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Wyniki wyszukiwania dla: in vitro metabolizm
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Fabrication and Characterization of Flexible Medical-Grade TPU Filament for Fused Deposition Modeling 3DP Technology
PublikacjaThe possibility of using additive manufacturing (AM) in the medicine area has created new opportunities in health care. This has contributed to a sharp increase in demand for 3D printers, their systems and materials that are adapted to strict medical requirements. We described herein a medical-grade thermoplastic polyurethane (S-TPU) which was developed and then formed into a filament for Fused Deposition Modeling (FDM) 3D printers...
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Properties of Different Varieties of Durian
PublikacjaDurian (Durio zibethinus Murr.), like many other exotic, tropical, and conventional fruits, is important in the prevention of different diseases. In this study, the characterization of the main bioactive compounds of the most popular cultivars of durian and their properties are described. The changes in the quality indices of the antioxidant status were determined by CUPRAC, ABTS, FRAP, DPPH, and ORAC assays. The profiling of phytochemicals...
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New generation of analytical tests based on the assessment of enzymatic and nuclear receptor activity changes induced by environmental pollutants
PublikacjaAnalytical methods show great potential in biological tests. The analysis of biological response that results from environmental pollutant exposure allows: (i) prediction of the risk of toxic effects and (ii) provision of the background for the development of markers of the toxicants presence. Bioanalytical tests based on changes in enzymatic activity and nuclear receptor action provide extremely high specificity and sensitivity....
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Construction of glucosamine-6-phosphate synthase with an internal hexahistydyl fragment
PublikacjaL-Glutamine: D-fructose-6-phosphate aminotransferase (EC 2.6.1.16), known also as glucosamine-6-phosphate (GlcN-6-P) syntase, catalyzes the first committed step in the amino sugar biosynthetic pathway in prokaryotic and eukaryotic organisms. The enzyme carries out a complex reaction involving ammonia transfer and sugar phosphate isomerization. GlcN-6-P syntase is an important point of metabolic control of amino sugar biosynthesis...
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Participatory co-design approach for Greencoin educational tool shaping urban green behaviors.
PublikacjaOur main goal is to prepare assumptions of the Greencoin (GC) cybernetic system, implying pro-ecological attitudes and behavior of city residents. We used qualitative methods, including a literature review and action research -workshops attended by academics, representatives of private and business sectors, urban movements, municipal institutions’ partners, and residents. Our results defined functionalities of the GC, identified...
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Splice variants of mitofusin 2 shape the endoplasmic reticulum and tether it to mitochondria
PublikacjaIn eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology,...
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The Occurrence and Role of Tetrasphaera in Enhanced Biological Phosphorus Removal Systems
PublikacjaThe application of enhanced biological phosphorus removal (EBPR) in wastewater treatment plants (WWTPs) has commonly been utilized worldwide. However, the optimum efficiency has not been realized over the past decades, prompting many studies and publications. The limitations, especially comprehension of the abundance and actual potential of polyphosphate-accumulating organisms (PAOs), are not fully understood. Recently identified...
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Pesticides in precipitation - a review of data from 1975 to 2001 = Stężenia pestycydów w opadach atmosferycznych w latach 1975-2001
PublikacjaZebrano i opracowano dane literaturowe dotyczące trwałości w środowisku, metodyki analitycznej i przede wszystkim wyników badań pestycydów występujących w próbkach opadów atmosferycznych w różnych regionach. Przeprowadzono analizę danych i przedstawiono stan zanieczyszczenia wód opadowych pestycydami w okresie 1975-2001 na terenie Europy i Ameryki Północnej. Z zebranych danych wynika, że pestycydy występują wszędzie, nie tylko...
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Ultimate biodegradability and ecotoxicity of orally administered antidiabetic drugs
PublikacjaHypoglycaemic pharmaceuticals are recently more and more frequently detected in the environment. In our previous study, we have shown that even though many of them undergo significant primary degradation some are transformed to stable products or undergo such transformation that a large part of the structure is still preserved. One of the main routes of elimination from wastewaters or surface waters is biodegradation and a lack...
