sensitive to this chemotherapy. In the validation cohort, preoperative serum samples were also available. N-glycans released from tumors and sera were permethylated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS analysis yielded a consecutive detection of 68 (tissue) and 63 (serum) N-glycan spectral signals. Eight of these were found to be differentially abundant in tissues of both independent cohorts including the cases with a postoperative cancer residue. One of these glycans was detected as differentially abundant in sera of the validation cohort. No statistically significant differences in intensities due to the same N-glycans were found in the cases without postoperative macroscopic residues in either the basic or validation cohort.
From the biochemical point of view, the statistically significant N-glycans correspond to the structures carrying bisecting (terminal) GlcNAc residue and tetra-antennary structures with sialic acid and/or fucose residues. Among them, six tissue N-glycans could be considered potential markers connected with a resistance to chemotherapy in ovarian cancer patients. The prediction of primary resistance to standard chemotherapy may identify the group of patients suitable for alternative treatment strategies.
Drug resistance has become a major impediment to a successful treatment of patients with advanced ovarian cancer. The glycomic measurements related to cancer are becoming increasingly popular in identification of the key molecules as potential diagnostic and prognostic indicators. Our report deals with identification of differences in N-glycosylation of proteins in tissue and serum samples from the individuals showing sensitivity or resistance to platinum/taxane-based chemotherapy. The detection sensitivity to chemotherapy is vitally important for these patients.
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•Molecular mechanisms of resistance to platinum derivate-based chemotherapy in patients with ovarian cancer is unclear.•Several N-glycans have been found differentially expressed in ovarian cancer.•Six tissue N-glycans could be considered as markers associated with a resistance to chemotherapy in ovarian cancer patients.
Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. ...Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.
A new β-elimination-based procedure has been devised for a microscale release of O-linked oligosaccharides from glycoproteins. Unlike the conventional Carlson degradation, which leads to formation of ...alditols, the procedure reported here renders the reducing end intact. Conversion of the liberated oligosaccharides to glycosylamines in ammonia medium is followed by the production of the reducing oligosaccharides through the addition of boric acid. The quantitatively generated oligosaccharides with the reducing end can subsequently be derivatized with a fluorophoric reagent for capillary electrophoresis or, alternatively, analyzed through MALDI mass spectrometry. The microscale version of these chemical steps permits us to investigate structurally O-linked oligosaccharides at very low levels.
To characterize the structures of N-glycans derived from human serum, we report a strategy that combines microchip electrophoresis, standard addition, enzymatic digestion, and matrix-assisted laser ...desorption/ionization-mass spectrometry (MALDI-MS). We compared (i) electrophoretic mobilities of known N-glycans from well-characterized (standard) glycoproteins through standard addition, (ii) the electrophoretic mobilities of N-glycans with their molecular weights determined by MALDI-MS, and (iii) electrophoretic profiles of N-glycans enzymatically treated with fucosidase. The key step to identify the sialylated N-glycans was to quantitatively neutralize the negative charge on both α2,3- and α2,6-linked sialic acids by covalent derivatization with methylamine. Both neutralized and nonsialylated N-glycans from these samples were then reacted with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) to provide a fluorescent label and a triple-negative charge, separated by microchip electrophoresis, and detected by laser-induced fluorescence. The methylamidation step leads to a 24% increase in the peak capacity of the separation and direct correlation of electrophoretic and MALDI-MS results. In total, 37 unique N-glycan structures were assigned to 52 different peaks recorded in the electropherograms of the serum samples. This strategy ensures the needed separation efficiency and detectability, easily resolves linkage and positional glycan isomers, and is highly reproducible.
Comparative glycan quantification has thus far been a challenging task due to the lack of sensitive and reproducible analytical techniques. We introduce here a combination of quantitative ...permethylation and isotope labeling of glycans as an approach (C-GlycoMAP) allowing precise comparison between different samples in a single MALDI-MS analysis. Samples are either methylated or deuteriomethylated prior to their mixing and mass spectrometric acquisitions. Comparative analyses are based on the ratio of the two isotopically distinct forms of the same glycan structure, thus allowing a direct absolute evaluation of the intensities of the two forms originating from two different biological samples (e.g., control and diseased). The direct comparison between the two forms eliminates a MALDI-MS low m/z bias commonly associated with this technique. These comparative analyses are highly reliable when the intensity ratios of the two forms lie between 0.125 and 6, an overall reproducibility better than 30% (RSD). The value of C-GlycoMAP is demonstrated here for N-glycans derived from human blood serum collected from a healthy individual and a breast cancer patient as well as for O-glycans derived from normal and cancer cell extracts.
