Development of Synthetic Platelet‐Activating Hydrogel Matrices to Induce Local Hemostasis

Several hemostatic strategies rely on the use of blood components such as fibrinogen and thrombin, which suffer from high cost and short shelf‐life. Here, a cost‐effective synthetic biomaterial is developed for rapid local hemostasis. Instead of using thrombin, thrombin‐receptor‐agonist‐peptide‐6 (TRAP6) is covalently engineered in polyvinyl alcohol (PVA) hydrogels. Soluble PVA‐TRAP6 is first prepared by covalent attachment of cysteine‐containing TRAP6 onto the backbone of PVA‐norbornenes (PVA‐NB) through photoconjugation. Cytotoxicity studies using C2C12 myoblasts indicate that PVA‐NB and PVA‐TRAP6 are nontoxic. Thromboelastography reveals that hemostatic activity of TRAP6 is retained in conjugated form, which is comparable to free TRAP6 solutions with equal concentrations. A 0.1% PVA‐TRAP6 solution can shorten the clotting time (CT) to ca. 45% of the physiological CT. High platelet‐activating efficiency is further confirmed by platelet aggregation assay and flow cytometry (FACS). For potential clinical applications, TRAP6‐presenting hydrogel particulates (PVA‐TRAP6‐P) are developed for local platelet activation and hemostasis. PVA‐TRAP6‐P is prepared by biofunctionalization of photopolymerized PVA‐NB hydrogel particulates (PVA‐NB‐P) with TRAP6. It is demonstrated that PVA‐TRAP6‐P can effectively shorten the CT to ca. 50%. FACS shows that PVA‐TRAP6‐P can activate platelets to a comparable extent as soluble TRAP6 control. Altogether, PVA‐TRAP6‐P represents a promising class of biomaterials for safe hemostasis and wound healing.     

Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.     

Influence of an additional carbon layer at the back contact-absorber interface in Cu(In,Ga)Se2 thin film solar cells

For paste-coated Cu(In,Ga)Se₂ (CIGS) absorber layers used for thin film solar cells one often gets a residual carbon layer between back contact and absorber layer. We investigate the influence of this layer on the solar cells’ performance with co-evaporated CIGS absorbers and find a beneficial effect. The power conversion efficiencies of thin chalcopyrite absorber layers are often limited by the influence of back contact recombination. It is assumed that the carbon layer between the back contact and the absorber layer helps lower this recombination and allows higher open circuit voltages and thus higher conversion efficiencies.     

Quality Control of Human Pluripotent Stem Cell Colonies by Computational Image Analysis Using Convolutional Neural Networks

Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes. Their maintenance in culture requires the deep control of their pluripotent and clonal status. A non-invasive method for such control involves day-to-day observation of the morphological changes, along with imaging colonies, with the subsequent automatic assessment of colony phenotype using image analysis by machine learning methods. We developed a classifier using a convolutional neural network and applied it to discriminate between images of human embryonic stem cell (hESC) colonies with “good” and “bad” morphological phenotypes associated with a high and low potential for pluripotency and clonality maintenance, respectively. The training dataset included the phase-contrast images of hESC line H9, in which the morphological phenotype of each colony was assessed through visual analysis. The classifier showed a high level of accuracy (89%) in phenotype prediction. By training the classifier on cropped images of various sizes, we showed that the spatial scale of ~144 μm was the most informative in terms of classification quality, which was an intermediate size between the characteristic diameters of a single cell (~15 μm) and the entire colony (~540 μm). We additionally performed a proteomic analysis of several H9 cell samples used in the computational analysis and showed that cells of different phenotypes differentiated at the molecular level. Our results indicated that the proposed approach could be used as an effective method of non-invasive automated analysis to identify undesirable developmental anomalies during the propagation of pluripotent stem cells.     

Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization,Multiplex RNA Hybridization,Sequencing and Subtyping

Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting <200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response.     

Aminopeptidase N Inhibitors as Pointers for Overcoming Antitumor Treatment Resistance

Aminopeptidase N (APN), also known as CD13 antigen or membrane alanyl aminopeptidase, belongs to the M1 family of the MA clan of zinc metallopeptidases. In cancer cells, the inhibition of aminopeptidases including APN causes the phenomenon termed the amino acid deprivation response (AADR), a stress response characterized by the upregulation of amino acid transporters and synthetic enzymes and activation of stress-related pathways such as nuclear factor kB (NFkB) and other pro-apoptotic regulators, which leads to cancer cell death by apoptosis. Recently, APN inhibition has been shown to augment DR4-induced tumor cell death and thus overcome resistance to cancer treatment with DR4-ligand TRAIL, which is available as a recombinant soluble form dulanermin. This implies that APN inhibitors could serve as potential weapons for overcoming cancer treatment resistance. In this study, a series of basically substituted acetamidophenones and the semicarbazones and thiosemicarbazones derived from them were prepared, for which APN inhibitory activity was determined. In addition, a selective anti-proliferative activity against cancer cells expressing APN was demonstrated. Our semicarbazones and thiosemicarbazones are the first compounds of these structural types of Schiff bases that were reported to inhibit not only a zinc-dependent aminopeptidase of the M1 family but also a metalloenzyme.     

COVID-19, Vaccination,and Female Fertility in the Czech Republic

The fast-track process to approve vaccines against COVID-19 has raised questions about their safety, especially in relation to fertility. Over the last 2 years, studies have appeared monitoring female fertility, especially from assisted reproduction centers or in animal experiments. However, studies monitoring healthy populations are still limited. The aim of our study was to monitor the relevant parameters of female fertility (sex and other steroids, LH, FSH, SHBG, Antimüllerian hormone and antral follicle count) before and then 2–4 months after the third dose of vaccination against COVID-19 in a group of 25 healthy fertile woman. In addition, anti-SARS-CoV-2 and anti-SARS-CoV-2S antibodies were determined. We did not observe significant changes in the measured parameters before and after the third dose of vaccination. By comparing levels of the analytes with antibodies indicating a prior COVID-19 infection, we found that women who had experienced the disease had statistically lower levels of estrone, estradiol, SHBG and 5α-dihydroprogesterone, and conversely, higher levels of androgen active dehydroepiandrosterone and dihydrotestosterone. Our results confirm that vaccination does not affect female fertility, and that what fertile women should be worried about is not vaccination, but rather COVID-19 infection itself.     

Approach versus avoidance: Different types of commitment in intimate relationships.

The major objective of the present study was to examine whether approach versus avoidance commitment to one's intimate relationship was differentially predictive of relationship quality parameters in the long run. In the 1st testing period, 134 participants (67 romantic couples) answered questions about approach- versus avoidance-related measures. Commitment and relationship quality parameters such as satisfaction and emotions depending on the partner's presence were assessed in all 3 testing periods. The proposed distinction between an approach and an avoidance type of commitment was validated through correlations with other approach- versus avoidance-related measures. Longitudinal analyses revealed that approach commitment predicted relationship quality parameters positively, whereas avoidance commitment predicted them negatively. The results are discussed in terms of the benefit of an approach-avoidance-based conceptualization of commitment. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Synthesis of Decorative Glass-Ceramic Material Based on Tinted Slag

A decorative glass-ceramic material was synthesized on the basis of tinted waste (slag). The resulting material has high chemical and thermal resistance, a saturated blue color, and can be used in various sectors of industry.