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Nuclear Magnetic Resonance Metabolomics Reveals Qualitative and Quantitative Differences in the Composition of Human Breast Milk and Milk Formulas
PublikacjaCommercial formula milk (FM) constitutes the best alternative to fulfill the nutritional requirements of infants when breastfeeding is precluded. Here, we present the comparative study of polar metabolite composition of human breast milk (HBM) and seven different brands of FM by nuclear magnetic resonance spectroscopy. The results of the multivariate data analysis exposed qualitative and quantitative differences between HBM and...
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Mitochondrial DNA copy number and trimethylamine levels in the blood: New insights on cardiovascular disease biomarkers
PublikacjaAmong cardiovascular disease (CVD) biomarkers, the mitochondrial DNA copy number (mtDNAcn) is a promising candidate. A growing attention has been also dedicated to trimethylamine-N-oxide (TMAO), an oxidative derivative of the gut metabolite trimethylamine (TMA). With the aim to identify biomarkers predictive of CVD, we investigated TMA, TMAO, and mtDNAcn in a population of 389 coronary artery disease...
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Bioaccumulation of metals in tissues of marine animals, Part II - metal concentrations in animal tissues
PublikacjaThe bioaccumulation of metals in an animal depends on a multitude of factors: biotic ones, like its body dimensions and mass, age, sex, diet, metabolism and position in the trophic web, as well as abiotic ones such as the distribution of metals in its environment, the salinity, temperature and pH of the water, habitat type, interactions with other metals. But it is diet that has the greatest influence on the accumulation of metals...
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Cabbage Juices and Indoles Modulate the Expression Profile of AhR, ERα, and Nrf2 in Human Breast Cell Lines
PublikacjaOur previous studies showed the diversified effect of cabbage juices and indoles on the estrogen metabolism key enzymes (CYP1A1, CYP1A2, CYP1B1) in breast epithelial cells differing in ER status, i.e., in tumorigenic—MCF7, MDA-MB-231 and non-tumorigenic—MCF10A cells. The aim of the present study was to further investigate the mechanism of chemopreventive action of cabbage juice and its active components by evaluating their effect...
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Glutathione-mediated conjugation of anticancer drugs: an overview of reaction mechanisms and biological significance for drug detoxification and bioactivation.
PublikacjaThe effectiveness of many anticancer drugs depends on the creation of specific metabolites that may alter their therapeutic or toxic properties. One significant route of biotransformation is a conjugation of electrophilic compounds with reduced glutathione, which can be non-enzymatic and/or catalyzed by glutathione-dependent enzymes. Glutathione usually combines with anticancer drugs and/or their metabolites to form more polar...
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Response of a New Low-Coherence Fabry-Perot Sensor to Hematocrit Levels in Human Blood
PublikacjaIn this paper, a low-coherence Fabry-Perot sensor with a spectrally measured signal processing response to the refractive index of liquids is presented. Optical fiber sensors are potentially capable of continuous measuring hematocrit levels in blood. Low-coherence Fabry-Perot interferometric sensors offer a robust solution, where information about the measurand is encoded in the full spectrum of light reflected from the sensing...
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Solid Phase Synthesis and Biological Activity of Tuftsin Conjugates
PublikacjaNew tuftsin/retro-tuftsin conjugates were designed and synthesized using a classical fluorenylmethoxycarbonyl (Fmoc) solid phase procedure. All the peptide conjugates were divided into three series: 1,4-dihydroxyanthraquinone (type A), 1-nitroacridine (type B), and 4-carboxyacridone (type C) derivatives. In type A conjugates, the N-terminal group of the peptide chain is directly connected to the anthraquinone ring at C1 (Scheme...
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Modulation of cellular response to anticancer treatment by caffeine:inhibition of cell cycle checkpoints, DNA repair and more
PublikacjaKofeina i inne metyloksantyny wywołują bardzo różne efekty fizjologiczne w organizmie człowieka. W pracy przedstawiamy, które z tych efektów mogą wpływać na skuteczność terapii przeciwnowotworowych. Kofeina może bezpośrednio wpływać na transport oraz aktywację metaboliczną cząsteczek leków do komórki, poprzez tworzenie kompleksów z lekami zawierającymi układy poliaromatyczne. Kofeina hamuje aktywność kinaz ATM/ATR co prowadzi do...