Extensive cross-ring fragmentation ions, which are very informative of the linkages of the monosaccharide residues constituting these molecules, were readily observed in the MALDI-TOF/TOF/MS/MS ...spectra of oligosaccharides. These ions, in some cases, were more intense than the commonly observed Y and B ions. The A-type ions observed for the simple oligosaccharides allowed the distinction between α(1−4)- and α(1−6)-linked isobaric structures. The distinction was based not merely on the differences in the type of ions formed, but also on the ion intensities. For example, both α(1−4)- and α(1−6)-linked isobaric structures produce ions resulting from the loss of ∼120 m/z units, but with different intensities, as a result of the fact that they correspond to two different ions (i.e., 0,4A- and 2,4A-ions), requiring different energies to be formed. Abundant A- and X-type ions were also observed for high-mannose N-glycans, allowing the determination of linkages. In addition, the high resolution furnished by MALDI-TOF/TOF allowed determination of certain ions that were commonly overlooked by MALDI-TOF or MALDI-magnetic sector instruments as a result of their lower resolution. Moreover, as a result of the fact that MS/MS spectra for parent ions and all fragment ions are acquired under the same experimental conditions, accurate determination of the molar ratios of isomeric glycans in a mixture analyzed simultaneously by MALDI-TOF/TOF tandem MS becomes possible.
As the role of O-linked oligosaccharides have been demonstrated to be increasingly important in numerous medical conditions, it is imperative to develop new techniques allowing their analysis at high ...sensitivity. While mass spectrometry (MS) provides adequate measurements of important O-linked oligosaccharides glycans and their profiles, the release from glycoproteins has not been sufficiently addressed for the needs of biomedical applications. This work describes a new strategy, involving the combination of a complete enzymatic degradation with a chemical release during the solid-phase permethylation of O-linked oligosaccharides. The analytical data implicate highly effective cleavage from the serine and threonine (but not arginine) residues, during permethylation. Tandem MS analyses confirmed these observations for model glycoproteins. Comparative measurements through isotopic labeling MS show this approach to be vastly superior over the previously used cleavage procedures.
Excessive home cage aggression often results in severe injury and subsequent premature euthanasia of male laboratory mice. Aggression can be reduced by transferring used nesting material during cage ...cleaning, which is thought to contain aggression appeasing odors from the plantar sweat glands. However, neither the composition of plantar sweat nor the deposits on used nesting material have been evaluated. The aims of this study were to (1) identify and quantify volatile compounds deposited in the nest site and (2) determine if nest and sweat compounds correlate with social behavior. Home cage aggression and affiliative behavior were evaluated in 3 strains: SJL, C57BL/6N, and A/J. Individual social rank was assessed via the tube test, because ranking may influence compound levels. Sweat and urine from the dominant and subordinate mouse in each cage, plus cage level nest samples were analyzed for volatile compound content using gas chromatography-mass spectrometry. Behavior data and odors from the nest, sweat, and urine were statistically analyzed with separate principal component analyses (PCA). Significant components, from each sample analysis, and strain were run in mixed models to test if odors were associated with behavior. Aggressive and affiliative behaviors were primarily impacted by strain. However, compound PCs were also impacted by strain, showing that strain accounts for any relationship between odors and behavior. C57BL/6N cages displayed the most allo-grooming behavior and had high scores on sweat PC1. SJL cages displayed the most aggression, with high scores on urine PC2 and low scores on nest PC1. These data show that certain compounds in nesting material, urine, and sweat display strain specific patterns which match strain specific behavior patterns. These results provide preliminary information about the connection between home cage compounds and behavior. Salient compounds will be candidates for future controlled studies to determine their direct effect on mouse social behavior.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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