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Steroid Sulfatase Inhibitors Based on Phosphate and Thiophosphate Flavone Analogs
PublikacjaA series of phosphate and thiophosphate flavone derivatives were synthesized and biologically evaluated in vitro for inhibition of steroid sulfatase (STS) activity. The described synthesis includes the straightforward preparation of 7-hydroxy-2-phenyl-4H-chromen-4-one 3a, 2-(4-fluorophenyl)-7-hydroxy-4H-chromen-4-one 3b, 7-hydroxy-2-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one 3c, 7-hydroxy-2-(p-tolyl)-4H-chromen-4-one 3d modified...
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Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1
PublikacjaIn this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules....
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Gelatin and gelatin/starch-based films modified with sorbitol for wound healing
PublikacjaGelatin-based films modified with sorbitol were produced from gelatin solution or gelatin/starch blends using a simple and low-cost solvent casting method, and subsequently, their physicochemical, mechanical, and biocompatibility properties were characterized. This work focused on developing and optimizing a biopolymeric blend to improve the pure biopolymers’ properties for potential biomedical applications such as wound dressing....
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Hyaluronic acid/tannic acid films for wound healing application
PublikacjaIn this study, thin films based on hyaluronic acid (HA) with tannic acid (TA) were investigated in three different weight ratios (80HA/20TA, 50HA/50TA, 20HA/80TA) for their application as materials for wound healing. Surface free energy, as well as their roughness, mechanical properties, water vapor permeability rate, and antioxidant activity were determined. Moreover, their compatibility with blood and osteoblast cells was investigated....
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Antibacterial Porous Systems Based on Polylactide Loaded with Amikacin
PublikacjaThree porous matrices based on poly(lactic acid) are proposed herein for the controlled release of amikacin. The materials were fabricated by the method of spraying a surface liquid. Description is given as to the possibility of employing a modifier, such as a silica nanocarrier, for prolonging the release of amikacin, in addition to using chitosan to improve the properties of the materials, e.g., stability and sorption capacity....
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Novel chalcone-derived pyrazoles as potential therapeutic agents for the treatment of non-small cell lung cancer
PublikacjaLung cancer is considered to account for approximately one-fifth of all malignant tumor-related deaths worldwide and is therefore one of the most lethal malignancies. Pyrazole scaffold possesses a wide range of biological and pharmacological activities, which play important roles in medicinal chemistry. The present study reports the synthesis and in vitro biological characterization of nine pyrazoles derived from chalcones as potential...
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Comparative Assessment of Bacteriophage and Antibiotic Activity against Multidrug-Resistant Staphylococcus aureus Biofilms
PublikacjaProblems connected with biofilm-related infections and antibiotic resistance necessitate the investigation and development of novel treatment strategies. Given their unique characteristics, one of the most promising alternatives to conventional antibiotics are bacteriophages. In the in vitro and in vivo larva model study, we demonstrate that phages vB_SauM-A, vB_SauM-C, and vB_SauM-D are effective antibiofilm agents. The exposure...
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Utilizing Genome-Wide mRNA Profiling to Identify the Cytotoxic Chemotherapeutic Mechanism of Triazoloacridone C-1305 as Direct Microtubule Stabilization
PublikacjaRational drug design and in vitro pharmacology profiling constitute the gold standard in drug development pipelines. Problems arise, however, because this process is often dicult due to limited information regarding the complete identification of a molecule’s biological activities. The increasing aordability of genome-wide next-generation technologies now provides an excellent opportunity to understand a compound’s diverse eects...
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Ruch w koncepcyjnej architekturze i urbanistyce XX wieku
PublikacjaArtykuł opisuje, zależność pomiędzy XX wiecznymi utopijnymi koncepcjami architektonicznymi zestawionymi z współcześnie realizowanymi obiektami lub prototypami. Poprzez analizy wykorzystania ruchu w sztuce, ze szczególnym uwzględnieniem rzeźby i architektury. Ruch i dynamika od zawsze stanowiły element, który fascynował człowieka. Ruch pojawia się najpierw w rzeźbie za sprawą Marcela Duchampa, w ślad z jego dziełami idą między innymi...
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The Application of Vibration Recording and Analysis in Tribological Research on Sliding Friction
PublikacjaThe paper reports on a tribological research on the macroscopic manifestation and characteristics of sliding friction. The aim of the task was to measure friction in lubricated sliding contacts and test the interactions between the environment (the test rig) and the experimental friction contact. Friction-induced vibrations were observed and studied as a manifestation of the process. The typical set of velocity and force/torque...
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Synthetic strategies in construction of organic macromolecular carrier–drug conjugates
PublikacjaMany metabolic inhibitors, considered potential antimicrobial or anticancer drug candidates, exhibit verylimited ability to cross the biological membranes of target cells. The restricted cellular penetration ofthose molecules is often due to their highhydrophilicity. One of the possible solutions to this problem is aconjugation of an inhibitor with a molecular organic nanocarrier. The conjugate thus formed should beable to penetrate...
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Profiling of polar ionogenic metabolites in Polish wines by capillary electrophoresis-mass spectrometry
PublikacjaThe composition of wine is determined by a complex interaction between environmental factors, genetic factors (i.e., grape varieties), and winemaking practices (including technology and storage). Metabolomics using NMR spec-troscopy, GC-MS, and/or LC-MS has shown to be a useful approach for assessing the origin, authenticity, and quality of various wines. Nonetheless, the use of additional analytical techniques with complementary...
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Hormony płciowe w środowisku - zagrożenia jakie niosą ze sobą
PublikacjaSpośród dużej ilości i różnorodności związków chemicznych dostających się do środowiska coraz większą uwagę i szczególne zainteresowanie poświęca się związkom endokrynnym (ang. Endocrine Disrupting Coumpounds - EDCs). Wśród związków endokrynnych to estrogeny zarówno naturalne jak i syntetyczne zostały wskazane i opisane jako związki niosące ze sobą największy potencjał aktywności estrogennej. Jednym z głównych źródeł estrogenów...
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Non-volatile molecular composition and discrimination of single grape white of chardonnay, riesling, sauvignon blanc and silvaner using untargeted GC–MS analysis
PublikacjaThis study developed and applied a GC–MS method aiming at molecular fingerprinting of 120 commercial single grape white wines (Chardonnay, Riesling, Sauvignon Blanc and Silvaner) for possible authentication according to grape variety. The method allowed detection of 372 peaks and tentative identification of 146 metabolites including alcohols, organic acids, esters, amino acids and sugars. The grape variety effect explained 8.3%...
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Zastosowanie immobilizowanego białka MutS do wychwytywania fragmentów DNA zawierających niesparowane zasady
PublikacjaBiałko MutS wiąże DNA zawierające błędnie sparowane oraz niesparowane zasady w DNA. Podjęto szereg prób wykorzystania tego białka jako narzędzia do detekcji nieznanych mutacji lub zagęszczania frakcji DNA z niesparowaną zasadą. Dotychczas żadna z metod oparta na użyciu białka MutS nie została wdrożona powszechnie do praktyki laboratoryjnej. Głównym powodem takiego stanu rzeczy jest stosunkowo silne powinowactwo MutS do DNA w pełni...
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Badanie przepuszczalności jelitowej - nowatorskie narzędzie diagnozy wielu chorób
PublikacjaPrzepuszczalność jelitowa jest przedmiotem badań od ponad dwóch dekad. Zbadano wpływ wielu chorób i schorzeń na działanie układu pokarmowego. Układ pokarmowy jest skomplikowanym systemem połączonych ze sobą narządów i spełnia różnorodne funkcje. Jedną z nich jest wchłanianie substancji odżywczych i wody z jelita cienkiego. Można wyróżnić dwie drogi, którymi są wchłanianie substancje odżywcze i woda: przez błonę komórkową i między...
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Koncepcja systemu do pomiaru temperatury podczas prób wysiłkowych u dzieci
PublikacjaOdpowiednia podaż tlenu i gwarancja prawidłowego metabolizmu tlenowego organizmu należą do głównych czynników determinujących wydolność fizyczną człowieka oraz możliwość kontynowania przez niego wysiłku fizycznego. Od lat trwają badania nad instrumentami pomocnymi w ocenie zmian przystosowawczych oraz adaptacyjnych, zachodzących pod wpływem wysiłku fizycznego oraz treningu fizycznego. W przypadku grup pediatrycznych poddawanych...
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Koncepcja systemu do pomiaru temperatury podczas prób wysiłkowych u dzieci
PublikacjaOdpowiednia podaż tlenu i gwarancja prawidłowego metabolizmu tlenowego organizmu należą do głównych czynników determinujących wydolność fizyczną człowieka oraz możliwość kontynuowania przez niego wysiłku fizycznego. Od lat trwają badania nad instrumentami pomocnymi w ocenie zmian przystosowawczych oraz adaptacyjnych, zachodzących pod wpływem wysiłku fizycznego oraz treningu fizycznego. W przypadku grup pediatrycznych poddawanych...
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Clinical studies of detecting COVID-19 from exhaled breath with electronic nose
PublikacjaThe COVID‑19 pandemic has attracted numerous research studies because of its impact on society and the economy. The pandemic has led to progress in the development of diagnostic methods, utilizing the polymerase chain reaction (PCR) as the gold standard for coronavirus SARS‑CoV‑2 detection. Numerous tests can be used at home within 15 min or so but of with lower accuracy than PCR. There is still a need for point‑of‑care tests available...
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In Silico Safety Assessment of Bacillus Isolated from Polish Bee Pollen and Bee Bread as Novel Probiotic Candidates
PublikacjaBacillus species isolated from Polish bee pollen (BP) and bee bread (BB) were characterized for in silico probiotic and safety attributes. A probiogenomics approach was used, and in-depth genomic analysis was performed using a wide array of bioinformatics tools to investigate the presence of virulence and antibiotic resistance properties, mobile genetic elements, and secondary metabolites. Functional annotation and Carbohydrate-Active...
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Evaluation of Partial Nitritation/Anammox (PN/A) Process Performance and Microorganisms Community Composition under Different C/N Ratio
PublikacjaA one-stage partial nitritation/anammox (PN/A) process with intermittent aeration is possible under sidestream conditions, but implementation in a mainstream is a challenge due to increased Carbon/Nitrogen (C/N) ratios in domestic wastewater. This study investigated the effect of C/N ratios on process efficiency and the effect of narrowing non-aeration time on process improvement at high chemical oxygen demand (COD) load. An increase...
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Bioavailability of Tannins and Other Oligomeric Polyphenols: a Still to Be Studied Phenomenon
PublikacjaPurpose of Review Polyphenols are a group of plant secondary metabolites characterized with the presence ofmultiples of phenol structural units. Tannins are plant originated phenolic substances, which play an important role in plant defense system as well as have found applications in medicine, industry, and other fields of human activity. Despite these, the mechanisms of bioavailability of these substances are poorly studied and...
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Anticancer Imidazoacridinone C-1311 is Effective in Androgen-Dependent and Androgen-Independent Prostate Cancer Cells
PublikacjaAndrogen receptor (AR) plays a crutial role in prostate cancer (PCa) development and metastasis. Here, we reported potent anti-PCa activity of a small molecule imidazoacridinone C-1311. In AR-positive PCa cells, C-1311 was found to inhibit the transcriptional activity of AR uncovering a novel mechanism that may be relevant for its anticancer effect. Mechanistically, C-1311 decreased AR binding to prostate-specific antigen (PSA)...
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Flap Monitoring Techniques: A Review
PublikacjaPostoperative tissue flap vitality monitoring enables early detection of clinical complications, allowing for intervention. Timely re-operation can prevent the need for extensive correction procedures, thus reducing healthcare costs and hospitalisation time. Statistics show that monitoring can increase the success rate of flap survival to 95\% or higher. However, despite the significant progress in monitoring techniques, major...
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Environmental fate of endocrine disrupting compounds - analytical problems and challenges
PublikacjaNarażenie na substancje wykazujące się aktywnością hormonów płciowych może negatywnie wpływać na układ endokrynny i rozrodczy u ludzi i innych organizmów żywych. Badania wykazały że obserwowane są znaczne wzrosty przypadków występowania nowotworów piersi, prostaty i jąder. Inne zaś wykazały obniżoną liczbę plemników w nasieniu mężczyzn i wydłużenie czasu oczekiwania na poczęcie. Odkrycia te znajdują potwierdzenie w badaniach polowych...
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Selective Cytostatic and Cytotoxic Anticancer Effects of Bisfunctional Agents: A Strategy for the Design of DNA Binding Agents
PublikacjaVarious agents have been synthesized and proved useful for the National Cancer Institute's anticancer testing as potential new drugs, but most agents suffer side effects from their limited selectivity against cancer cells over healthy ones. Therefore, this paper attempts to describe drugs in terms of the level of tumor cell selectivity which they possess to define the features of mols. that are essential for useful cytotoxicity....
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Biocompatibility and potential functionality of lanthanum-substituted cobalt ferrite spinels
PublikacjaBulk and nanostructurized lanthanum-cobalt spinels have attracted a lot of interest from researchers, due to their unique physical and chemical properties as well as functionalities, which are interesting for biomedical and electronic industries. In this manuscript we show that introducing small lanthanum (La3+) content can tune magnetic, electronic and cytotoxic properties of the CoFe2− xLaxO4 system (x ≤ 0.1). The mechanisms...
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Recent advances in 3D bioprinted tumor models for personalized medicine
PublikacjaCancerous tumors are among the most fatal diseases worldwide, claiming nearly 10 million lives in 2020. Due to their complex and dynamic nature, modeling tumors accurately is a challenging task. Current models suffer from inadequate translation between in vitro and in vivo results, primarily due to the isotropic nature of tumors and their microenvironment's relationship. To address these limitations, hydrogel-based 3D bioprinting...
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Antibacterial Activity and Cytocompatibility of Bone Cement Enriched with Antibiotic, Nanosilver, and Nanocopper for Bone Regeneration
PublikacjaBacterial infections due to bone replacement surgeries require modifications of bone cement with antibacterial components. This study aimed to investigate whether the incorporation of gentamicin or nanometals into bone cement may reduce and to what extent bacterial growth without the loss of overall cytocompatibility and adverse effects in vitro. The bone cement Cemex was used as the base material, modified either with gentamicin...
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Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant
PublikacjaBionanocellulose (BNC) is a clear polymer produced by the bacterium Gluconacetobacter xylinus. In our current study, “Research on the use of bacterial nanocellulose (BNC) in regenerative medicine as a function of the biological implants in cardiac and vascular surgery”, we carried out material analysis, biochemical analysis, in vitro tests and in vivo animal model testing. In stage 1 of the project, we carried out physical and...
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Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA
PublikacjaAn essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA–protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the...
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Evaluating the antibacterial activity of muramyl dipeptide derivatives, retro-tuftsin derivatives, and anthraquinone oligopeptides against a range of pathogenic bacteria
PublikacjaSearch for new and efficient antibiotic is crucial because of microbial drug resistance and problems with side effects of the administered medication. In this study, we evaluate the in vitro microbiological activity of muramyl dipeptide derivatives, retro-tuftsin derivatives (i.e., tuftsin with reversed amino acid sequences), and combinations of retro-tuftsin derivatives with substituted anthraquinones. The potency of the investigated...
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Diagnostic Accuracy of Liquid Biopsy in Endometrial Cancer
PublikacjaBackground: Liquid biopsy is a minimally invasive collection of a patient body fluid sample. In oncology, they offer several advantages compared to traditional tissue biopsies. However, the potential of this method in endometrial cancer (EC) remains poorly explored. We studied the utility of tumor educated platelets (TEPs) and circulating tumor DNA (ctDNA) for preoperative EC diagnosis, including histology determination. Methods:...